Advertisement

Aptamers targeting amyloidogenic proteins and their emerging role in neurodegenerative diseases

Open AccessPublished:December 08, 2021DOI:https://doi.org/10.1016/j.jbc.2021.101478
      Aptamers are oligonucleotides selected from large pools of random sequences based on their affinity for bioactive molecules and are used in similar ways to antibodies. Aptamers provide several advantages over antibodies, including their small size, facile, large-scale chemical synthesis, high stability, and low immunogenicity. Amyloidogenic proteins, whose aggregation is relevant to neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and prion diseases, are among the most challenging targets for aptamer development due to their conformational instability and heterogeneity, the same characteristics that make drug development against amyloidogenic proteins difficult. Recently, chemical tethering of aptagens (equivalent to antigens) and advances in high-throughput sequencing-based analysis have been used to overcome some of these challenges. In addition, internalization technologies using fusion to cellular receptors and extracellular vesicles have facilitated central nervous system (CNS) aptamer delivery. In view of the development of these techniques and resources, here we review antiamyloid aptamers, highlighting preclinical application to CNS therapy.

      Keywords

      Abbreviations:

      αSyn (α-synuclein), (amyloid β-protein), AD (Alzheimer's disease), ADDLs (Aβ-derived diffusible ligands), AI (artificial intelligence), CD (circular dichroism), HT-SELEX (high throughput-systematic evolution of ligands by exponential enrichment), PD (Parkinson's disease), PICUP (photo-induced cross-linking of unmodified protein), RVG (rabies viral glycoprotein), SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis), SELEX (systematic evolution of ligands by exponential enrichment)
      Neurodegenerative diseases are characterized by a progressive loss of neuronal function. Most of these diseases are age-related, and self-assembly of amyloidogenic proteins is thought to be a cause or a major deleterious mechanism in many of them. Examples include Alzheimer’s disease (AD) and various other tauopathies, Parkinson’s diseases (PD) and other synucleinopathies, prion diseases, and many other sporadic or genetic proteinopathies. The amyloidogenic proteins involved in these diseases are prone to self-association into neurotoxic oligomers and amyloid fibrils (Fig. 1). In many cases, the oligomers, which have metastable structures, have been shown to play pivotal roles in the pathogenesis of the associated diseases and to be more toxic than the structurally stable fibrils (
      • Iadanza M.G.
      • Jackson M.P.
      • Hewitt E.W.
      • Ranson N.A.
      • Radford S.E.
      A new era for understanding amyloid structures and disease.
      ). The abnormal protein assemblies cause neurotoxicity by a variety of mechanisms, including apoptosis, oxidative stress, inflammation, and disruption of proteostasis through blockage of proteasomal and lysosomal protein degradation. Given the significance of oligomers in the pathogenesis of many neurodegenerative diseases (
      • Hartl F.U.
      Protein misfolding diseases.
      ,
      • Chuang E.
      • Hori A.M.
      • Hesketh C.D.
      • Shorter J.
      Amyloid assembly and disassembly.
      ,
      • Otzen D.
      • Riek R.
      Functional amyloids.
      ), they have been the focus of attention as molecular targets of both diagnostic and therapeutic research and development. Therefore, generation of specific oligomer-binding reagents is a promising approach for development of early detection tools and redirecting the self-assembly process into dissociation back to nontoxic monomers, formation of nontoxic and nonamyloidogenic assemblies amenable to degradation, or in some cases, accelerating the aggregation process to reduce steady-state levels of oligomers in favor of less-toxic fibrils.
      Figure thumbnail gr1
      Figure 1A Schematic diagram of amyloidogenic proteins aggregation. Amyloidogenic proteins can be naturally structured or unstructured. Naturally structured proteins (a), e.g., PrP undergo partial unfolding, whereas naturally unstructured proteins (b), such as Aβ, αSyn, or tau, undergo partial folding under pathological conditions, initiating the self-assembly process. Both cases lead to formation of partially (un)folded monomers, which self-associate into increasing-size oligomers until a quasi-stable nucleus forms leading to the elongation phase. Elongation typically proceeds at a fast rate compared with the nucleation and may involve formation of quasi-stable high-molecular-weight oligomers, protofibrils, and eventually fibrils. Finally, the monomers are consumed and the system reaches a stationary phase in which no more growth is observed.
      Aptamers are molecular-recognition agents comprising single-stranded DNA or RNA oligonucleotides that similar to antibodies, bind specifically to diverse targets, including small molecules, peptides, proteins, and nucleic acids (
      • Zhou J.
      • Rossi J.
      Aptamers as targeted therapeutics: Current potential and challenges.
      ). Amyloidogenic proteins inherently tend to bind nucleic acids (
      • Ginsberg S.D.
      • Galvin J.E.
      • Chiu T.S.
      • Lee V.M.
      • Masliah E.
      • Trojanowski J.Q.
      RNA sequestration to pathological lesions of neurodegenerative diseases.
      ,
      • Ginsberg S.D.
      • Crino P.B.
      • Hemby S.E.
      • Weingarten J.A.
      • Lee V.M.
      • Eberwine J.H.
      • Trojanowski J.Q.
      Predominance of neuronal mRNAs in individual Alzheimer's disease senile plaques.
      ,
      • Hua Q.
      • He R.Q.
      • Haque N.
      • Qu M.H.
      • del Carmen Alonso A.
      • Grundke-Iqbal I.
      • Iqbal K.
      Microtubule associated protein tau binds to double-stranded but not single-stranded DNA.
      ) making it difficult to select aptamers specific for just one protein and even more so for distinct assembly states of these proteins. Nonetheless, aptamers offer several advantages compared with antibodies including their small size, facile chemical synthesis, including in large scale, high stability, and low immunogenicity, making them attractive for researchers aiming at developing molecular recognition tools for amyloidogenic proteins. Aptamers typically are obtained by selection from a random-sequence oligonucleotide library based on their affinity for the target of interest using a method called systematic evolution of ligands by exponential enrichment (SELEX, Fig. 2). The oligonucleotides usually span 30 to 100 nucleotides in length and their dissociation constants in complexes with their targets range from pM to mM. The sequence also contains constant regions required for enzymatic manipulation, such as PCR-primer binding and in vitro transcription.
      Figure thumbnail gr2
      Figure 2Generation of aptamers by SELEX. The process of SELEX can be used for selection of DNA or RNA aptamers. After the initial PCR amplification of the template DNA, single-strand nucleic acid sequences need to be prepared from the double-strand DNA for binding to the target and selection of high-affinity sequences. This is done using NaOH denaturation or enzyme digestion for DNA aptamers and by in vitro transcription for RNA aptamers. Unbound sequences are discarded and the bound oligonucleotide pool is released from the target. DNA sequences are subjected to PCR amplification to produce double-strand DNA for the next round of selection, whereas RNA sequences are reverse-transcribed first and then amplified by PCR for the next cycle.
      A systematic survey by Dumontier, DeRosa, and their colleagues (
      • McKeague M.
      • McConnell E.M.
      • Cruz-Toledo J.
      • Bernard E.D.
      • Pach A.
      • Mastronardi E.
      • Zhang X.
      • Beking M.
      • Francis T.
      • Giamberardino A.
      • Cabecinha A.
      • Ruscito A.
      • Aranda-Rodriguez R.
      • Dumontier M.
      • DeRosa M.C.
      Analysis of in vitro aptamer selection parameters.
      ) based on 492 published aptamer-related papers has found that the use of DNA aptamers is increasing compared with RNA aptamers. Each nucleic acid has its own advantages and limitations. DNA oligonucleotides are more stable than their RNA counterparts to enzymatic and chemical degradation and are therefore easier to work with. On the other hand, the presence of the 2′-OH in ribose, as opposed to deoxyribose, and the absence of the 5′-methyl group in uracil compared with thymine allow higher conformational stability of RNA, potentially increasing their affinity for the target (
      • Shu Y.
      • Pi F.
      • Sharma A.
      • Rajabi M.
      • Haque F.
      • Shu D.
      • Leggas M.
      • Evers B.M.
      • Guo P.
      Stable RNA nanoparticles as potential new generation drugs for cancer therapy.
      ). Deciding whether to use DNA or RNA aptamers is thus a crucial step in the beginning of every aptamer-based research project.
      The application of aptamers in the neurodegenerative-disease field has been reviewed in the past (
      • Qu J.
      • Yu S.
      • Zheng Y.
      • Zheng Y.
      • Yang H.
      • Zhang J.
      Aptamer and its applications in neurodegenerative diseases.
      ,
      • Bouvier-Muller A.
      • Duconge F.
      Nucleic acid aptamers for neurodegenerative diseases.
      ,
      • Rahimi F.
      Aptamers selected for recognizing amyloid β-protein-a case for cautious optimism.
      ), yet to our knowledge, there are no systematic reviews on aptamers targeting amyloidogenic proteins. The metastable nature of intrinsically disordered or misfolded proteins, which are prone to form toxic oligomers and eventually amyloid, makes these proteins one of the most challenging targets for aptamer generation. In the case of oligomers, the aptagens (equivalent to antigens) presented to the oligonucleotide library constantly change, whereas in the amyloid fibrils, the conformation of the aptagens can be both variable for the same protein due to formation of different strains and similar for different proteins in the core cross-β structure shared by most amyloid fibrils.
      Aptamers selected against amyloidogenic proteins can have various applications, including sensitive detection of biomarkers, as selective inhibitors of the self-assembly process, and as tools for probing molecular mechanisms. The first application—using aptamers as probes for biomarkers—has been developing rapidly and is too large to include in this review. Therefore, we focus here on the challenges and potential solutions in this field and on preclinical therapeutic applications of aptamers specific for amyloidogenic proteins.
      To overcome the difficulty in selecting aptamers against metastable protein assemblies, approaches using stable mimics of these assemblies could be useful if the stabilized molecules represent accurately the metastable target. Here, we provide an update on the development of specific aptamers against amyloidogenic proteins, including amyloid β-protein (Aβ), tau, α-synuclein (αSyn), and prion protein (PrP). These proteins represent a range of sizes spanning an order of magnitude—from 40- to 441-amino acid residues, and the two main mechanisms of initial misfolding and aggregation—partial folding of an unstructured protein, such as Aβ, αSyn, or tau, and partial unfolding of a structured protein—PrP (Fig. 1) (
      • Fezoui Y.
      • Teplow D.B.
      Kinetic studies of amyloid β-protein fibril assembly. Differential effects of α-helix stabilization.
      ). We discuss how bioinformatics-assisted approaches or artificial-intelligence-based technologies are used to assist the aptamer selection and optimization processes. We also examine approaches for analysis of the secondary and tertiary structures of aptamers and aptamer–target interaction and strategies for CNS-targeting delivery of aptamers for future development of therapeutics in neurodegenerative diseases.

      Development of aptamers against amyloidogenic proteins

      Several dozen amyloidogenic proteins play major deleterious roles in over 50 proteinopathies. However, studies of aptamers against these proteins have concentrated primarily on four proteins, Aβ, tau, αSyn, and PrP, and therefore, these proteins are also the focus of this review.

      Aptamers against Aβ

      AD is the most common neurodegenerative disease. Amyloid fibrils in senile plaques in the AD brain consist mainly of the 42-amino acid residue form of Aβ, Aβ42, whereas vascular deposits comprise predominantly the 40-residue form, Aβ40. Both forms are generated from the Aβ-protein precursor by the somewhat promiscuous protease, γ-secretase (
      • Glenner G.G.
      • Wong C.W.
      Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein.
      ,
      • Masters C.L.
      • Simms G.
      • Weinman N.A.
      • Multhaup G.
      • McDonald B.L.
      • Beyreuther K.
      Amyloid plaque core protein in Alzheimer disease and Down syndrome.
      ,
      • Karran E.
      • Mercken M.
      • De Strooper B.
      The amyloid cascade hypothesis for Alzheimer's disease: An appraisal for the development of therapeutics.
      ). Aβ42 aggregates faster (
      • Jarrett J.T.
      • Berger E.P.
      • Lansbury Jr., P.T.
      The carboxy terminus of the β amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer's disease.
      ), forms higher-molecular-weight oligomers (
      • Bitan G.
      • Kirkitadze M.D.
      • Lomakin A.
      • Vollers S.S.
      • Benedek G.B.
      • Teplow D.B.
      Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways.
      ), and is more neurotoxic than Aβ40 (
      • Haass C.
      • Selkoe D.J.
      Soluble protein oligomers in neurodegeneration: Lessons from the Alzheimer's amyloid β-peptide.
      ), and the oligomers of both isoforms are more neurotoxic than the corresponding fibrils (
      • Dahlgren K.N.
      • Manelli A.M.
      • Stine Jr., W.B.
      • Baker L.K.
      • Krafft G.A.
      • LaDu M.J.
      Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability.
      ,
      • Roychaudhuri R.
      • Yang M.
      • Hoshi M.M.
      • Teplow D.B.
      Amyloid β-protein assembly and Alzheimer disease.
      ), making Aβ42 oligomers a primary target for therapy development. In this context, it is important to consider, however, that fibrils might sequester the more toxic Aβ oligomers and possibly are a way cells attempt to reduce the damage caused by the oligomers (
      • Meyer-Luehmann M.
      • Spires-Jones T.L.
      • Prada C.
      • Garcia-Alloza M.
      • de Calignon A.
      • Rozkalne A.
      • Koenigsknecht-Talboo J.
      • Holtzman D.M.
      • Bacskai B.J.
      • Hyman B.T.
      Rapid appearance and local toxicity of amyloid-β plaques in a mouse model of Alzheimer's disease.
      ). Thus, if strategies targeting fibril dissociation are considered, inadvertent increase in the concentration of the toxic oligomers, leading to exacerbation, rather than amelioration, of the disease might occur, and one must ensure that this is not the case.
      A complicating factor is that “oligomer” is a loosely defined term used for anything from a dimer to large assemblies consisting of hundreds of monomers (
      • Roychaudhuri R.
      • Yang M.
      • Hoshi M.M.
      • Teplow D.B.
      Amyloid β-protein assembly and Alzheimer disease.
      ,
      • Benilova I.
      • Karran E.
      • De Strooper B.
      The toxic Aβ oligomer and Alzheimer's disease: An emperor in need of clothes.
      ,
      • Murakami K.
      Conformation-specific antibodies to target amyloid β oligomers and their application to immunotherapy for Alzheimer's disease.
      ,
      • Bitan G.
      • Fradinger E.A.
      • Spring S.M.
      • Teplow D.B.
      Neurotoxic protein oligomers--what you see is not always what you get.
      ) as long as these assemblies are soluble in aqueous solutions, as opposed to fibrils and other insoluble aggregates. Many different types of Aβ oligomers have been reported, including paranuclei (5,6-mers) (
      • Bitan G.
      • Kirkitadze M.D.
      • Lomakin A.
      • Vollers S.S.
      • Benedek G.B.
      • Teplow D.B.
      Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways.
      ), Aβ∗56 (56 kDa, 12-mer) (
      • Lesnè S.
      • Koh M.T.
      • Kotilinek L.
      • Kayed R.
      • Glabe C.G.
      • Yang A.
      • Gallagher M.
      • Ashe K.H.
      A specific amyloid-β protein assembly in the brain impairs memory.
      ), protofibrils (PFs, 24–700-mer) (
      • Walsh D.M.
      • Lomakin A.
      • Benedek G.B.
      • Condron M.M.
      • Teplow D.B.
      Amyloid β-protein fibrillogenesis. Detection of a protofibrillar intermediate.
      ,
      • Walsh D.M.
      • Hartley D.M.
      • Kusumoto Y.
      • Fezoui Y.
      • Condron M.M.
      • Lomakin A.
      • Benedek G.B.
      • Selkoe D.J.
      • Teplow D.B.
      Amyloid β-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates.
      ,
      • Harper J.D.
      • Wong S.S.
      • Lieber C.M.
      • Lansbury P.T.
      Observation of metastable Aβ amyloid protofibrils by atomic force microscopy.
      ), globulomers (38/48 kDa, ∼12-mer) (
      • Barghorn S.
      • Nimmrich V.
      • Striebinger A.
      • Krantz C.
      • Keller P.
      • Janson B.
      • Bahr M.
      • Schmidt M.
      • Bitner R.S.
      • Harlan J.
      • Barlow E.
      • Ebert U.
      • Hillen H.
      Globular amyloid β-peptide oligomer - a homogenous and stable neuropathological protein in Alzheimer's disease.
      ), AβO (∼90 kDa, 15–20-mer) (
      • Deshpande A.
      • Mina E.
      • Glabe C.
      • Busciglio J.
      Different conformations of amyloid β induce neurotoxicity by distinct mechanisms in human cortical neurons.
      ), Aβ-derived diffusible ligands (ADDLs, ∼90 kDa, ∼24-mer) (
      • Lambert M.P.
      • Barlow A.K.
      • Chromy B.A.
      • Edwards C.
      • Freed R.
      • Liosatos M.
      • Morgan T.E.
      • Rozovsky I.
      • Trommer B.
      • Viola K.L.
      • Wals P.
      • Zhang C.
      • Finch C.E.
      • Krafft G.A.
      • Klein W.L.
      Diffusible, nonfibrillar ligands derived from Aβ1-42 are potent central nervous system neurotoxins.
      ), annuli (150–250 kDa, ∼50-mer) (
      • Caughey B.
      • Lansbury P.T.
      Protofibrils, pores, fibrils, and neurodegeneration: Separating the responsible protein aggregates from the innocent bystanders.
      ), and amylospheroids (ASPD; 158–669 kDa, ∼100-mer) (
      • Hoshi M.
      • Sato M.
      • Matsumoto S.
      • Noguchi A.
      • Yasutake K.
      • Yoshida N.
      • Sato K.
      Spherical aggregates of β-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3β.
      ). Thus, when one contemplates selection of aptamers against Aβ oligomers, an important step is definition of the oligomers used as a target for the selection process.
      Protofibrils and ADDLs were the first types of Aβ oligomers described, in 1997 (
      • Walsh D.M.
      • Lomakin A.
      • Benedek G.B.
      • Condron M.M.
      • Teplow D.B.
      Amyloid β-protein fibrillogenesis. Detection of a protofibrillar intermediate.
      ,
      • Harper J.D.
      • Wong S.S.
      • Lieber C.M.
      • Lansbury P.T.
      Observation of metastable Aβ amyloid protofibrils by atomic force microscopy.
      ) and 1998 (
      • Lambert M.P.
      • Barlow A.K.
      • Chromy B.A.
      • Edwards C.
      • Freed R.
      • Liosatos M.
      • Morgan T.E.
      • Rozovsky I.
      • Trommer B.
      • Viola K.L.
      • Wals P.
      • Zhang C.
      • Finch C.E.
      • Krafft G.A.
      • Klein W.L.
      Diffusible, nonfibrillar ligands derived from Aβ1-42 are potent central nervous system neurotoxins.
      ), respectively. Follow-up studies have generated antibodies against these assemblies, including mAb158 (
      • Tucker S.
      • Moller C.
      • Tegerstedt K.
      • Lord A.
      • Laudon H.
      • Sjodahl J.
      • Soderberg L.
      • Spens E.
      • Sahlin C.
      • Waara E.R.
      • Satlin A.
      • Gellerfors P.
      • Osswald G.
      • Lannfelt L.
      The murine version of BAN2401 (mAb158) selectively reduces amyloid-β protofibrils in brain and cerebrospinal fluid of tg-ArcSwe mice.
      ) and NU-1 (
      • Lambert M.P.
      • Velasco P.T.
      • Chang L.
      • Viola K.L.
      • Fernandez S.
      • Lacor P.N.
      • Khuon D.
      • Gong Y.
      • Bigio E.H.
      • Shaw P.
      • De Felice F.G.
      • Krafft G.A.
      • Klein W.L.
      Monoclonal antibodies that target pathological assemblies of Aβ.
      ), which bind protofibrils and ADDLs, respectively. Many other oligomer-selective antibodies have been reported over the last 2 decades (
      • Tucker S.
      • Moller C.
      • Tegerstedt K.
      • Lord A.
      • Laudon H.
      • Sjodahl J.
      • Soderberg L.
      • Spens E.
      • Sahlin C.
      • Waara E.R.
      • Satlin A.
      • Gellerfors P.
      • Osswald G.
      • Lannfelt L.
      The murine version of BAN2401 (mAb158) selectively reduces amyloid-β protofibrils in brain and cerebrospinal fluid of tg-ArcSwe mice.
      ,
      • Lambert M.P.
      • Velasco P.T.
      • Chang L.
      • Viola K.L.
      • Fernandez S.
      • Lacor P.N.
      • Khuon D.
      • Gong Y.
      • Bigio E.H.
      • Shaw P.
      • De Felice F.G.
      • Krafft G.A.
      • Klein W.L.
      Monoclonal antibodies that target pathological assemblies of Aβ.
      ,
      • Kayed R.
      • Head E.
      • Thompson J.L.
      • McIntire T.M.
      • Milton S.C.
      • Cotman C.W.
      • Glabe C.G.
      Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.
      ,
      • Hillen H.
      • Barghorn S.
      • Striebinger A.
      • Labkovsky B.
      • Muller R.
      • Nimmrich V.
      • Nolte M.W.
      • Perez-Cruz C.
      • van der Auwera I.
      • van Leuven F.
      • van Gaalen M.
      • Bespalov A.Y.
      • Schoemaker H.
      • Sullivan J.P.
      • Ebert U.
      Generation and therapeutic efficacy of highly oligomer-specific β-amyloid antibodies.
      ,
      • Goñi F.
      • Marta-Ariza M.
      • Peyser D.
      • Herline K.
      • Wisniewski T.
      Production of monoclonal antibodies to pathologic β-sheet oligomeric conformers in neurodegenerative diseases.
      ,
      • Kayed R.
      • Canto I.
      • Breydo L.
      • Rasool S.
      • Lukacsovich T.
      • Wu J.
      • Albay 3rd, R.
      • Pensalfini A.
      • Yeung S.
      • Head E.
      • Marsh J.L.
      • Glabe C.
      Conformation dependent monoclonal antibodies distinguish different replicating strains or conformers of prefibrillar Aβ oligomers.
      ) as reviewed elsewhere (
      • Murakami K.
      Conformation-specific antibodies to target amyloid β oligomers and their application to immunotherapy for Alzheimer's disease.
      ), though due to the metastable nature of the targets, in all cases the specificity of the antibody was lower than what is typically expected for antibodies against stable antigens. Thus, stabilization of the antigens, for example, by attachment of Aβ to gold particles (
      • Kayed R.
      • Head E.
      • Thompson J.L.
      • McIntire T.M.
      • Milton S.C.
      • Cotman C.W.
      • Glabe C.G.
      Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.
      ), has been an important strategy for generation of antioligomer antibodies. However, an important consideration is that the relevance of the stabilized antigens to the pathological species in the AD brain must be established in each case. Of the many clinical trials using anti-Aβ antibodies, more recent ones have examined antibodies selective against Aβ oligomers, including Biogen’s aducanumab, which recently was approved by the Food and Drug Administration (FDA), and BAN2401, a humanized version of mAb158.
      The first aptamers targeting Aβ were reported in 2002 by Ylera et al. The aptamers were RNA oligonucleotides screened against Aβ40 monomers tethered to a Sepharose support. All the selected aptamers recognized Aβ fibrils but not monomers (
      • Ylera F.
      • Lurz R.
      • Erdmann V.A.
      • Furste J.P.
      Selection of RNA aptamers to the Alzheimer's disease amyloid peptide.
      ). In a later study by Hyman and coworkers (
      • Farrar C.T.
      • William C.M.
      • Hudry E.
      • Hashimoto T.
      • Hyman B.T.
      RNA aptamer probes as optical imaging agents for the detection of amyloid plaques.
      ), one of the aptamers reported by Ylera et al., β55, was used to visualize senile plaques using multiphoton microscopy in brain tissue from patients with AD and the APP/PS1 mouse model (
      • Jankowsky J.L.
      • Fadale D.J.
      • Anderson J.
      • Xu G.M.
      • Gonzales V.
      • Jenkins N.A.
      • Copeland N.G.
      • Lee M.K.
      • Younkin L.H.
      • Wagner S.L.
      • Younkin S.G.
      • Borchelt D.R.
      Mutant presenilins specifically elevate the levels of the 42 residue β-amyloid peptide in vivo: Evidence for augmentation of a 42-specific γ secretase.
      ). The characteristics of these anti-Aβ aptamers and all the subsequent aptamers against amyloidogenic proteins included in this review are summarized in Table 1.
      Table 1Characteristics of aptamers against Aβ, tau, αSyn, and PrP
      NameTargetNucleic acidKD (testing method)SelectivityYearRef.
      Anti-Aβ aptamers
       β55Aβ40 monomersRNA29 nM (affinity chromatography)fibrils2002(
      • Ylera F.
      • Lurz R.
      • Erdmann V.A.
      • Furste J.P.
      Selection of RNA aptamers to the Alzheimer's disease amyloid peptide.
      )
       KM33Aβ40 trimersRNAN.T.
      Not tested.
      fibrils2009(
      • Rahimi F.
      • Murakami K.
      • Summers J.L.
      • Chen C.H.
      • Bitan G.
      RNA aptamers generated against oligomeric Aβ40 recognize common amyloid aptatopes with low specificity but high sensitivity.
      )
       T-SO508αSyn oligomersDNA25 nM for Aβ40 oligomers (ELONA
      Enzyme-linked oligonucleotide assay.
      )
      oligomers
      Size is unspecified, unless stated otherwise.
      of αSyn and Aβ40
      2012(
      • Tsukakoshi K.
      • Abe K.
      • Sode K.
      • Ikebukuro K.
      Selection of DNA aptamers that recognize α-synuclein oligomers using a competitive screening method.
      )
       E2Aβ40RNA10.9 μM (fluorescence anisotropy)fibrils2009(
      • Takahashi T.
      • Tada K.
      • Mihara H.
      RNA aptamers selected against amyloid β-peptide (Aβ) inhibit the aggregation of Aβ.
      )
       RNV95Aβ40DNAN.T.oligomers (∼75 kDa and ∼150 kDa)2018(
      • Chakravarthy M.
      • AlShamaileh H.
      • Huang H.
      • Tannenberg R.K.
      • Chen S.
      • Worrall S.
      • Dodd P.R.
      • Veedu R.N.
      Development of DNA aptamers targeting low-molecular-weight amyloid-β peptide aggregates in vitro.
      )
       E22P-AbD43Aβ42 protofibrilsRNA20 nM (BLI
      BioLayer interferometry.
      )
      dimers2020(
      • Murakami K.
      • Obata Y.
      • Sekikawa A.
      • Ueda H.
      • Izuo N.
      • Awano T.
      • Takabe K.
      • Shimizu T.
      • Irie K.
      An RNA aptamer with potent affinity for a toxic dimer of amyloid β42 has potential utility for histochemical studies of Alzheimer's disease.
      )
       Aβ7-92-1H1Aβ42DNA53.3 nM (SPR
      Surface plasmon resonance.
      )
      oligomers2020(
      • Zheng Y.
      • Wang P.
      • Li S.
      • Geng X.
      • Zou L.
      • Jin M.
      • Zou Q.
      • Wang Q.
      • Yang X.
      • Wang K.
      Development of DNA aptamer as a β-amyloid aggregation inhibitor.
      )
      Antitau aptamers
       ssDNA11N3R-tauDNA190 nM (capillary electrophoresis)monomers2005(
      • Krylova S.M.
      • Musheev M.
      • Nutiu R.
      • Li Y.
      • Lee G.
      • Krylov S.N.
      Tau protein binds single-stranded DNA sequence specifically--the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures.
      )
       31462N4R-tauDNA13 nM (SPR)monomers2018(
      • Lisi S.
      • Fiore E.
      • Scarano S.
      • Pascale E.
      • Boehman Y.
      • Duconge F.
      • Chierici S.
      • Minunni M.
      • Peyrin E.
      • Ravelet C.
      Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau proteins.
      )
       tau-12N4R-tauRNAN.T.dimers and trimers2016(
      • Kim J.H.
      • Kim E.
      • Choi W.H.
      • Lee J.
      • Lee J.H.
      • Lee H.
      • Kim D.E.
      • Suh Y.H.
      • Lee M.J.
      Inhibitory RNA aptamers of tau oligomerization and their neuroprotective roles against proteotoxic stress.
      )
      Anti-αSyn aptamers
       M5-15αSynDNAN.T.monomer and oligomers2010(
      • Tsukakoshi K.
      • Harada R.
      • Sode K.
      • Ikebukuro K.
      Screening of DNA aptamer which binds to α-synuclein.
      )
       T-SO530αSyn oligomersDNA63 nM for αSyn oligomers (ELONA)oligomers of αSyn and Aβ402012(
      • Tsukakoshi K.
      • Abe K.
      • Sode K.
      • Ikebukuro K.
      Selection of DNA aptamers that recognize α-synuclein oligomers using a competitive screening method.
      )
       F5R1αSynDNA2.4 nM (SPR)N.T.2018(
      • Zheng Y.
      • Qu J.
      • Xue F.
      • Zheng Y.
      • Yang B.
      • Chang Y.
      • Yang H.
      • Zhang J.
      Novel DNA aptamers for Parkinson's disease treatment inhibit α-synuclein aggregation and facilitate its degradation.
      )
      Antiprion aptamers
       Ap1hamster PrP23–231RNAN.T.hamster, mouse, cattle PrPC1997(
      • Weiss S.
      • Proske D.
      • Neumann M.
      • Groschup M.H.
      • Kretzschmar H.A.
      • Famulok M.
      • Winnacker E.L.
      RNA aptamers specifically interact with the prion protein PrP.
      )
       60–3mouse PrP23–230RNA5.6 nM for PrPC (competitive assay)mouse and bovine PrPC2006(
      • Sekiya S.
      • Noda K.
      • Nishikawa F.
      • Yokoyama T.
      • Kumar P.K.
      • Nishikawa S.
      Characterization and application of a novel RNA aptamer against the mouse prion protein.
      )
       RM312sheep PrP23–234RNA15 nM for PrPC (SPR)N.T.2006(
      • Mercey R.
      • Lantier I.
      • Maurel M.C.
      • Grosclaude J.
      • Lantier F.
      • Marc D.
      Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns.
      )
       4–9mouse PrP23–230DNA113 nM for PrPC, 100 nM for PrPSc (SPR)PrPC ≅ β-isoform of PrPSc2007(
      • Ogasawara D.
      • Hasegawa H.
      • Kaneko K.
      • Sode K.
      • Ikebukuro K.
      Screening of DNA aptamer against mouse prion protein by competitive selection.
      )
       4C26mouse PrP90–231DNA18 nM for PrPC (affinity assay)PrPC (N.T. for PrPSc)2008(
      • Bibby D.F.
      • Gill A.C.
      • Kirby L.
      • Farquhar C.F.
      • Bruce M.E.
      • Garson J.A.
      Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins.
      )
       SAF-93PrP fibris from infected hamster brainRNA23.4 nM for PrPSc (affinity assay)PrPSc > PrPC2003(
      • Rhie A.
      • Kirby L.
      • Sayer N.
      • Wellesley R.
      • Disterer P.
      • Sylvester I.
      • Gill A.
      • Hope J.
      • James W.
      • Tahiri-Alaoui A.
      Characterization of 2'-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion.
      )
       DP7human PrP90–129RNA1.7 μM for PrPC (affinity assay)human, hamster, mouse PrPC2002(
      • Proske D.
      • Gilch S.
      • Wopfner F.
      • Schatzl H.M.
      • Winnacker E.L.
      • Famulok M.
      Prion-protein-specific aptamer reduces PrPSc formation.
      )
       SSAP3-10human PrP23–231DNAN.T.human, sheep, calf, piglet, deer PrPC, but not PrPSc2006(
      • Takemura K.
      • Wang P.
      • Vorberg I.
      • Surewicz W.
      • Priola S.A.
      • Kanthasamy A.
      • Pottathil R.
      • Chen S.G.
      • Sreevatsan S.
      DNA aptamers that bind to PrP(C) and not PrP(Sc) show sequence and structure specificity.
      )
       R14 (from apt#1)bovine PrP25–241RNA8.5 nM for PrPC, 280 nM for PrPSc (affinity assay)PrPC > PrPSc2008(
      • Murakami K.
      • Nishikawa F.
      • Noda K.
      • Yokoyama T.
      • Nishikawa S.
      Anti-bovine prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its β isoform with high affinity.
      )
      a Not tested.
      b Enzyme-linked oligonucleotide assay.
      c Size is unspecified, unless stated otherwise.
      d BioLayer interferometry.
      e Surface plasmon resonance.
      To select aptamers against Aβ oligomers, several chemical tethering approaches have been used in later studies. Rahimi et al. prepared covalently cross-linked Aβ40 oligomers using Photo-Induced Cross-linking of Unmodified Proteins (PICUP) (
      • Bitan G.
      Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.
      ,
      • Bitan G.
      • Teplow D.B.
      Rapid photochemical cross-linking--a new tool for studies of metastable, amyloidogenic protein assemblies.
      ), which creates covalent bonds at unspecified positions, primarily at Tyr10 in Aβ (Fig. 3A) (
      • Maji S.K.
      • Ogorzalek Loo R.R.
      • Inayathullah M.
      • Spring S.M.
      • Vollers S.S.
      • Condron M.M.
      • Bitan G.
      • Loo J.A.
      • Teplow D.B.
      Amino acid position-specific contributions to amyloid β-protein oligomerization.
      ). Of several Aβ40 oligomers stabilized by this technique, they then isolated the most abundant type, trimers, using SDS-PAGE and used the cross-linked trimers to isolate RNA aptamers out of a 77-nucleotide library including 49 randomized nucleotides (A:U:G:C at equal ratios). This size library contains 3.2 × 1029 unique sequences theoretically, yet in reality, if every sequence indeed was included in the library, the mass of such a library would be too high, and the actual number of unique sequences is lower.
      Figure thumbnail gr3
      Figure 3Strategies for stabilization of Aβ oligomers for generation of aptamers. A, photo-cross-linking of Aβ40 by PICUP. Cross-linking is induced by visible-light irradiation in the presence of the photocatalyst tris(bipyridyl)ruthenium(II) ([Ru(bpy)3]2+) and the electron acceptor ammonium persulfate. This method leads to “zero-length” cross-linking directly between amino acid residues, primarily tryptophane and tyrosine. In Aβ, the main cross-link is at Y10, though other bonds also can form. B, a turn structure in Aβ42 is stabilized by an E22P substitution. Dimer stabilization is achieved through substituting V40 by a divalent amino acid (e.g., 2,6-diaminopimelic acid).
      Although the selection of the RNA aptamers was against Aβ40 trimers, the final aptamers, KM33 and KM41, were not selective for trimers or other oligomers, but bound to Aβ40 fibrils and did not display a higher affinity for the fibrils than the naive oligonucleotide library used for the selection before enrichment (
      • Rahimi F.
      • Murakami K.
      • Summers J.L.
      • Chen C.H.
      • Bitan G.
      RNA aptamers generated against oligomeric Aβ40 recognize common amyloid aptatopes with low specificity but high sensitivity.
      ,
      • Rahimi F.
      • Bitan G.
      Selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.
      ). Part of the explanation of these results was that the PICUP-immobilized Aβ40 trimers themselves might have aggregated during the selection process to form fibrillar structures. Further analysis suggested that oligonucleotides have a high, nonspecific affinity for amyloid fibrils, as KM33 and KM41 also recognized fibrils of other amyloidogenic proteins, including calcitonin, islet amyloid polypeptide, insulin, lysozyme, and PrP106–126 (
      • Rahimi F.
      • Murakami K.
      • Summers J.L.
      • Chen C.H.
      • Bitan G.
      RNA aptamers generated against oligomeric Aβ40 recognize common amyloid aptatopes with low specificity but high sensitivity.
      ), which share a cross-β structure and fibrillar morphology with Aβ fibrils (
      • Sipe J.D.
      • Cohen A.S.
      Review: History of the amyloid fibril.
      ). Thus, Rahimi et al. (
      • Rahimi F.
      • Murakami K.
      • Summers J.L.
      • Chen C.H.
      • Bitan G.
      RNA aptamers generated against oligomeric Aβ40 recognize common amyloid aptatopes with low specificity but high sensitivity.
      ) demonstrated that the aptamers could be used to monitor fibril formation in a similar manner to thioflavin T (ThT) fluorescence.
      Interestingly, a DNA aptamer called T-SO517, originally developed by Ikebukuro and colleagues against oligomeric αSyn, a key protein involved in PD and other synucleinopathies, bound not only to their intended target, αSyn oligomers, but also to Aβ40 oligomers (
      • Tsukakoshi K.
      • Abe K.
      • Sode K.
      • Ikebukuro K.
      Selection of DNA aptamers that recognize α-synuclein oligomers using a competitive screening method.
      ), demonstrating the difficulty in obtaining highly specific aptamers against assemblies of amyloidogenic proteins. Considering the high affinity of the KM aptamers mentioned above for fibrils, these findings likely reflect the presence of common structures in such assemblies, i.e., not only the common cross-β structure of the fibrils, but also in the oligomers, as has been suggested by binding of antioligomer antibodies, such as A11, to oligomers of multiple amyloidogenic proteins (
      • Kayed R.
      • Head E.
      • Thompson J.L.
      • McIntire T.M.
      • Milton S.C.
      • Cotman C.W.
      • Glabe C.G.
      Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.
      ). In an attempt to develop a detection system for Aβ40 oligomers, T-SO517 was applied to a fluorescence detection system using abasic site-containing DNA oligonucleotides (
      • Zhu L.
      • Zhang J.
      • Wang F.
      • Wang Y.
      • Lu L.
      • Feng C.
      • Xu Z.
      • Zhang W.
      Selective amyloid β oligomer assay based on abasic site-containing molecular beacon and enzyme-free amplification.
      ). When monitoring the Aβ40 aggregation process, this system showed preference for detecting oligomers over monomers or fibrils in vitro, which was confirmed by transmission electron microscopy.
      Using a different selection approach, Takahashi et al. (
      • Takahashi T.
      • Tada K.
      • Mihara H.
      RNA aptamers selected against amyloid β-peptide (Aβ) inhibit the aggregation of Aβ.
      ) tethered Aβ40 to colloidal gold nanoparticles as a target for selection of RNA aptamers using the same method previously developed by Kayed et al. (
      • Kayed R.
      • Head E.
      • Thompson J.L.
      • McIntire T.M.
      • Milton S.C.
      • Cotman C.W.
      • Glabe C.G.
      Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.
      ) for preparation of oligomer-specific antibodies. The RNA aptamers, N2 and E2, bound to Aβ40 monomers and inhibited Aβ40 fibril formation assessed using the ThT fluorescence assay and transmission electron micrography. However, as was found by Rahimi et al. (
      • Rahimi F.
      • Murakami K.
      • Summers J.L.
      • Chen C.H.
      • Bitan G.
      RNA aptamers generated against oligomeric Aβ40 recognize common amyloid aptatopes with low specificity but high sensitivity.
      ), Babu et al. (
      • Babu E.
      • Muthu Mareeswaran P.
      • Sathish V.
      • Singaravadivel S.
      • Rajagopal S.
      Sensing and inhibition of amyloid-β based on the simple luminescent aptamer-ruthenium complex system.
      ) demonstrated later by using atomic force microscopy that these aptamers also bound to Aβ40 fibrils. Binding to Aβ- or other protein oligomers was not described in either study. A DNA aptamer named RNV95, selected using column-immobilized Aβ40 by Chakravarthy et al. with the goal of detecting low-molecular-weight Aβ40, recognized human brain Aβ oligomers at both ∼75 kDa and ∼150 kDa. However, it was not tested for binding to Aβ fibrils or fibrils of other amyloidogenic proteins so its selectivity for oligomers was not established (
      • Chakravarthy M.
      • AlShamaileh H.
      • Huang H.
      • Tannenberg R.K.
      • Chen S.
      • Worrall S.
      • Dodd P.R.
      • Veedu R.N.
      Development of DNA aptamers targeting low-molecular-weight amyloid-β peptide aggregates in vitro.
      ).
      To our knowledge, to date, only two studies have explored development of aptamers against Aβ42, likely because it is an even more difficult target than Aβ40. Aβ42 has been shown to aggregate faster (
      • Jarrett J.T.
      • Berger E.P.
      • Lansbury Jr., P.T.
      The carboxy terminus of the β amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer's disease.
      ), form distinct oligomers (
      • Bitan G.
      • Kirkitadze M.D.
      • Lomakin A.
      • Vollers S.S.
      • Benedek G.B.
      • Teplow D.B.
      Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways.
      ,
      • Bernstein S.L.
      • Dupuis N.F.
      • Lazo N.D.
      • Wyttenbach T.
      • Condron M.M.
      • Bitan G.
      • Teplow D.B.
      • Shea J.E.
      • Ruotolo B.T.
      • Robinson C.V.
      • Bowers M.T.
      Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease.
      ), and cause stronger neurotoxicity than Aβ40 (
      • Dahlgren K.N.
      • Manelli A.M.
      • Stine Jr., W.B.
      • Baker L.K.
      • Krafft G.A.
      • LaDu M.J.
      Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability.
      ,
      • Murakami K.
      • Irie K.
      • Morimoto A.
      • Ohigashi H.
      • Shindo M.
      • Nagao M.
      • Shimizu T.
      • Shirasawa T.
      Neurotoxicity and physicochemical properties of Aβ mutant peptides from cerebral amyloid angiopathy: Implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease.
      ). The first characteristic, substantially faster aggregation, makes working with Aβ42 particularly difficult because the preparation of the protein changes during the experiment making obtaining reproducible data highly challenging.
      The first example of a successful generation of aptamers against Aβ oligomers we are aware of is by Murakami et al., who developed RNA aptamers termed E22P-AbD4, -AbD31, and -AbD43 against Aβ42 protofibrils (
      • Murakami K.
      • Obata Y.
      • Sekikawa A.
      • Ueda H.
      • Izuo N.
      • Awano T.
      • Takabe K.
      • Shimizu T.
      • Irie K.
      An RNA aptamer with potent affinity for a toxic dimer of amyloid β42 has potential utility for histochemical studies of Alzheimer's disease.
      ,
      • Obata Y.
      • Murakami K.
      • Kawase T.
      • Hirose K.
      • Izuo N.
      • Shimizu T.
      • Irie K.
      Detection of amyloid β oligomers with RNA aptamers in AppNL-G-F/NL-G-F mice: A model of Arctic Alzheimer's disease.
      ). To select the aptamers, a dimer of E22P-Aβ42 was used in which the monomers were tethered covalently by a bivalent amino acid linker in place of Val40, within the C-terminal hydrophobic region of Aβ42 (Fig. 3B) (
      • Murakami K.
      • Tokuda M.
      • Suzuki T.
      • Irie Y.
      • Hanaki M.
      • Izuo N.
      • Monobe Y.
      • Akagi K.
      • Ishii R.
      • Tatebe H.
      • Tokuda T.
      • Maeda M.
      • Kume T.
      • Shimizu T.
      • Irie K.
      Monoclonal antibody with conformational specificity for a toxic conformer of amyloid β42 and its application toward the Alzheimer's disease diagnosis.
      ). Upon incubation in phosphate buffer at 37 °C without agitation for 48 h, this dimer construct, in which a turn near Glu22 was stabilized by an E22P substitution (
      • Irie K.
      New diagnostic method for Alzheimer's disease based on the toxic conformation theory of amyloid β.
      ), formed protofibrils, the morphology of which was confirmed by transmission electron microscopy. The protofibrils then were used in SELEX to obtain the aptamers from a 77-nucleotide RNA library including a 49-nucleutide random sequence. When used in histological experiments, all three aptamers stained diffuse oligomeric aggregates in two mouse models of AD, Tg2576/PS2 (
      • Toda T.
      • Noda Y.
      • Ito G.
      • Maeda M.
      • Shimizu T.
      Presenilin-2 mutation causes early amyloid accumulation and memory impairment in a transgenic mouse model of Alzheimer's disease.
      ) and AppNL-G-F/NL-G-F (
      • Saito T.
      • Matsuba Y.
      • Mihira N.
      • Takano J.
      • Nilsson P.
      • Itohara S.
      • Iwata N.
      • Saido T.C.
      Single app knock-in mouse models of Alzheimer's disease.
      ), suggesting that the Aβ assemblies formed in the brain of these two mouse models contained similar protofibril-derived aptatopes. Incubation of the E22P-Aβ42 dimer in the presence of aptamer E22P-AbD43 showed that the aptamer inhibited the nucleation phase of the protofibril formation. The aptamer also inhibited dose-dependently the neurotoxicity of both the E22P-Aβ42 dimer and Aβ42 in the neuroblastoma cell line SH-SY5Y cells. Computational and two-dimensional structure analysis of E22P-AbD43 suggested that preferential binding of the aptamer to Aβ42 protofibrils compared with fibrils might be related to formation of a G-quadruplex structure, implying the presence of a common structure in protofibrils made of either the synthetic dimer or native Aβ42 protofibrils (
      • Obata Y.
      • Murakami K.
      • Kawase T.
      • Hirose K.
      • Izuo N.
      • Shimizu T.
      • Irie K.
      Detection of amyloid β oligomers with RNA aptamers in AppNL-G-F/NL-G-F mice: A model of Arctic Alzheimer's disease.
      ).
      For the purpose of developing inhibitors of Aβ42 aggregation, Zheng et al. selected an anti-Aβ42 DNA aptamer (Aβ7-92-1H1) by incubating a library of DNA-oligonucleotide-coated beads with nonaggregated Aβ42. This library consisted of 100-nucleotide-long sequences including two separate 18-nucleotide random sequences. Aβ7-92-1H1 bound Aβ42 and neither Aβ40 nor other amyloids. The affinity of the aptamer for Aβ42 oligomers was slightly higher than for Aβ42 monomers, measured by surface plasmon resonance (SPR). The aptamer inhibited Aβ42 fibril formation as evidenced by atomic force microscopy and β-sheet formation measured using CD spectra (
      • Zheng Y.
      • Wang P.
      • Li S.
      • Geng X.
      • Zou L.
      • Jin M.
      • Zou Q.
      • Wang Q.
      • Yang X.
      • Wang K.
      Development of DNA aptamer as a β-amyloid aggregation inhibitor.
      ).

      Aptamers against tau protein

      Tauopathies are neurodegenerative diseases caused by formation of toxic oligomers and aggregates of the microtubule-associated protein tau. Tau is produced as six different isoforms in humans, due to alternative splicing of exons 2, 3, and 10 leading to polypeptides ranging from 352 to 441 amino acid residues. Translation of neither, one, or both exons 2 and 3 is marked as 0N, 1N, or 2N isoforms of tau, whereas the alternative splicing of exon 10, which encodes part of the microtubule binding, repeat domain of tau leads to isoforms containing three (3R) or four (4R) repeats.
      The most prevalent tauopathy is AD (
      • Arendt T.
      • Stieler J.T.
      • Holzer M.
      Tau and tauopathies.
      ). Other examples of tauopathies include frontotemporal lobar degeneration, progressive supranuclear palsy, and chronic traumatic encephalopathy. Aggregation and deposition of tau, e.g., as neurofibrillary tangles in AD, are associated with hyperphosphorylation and other aberrant posttranslational modifications of the protein.
      In the course of testing whether tau binds single-strand DNA, Krylova et al. (
      • Krylova S.M.
      • Musheev M.
      • Nutiu R.
      • Li Y.
      • Lee G.
      • Krylov S.N.
      Tau protein binds single-stranded DNA sequence specifically--the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures.
      ) first found DNA sequences binding recombinant 1N3R-tau or 2N3R-tau using nonequilibrium capillary electrophoresis of equilibrium mixtures, in which the gel shift caused by binding of the DNA oligonucleotides to tau was evaluated. In follow-up studies, these DNA sequences were used as aptamers for detection of 1N3R-tau in human plasma using SPR and were shown to reach femtomolar level sensitivity (
      • Kim S.
      • Wark A.W.
      • Lee H.J.
      Femtomolar detection of tau proteins in undiluted plasma using surface plasmon resonance.
      ). Selection of aptamers without amplification between selection rounds shortens the time and saves costs compared with conventional SELEX (
      • Berezovski M.V.
      • Musheev M.U.
      • Drabovich A.P.
      • Jitkova J.V.
      • Krylov S.N.
      Non-SELEX: Selection of aptamers without intermediate amplification of candidate oligonucleotides.
      ). However, this is not a standard method in the amyloid field. Lisi et al. applied this method coupled with capillary electrophoresis for partitioning of bound DNA from unbound DNA to isolate DNA aptamers targeting several tau isoforms. The aptamer 3146 was obtained from a 5 × 1012 DNA oligonucleotide library in only three rounds within 1 day. 3146 bound tau isoforms in the order 2N4R (KD = 13 nM) > 0N4R (49 nM) > 0N3R (84 nM) > 1N3R (116 nM) (
      • Lisi S.
      • Fiore E.
      • Scarano S.
      • Pascale E.
      • Boehman Y.
      • Duconge F.
      • Chierici S.
      • Minunni M.
      • Peyrin E.
      • Ravelet C.
      Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau proteins.
      ).
      An RNA aptamer called tau-1 was obtained against 2N4R-tau, by applying SELEX to a 90-nucleotide RNA library containing a 40-nucleotide random region. This aptamer prevented the formation of 2N4R-tau dimers and trimers assessed by SDS-PAGE/western blotting (
      • Kim J.H.
      • Kim E.
      • Choi W.H.
      • Lee J.
      • Lee J.H.
      • Lee H.
      • Kim D.E.
      • Suh Y.H.
      • Lee M.J.
      Inhibitory RNA aptamers of tau oligomerization and their neuroprotective roles against proteotoxic stress.
      ). It is important to note, however, that SDS-PAGE is not a reliable method for assessing oligomer formation by amyloidogenic proteins, as SDS perturbs protein conformation and can both dissociate existing assemblies and induce formation of different assemblies (
      • Bitan G.
      • Fradinger E.A.
      • Spring S.M.
      • Teplow D.B.
      Neurotoxic protein oligomers--what you see is not always what you get.
      ). Nonetheless, in an experiment using a HEK293-derived cell line expressing human 2N4R-tau under control of doxycycline induction (
      • Bandyopadhyay B.
      • Li G.
      • Yin H.
      • Kuret J.
      Tau aggregation and toxicity in a cell culture model of tauopathy.
      ), reduced cell viability resulting from tau expression was recovered by treating with tau-1 compared to a random RNA library (
      • Kim J.H.
      • Kim E.
      • Choi W.H.
      • Lee J.
      • Lee J.H.
      • Lee H.
      • Kim D.E.
      • Suh Y.H.
      • Lee M.J.
      Inhibitory RNA aptamers of tau oligomerization and their neuroprotective roles against proteotoxic stress.
      ).

      Aptamers against α-synuclein

      αSyn, a causative agent of synucleinopathies, such as PD, dementia with Lewy bodies, and multiple system atrophy, oligomerizes and aggregates in the brain of patients leading to neurotoxicity and neurodegeneration (
      • Breydo L.
      • Wu J.W.
      • Uversky V.N.
      α-Synuclein misfolding and Parkinson's disease.
      ). αSyn is a 140-amino-acid long protein mainly located at presynaptic terminals. The amphipathic N-terminal region (residues 1–60), which includes four 11-residue imperfect repeats, and the hydrophobic middle region (residues 61–95) are more important for aggregation than the acidic and proline-rich C-terminal region (residues 96–140) of αSyn (
      • Tripathi T.
      A master regulator of α-synuclein aggregation.
      ). The C-terminal region of αSyn is susceptible to cleavage upon aggregation (
      • Sorrentino Z.A.
      • Giasson B.I.
      The emerging role of α-synuclein truncation in aggregation and disease.
      ), yet most antibodies bind to this region and therefore may miss αSyn aggregates in pathological analysis of patient brains or animal models. This problem is particularly important because the most common form of αSyn used to detect pathological aggregates is phosphorylated at Ser129 (pS129-αSyn), yet some cleavage sites are N-terminal to position 129 and eliminate the epitope for antibodies against pS129-αSyn.
      Similar to Aβ and tau, αSyn oligomers, rather than fibrils, are thought to be the primary neurotoxic form of the protein. αSyn oligomers cause neurotoxicity, synaptic impairment, mitochondrial dysfunction, endoplasmic reticulum stress, neuroinflammation, proteostasis dysregulation, and apoptosis, culminating in neuronal death (
      • Du X.Y.
      • Xie X.X.
      • Liu R.T.
      The role of α-synuclein oligomers in Parkinson's disease.
      ). Recent cryo-electron microscopy analyses have deciphered the atomic structure of αSyn filaments (
      • Schweighauser M.
      • Shi Y.
      • Tarutani A.
      • Kametani F.
      • Murzin A.G.
      • Ghetti B.
      • Matsubara T.
      • Tomita T.
      • Ando T.
      • Hasegawa K.
      • Murayama S.
      • Yoshida M.
      • Hasegawa M.
      • Scheres S.H.W.
      • Goedert M.
      Structures of α-synuclein filaments from multiple system atrophy.
      ) and structure–activity analysis suggested that the N-terminus controls αSyn aggregation (
      • McGlinchey R.P.
      • Ni X.
      • Shadish J.A.
      • Jiang J.
      • Lee J.C.
      The N terminus of α-synuclein dictates fibril formation.
      ). The details of αSyn oligomer structures are not known, yet β-sheet structure has been reported in rigid regions of toxic αSyn oligomers, whereas in nontoxic oligomers, these regions are unstructured (
      • Fusco G.
      • Chen S.W.
      • Williamson P.T.F.
      • Cascella R.
      • Perni M.
      • Jarvis J.A.
      • Cecchi C.
      • Vendruscolo M.
      • Chiti F.
      • Cremades N.
      • Ying L.
      • Dobson C.M.
      • De Simone A.
      Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers.
      ). The mechanism underlying oligomer toxicity may involve insertion into lipid bilayers, disrupting membrane integrity (
      • Fusco G.
      • Chen S.W.
      • Williamson P.T.F.
      • Cascella R.
      • Perni M.
      • Jarvis J.A.
      • Cecchi C.
      • Vendruscolo M.
      • Chiti F.
      • Cremades N.
      • Ying L.
      • Dobson C.M.
      • De Simone A.
      Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers.
      ). Bioinformatic and NMR studies have supported an important role for the N-terminus in modulating the aggregation of αSyn (
      • Doherty C.P.A.
      • Ulamec S.M.
      • Maya-Martinez R.
      • Good S.C.
      • Makepeace J.
      • Khan G.N.
      • van Oosten-Hawle P.
      • Radford S.E.
      • Brockwell D.J.
      A short motif in the N-terminal region of α-synuclein is critical for both aggregation and function.
      ).
      Ikebukuro and colleagues reported a first DNA aptamer against αSyn, called M5-15, which showed affinity for both monomers and oligomers (
      • Tsukakoshi K.
      • Harada R.
      • Sode K.
      • Ikebukuro K.
      Screening of DNA aptamer which binds to α-synuclein.
      ). In follow-up studies, the same group applied counterselection of monomers and fibrils, leading to a more selective aptamer, T-SO530, against αSyn oligomers, whose selectivity was confirmed by dot blots (
      • Tsukakoshi K.
      • Abe K.
      • Sode K.
      • Ikebukuro K.
      Selection of DNA aptamers that recognize α-synuclein oligomers using a competitive screening method.
      ). CD analysis suggested that T-SO530 formed a G-quadruplex structure, which might have contributed to its affinity. Indeed, G-quadruplex stabilizers, such as L1H1-7OTD and TmPyP4 (Fig. 4), enhanced the binding of T-SO530 to αSyn oligomers (
      • Tsukakoshi K.
      • Ikuta Y.
      • Abe K.
      • Yoshida W.
      • Iida K.
      • Ma Y.
      • Nagasawa K.
      • Sode K.
      • Ikebukuro K.
      Structural regulation by a G-quadruplex ligand increases binding abilities of G-quadruplex-forming aptamers.
      ), suggesting that such stabilizers can be promising synthetic modulators/cofactors for applications of aptamers against amyloidogenic proteins in neurodegenerative diseases.
      Figure thumbnail gr4
      Figure 4G-quadruplex structures in aptamers and their stabilization. A, a schematic structure of a G-quadruplex, which can be stabilized by a metal ion, e.g., K+, or flat molecules, for example those shown in panel B. B, structures of L1H1-7OTD and TmPyP4, which have been used as stabilizers of a G-quadruplex in the anti-αSyn aptamer T-SO530.
      Zheng et al. (
      • Zheng Y.
      • Qu J.
      • Xue F.
      • Zheng Y.
      • Yang B.
      • Chang Y.
      • Yang H.
      • Zhang J.
      Novel DNA aptamers for Parkinson's disease treatment inhibit α-synuclein aggregation and facilitate its degradation.
      ) developed 58-nucleotide DNA aptamers they termed F5R1 and F5R2, starting with a 40-nucleotide random single-strand DNA library, which bound αSyn with a high affinity and inhibited αSyn aggregation. To enhance cell membrane permeability of the aptamers, they were modified by attachment of a peptide carrier, CADY, which had been reported previously to form stable complexes with nucleic acids, leading to improvement of their delivery into cultured cells (
      • Crombez L.
      • Aldrian-Herrada G.
      • Konate K.
      • Nguyen Q.N.
      • McMaster G.K.
      • Brasseur R.
      • Heitz F.
      • Divita G.
      A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells.
      ). The modified aptamers reduced intracellular aggregation of αSyn, synaptic protein loss, and neuronal death caused by αSyn overexpression.

      Aptamers against prion protein

      The accumulation of misfolded PrP characterizes prionoses, such as Creutzfeldt-Jakob disease, Kuru, Gerstmann–Sträussler–Scheinker Syndrome, and fatal familial insomnia in humans, bovine spongiform encephalopathy in cattle, scrapie in sheep, and chronic wasting disease in deer and elk (
      • Prusiner S.B.
      • Scott M.R.
      • DeArmond S.J.
      • Cohen F.E.
      Prion protein biology.
      ,
      • Collinge J.
      Prion diseases of humans and animals: Their causes and molecular basis.
      ,
      • Aguzzi A.
      • Polymenidou M.
      Mammalian prion biology: One century of evolving concepts.
      ). PrP exists normally as PrPC (cellular form), which is involved in neuroprotection and trophic signaling (
      • McLennan N.F.
      • Brennan P.M.
      • McNeill A.
      • Davies I.
      • Fotheringham A.
      • Rennison K.A.
      • Ritchie D.
      • Brannan F.
      • Head M.W.
      • Ironside J.W.
      • Williams A.
      • Bell J.E.
      Prion protein accumulation and neuroprotection in hypoxic brain damage.
      ,
      • Spudich A.
      • Frigg R.
      • Kilic E.
      • Kilic U.
      • Oesch B.
      • Raeber A.
      • Bassetti C.L.
      • Hermann D.M.
      Aggravation of ischemic brain injury by prion protein deficiency: Role of ERK-1/-2 and STAT-1.
      ,
      • Mitteregger G.
      • Vosko M.
      • Krebs B.
      • Xiang W.
      • Kohlmannsperger V.
      • Nolting S.
      • Hamann G.F.
      • Kretzschmar H.A.
      The role of the octarepeat region in neuroprotective function of the cellular prion protein.
      ), yet this form can misfold due to genetic, environmental, or yet unknown causes into a toxic and infectious form called PrPSc (scrapie form). The term PrPSc does not describe one particular structure but refers to many conformational strains that are self-propagating, transmissible from cell to cell, and are infectious within the same species and sometimes across species. PrPSc infectivity to other organisms depends on the specific strain and species barrier. PrPC is rich in α-helix, whereas PrPSc contains the typical cross-β structure of amyloid fibrils. The aggregation process, including oligomerization of PrPSc, plays a central role in the prion’s propagation and neurotoxicity (
      • Sengupta I.
      • Udgaonkar J.B.
      Structural mechanisms of oligomer and amyloid fibril formation by the prion protein.
      ). Biochemically, PrPSc is a highly stable form of the protein, which is resistant to proteinases and denaturing agents.
      Interestingly, one of the biological roles of PrPC is a receptor of Aβ oligomers. Binding of Aβ oligomers to PrPC causes synaptotoxicity and neuritic dystrophy, possibly also leading to tauopathy (
      • Lauren J.
      Cellular prion protein as a therapeutic target in Alzheimer's disease.
      ,
      • Lauren J.
      • Gimbel D.A.
      • Nygaard H.B.
      • Gilbert J.W.
      • Strittmatter S.M.
      Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers.
      ). Thus, both PrPC and PrPSc are potential therapeutic targets for development of various drug modalities, including aptamers.
      Weiss et al. reported the first RNA aptamer, Ap1, against a recombinant Syrian golden hamster prion protein PrP23–231, a common model of full-length PrPC. The aptamer recognized PrPC in brain extracts of scrapie-infected mice, hamsters, and cattle, though its binding affinity was not measured (
      • Weiss S.
      • Proske D.
      • Neumann M.
      • Groschup M.H.
      • Kretzschmar H.A.
      • Famulok M.
      • Winnacker E.L.
      RNA aptamers specifically interact with the prion protein PrP.
      ). An RNA aptamer called “60-3”, selected against mouse PrP23–230, was reported by Sekiya et al. (
      • Sekiya S.
      • Noda K.
      • Nishikawa F.
      • Yokoyama T.
      • Kumar P.K.
      • Nishikawa S.
      Characterization and application of a novel RNA aptamer against the mouse prion protein.
      ) and also bound bovine PrP25–241. Mercey et al. (
      • Mercey R.
      • Lantier I.
      • Maurel M.C.
      • Grosclaude J.
      • Lantier F.
      • Marc D.
      Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns.
      ) developed an RNA aptamer, RM312, against sheep PrP23–234, which bound with 15 nM affinity to PrPC. In another study, Ikebukuro and colleagues developed a DNA aptamer, termed “4-9”, against mouse PrP23–230 that bound to both PrPC and PrPSc equally (
      • Ogasawara D.
      • Hasegawa H.
      • Kaneko K.
      • Sode K.
      • Ikebukuro K.
      Screening of DNA aptamer against mouse prion protein by competitive selection.
      ). A DNA aptamer called 4C26 was prepared by Garson and colleagues against mouse PrP90–231 and exhibited a higher binding affinity for mouse prion than “4-9” (
      • Bibby D.F.
      • Gill A.C.
      • Kirby L.
      • Farquhar C.F.
      • Bruce M.E.
      • Garson J.A.
      Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins.
      ) suggesting that shortening the target length in this case improved the affinity.
      To pursue aptamers selective for PrPSc versus PrPC, an RNA aptamer, SAF-93, was developed against scrapie-associated fibrils from the brains of infected hamsters. In a competitive binding assay using bovine PrPSc fibrils, SAF-93 showed >10-fold higher affinity for PrPSc than for PrPC (
      • Rhie A.
      • Kirby L.
      • Sayer N.
      • Wellesley R.
      • Disterer P.
      • Sylvester I.
      • Gill A.
      • Hope J.
      • James W.
      • Tahiri-Alaoui A.
      Characterization of 2'-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion.
      ). Uniquely, SAF-93 was obtained by SELEX using 2′-fluoro-modified pyrimidine triphosphate nucleotides together with unmodified purine nucleotides. Enzymatic probing and gel footprinting, in addition to computer-assisted secondary structure analysis, identified multiple binding sites of SAF-93 on PrPSc (
      • Sayer N.M.
      • Cubin M.
      • Rhie A.
      • Bullock M.
      • Tahiri-Alaoui A.
      • James W.
      Structural determinants of conformationally selective, prion-binding aptamers.
      ).
      Several aptamers also have been selected against human prion proteins. An RNA aptamer termed DP7 was prepared by Proske et al. against human PrP90–129, an important domain for the conversion of PrPC into PrPSc. The aptamer also bound PrP derived from hamster and mouse. The utility of this aptamer for reducing the ratio of PrPSc to PrPC was demonstrated in prion-infected mouse neuroblastoma N2a cells (
      • Proske D.
      • Gilch S.
      • Wopfner F.
      • Schatzl H.M.
      • Winnacker E.L.
      • Famulok M.
      Prion-protein-specific aptamer reduces PrPSc formation.
      ). Takemura et al. (
      • Takemura K.
      • Wang P.
      • Vorberg I.
      • Surewicz W.
      • Priola S.A.
      • Kanthasamy A.
      • Pottathil R.
      • Chen S.G.
      • Sreevatsan S.
      DNA aptamers that bind to PrP(C) and not PrP(Sc) show sequence and structure specificity.
      ) reported a DNA aptamer called SSAP3-10 against human PrP23–231, which bound other mammalian PrPC (sheep, calf, piglet, and deer) in addition to human PrPC, but not to PrPSc.
      Structural studies of prion aptamers have been advanced compared with other amyloidogenic proteins. Murakami et al. developed an RNA aptamer, apt#1, selected against bovine PrP25–241. The binding was deduced to be due to a G-quadruplex structure using circular dichroism (CD) spectroscopy. An extracted sequence from apt#1 (R14 aptamer, GGAGGUUUUGGAGG) was identified using mutagenesis and showed improved binding affinity and ∼30-fold selectivity for PrPC compared with PrPSc (
      • Murakami K.
      • Nishikawa F.
      • Noda K.
      • Yokoyama T.
      • Nishikawa S.
      Anti-bovine prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its β isoform with high affinity.
      ). Katahira and colleagues further optimized the sequence to deduce a tandem repeat sequence (R12 aptamer, (GGA)4), and found using NMR measurements that it formed an intramolecular parallel G-quadruplex (
      • Mashima T.
      • Matsugami A.
      • Nishikawa F.
      • Nishikawa S.
      • Katahira M.
      Unique quadruplex structure and interaction of an RNA aptamer against bovine prion protein.
      ). R12 bound to two sites in the N-terminal half of PrPC by forming a dimer (
      • Mashima T.
      • Nishikawa F.
      • Kamatari Y.O.
      • Fujiwara H.
      • Saimura M.
      • Nagata T.
      • Kodaki T.
      • Nishikawa S.
      • Kuwata K.
      • Katahira M.
      Anti-prion activity of an RNA aptamer and its structural basis.
      ,
      • Hayashi T.
      • Oshima H.
      • Mashima T.
      • Nagata T.
      • Katahira M.
      • Kinoshita M.
      Binding of an RNA aptamer and a partial peptide of a prion protein: Crucial importance of water entropy in molecular recognition.
      ) (Fig. 5). Treatment with R12 reduced the level of PrPC (
      • Mashima T.
      • Nishikawa F.
      • Kamatari Y.O.
      • Fujiwara H.
      • Saimura M.
      • Nagata T.
      • Kodaki T.
      • Nishikawa S.
      • Kuwata K.
      • Katahira M.
      Anti-prion activity of an RNA aptamer and its structural basis.
      ) and blocked the pathological conformational conversion of PrPC into PrPSc in scrapie-infected mouse neuronal GT1-7 cells (
      • Mashima T.
      • Lee J.H.
      • Kamatari Y.O.
      • Hayashi T.
      • Nagata T.
      • Nishikawa F.
      • Nishikawa S.
      • Kinoshita M.
      • Kuwata K.
      • Katahira M.
      Development and structural determination of an anti-PrP(C) aptamer that blocks pathological conformational conversion of prion protein.
      ).
      Figure thumbnail gr5
      Figure 5Stabilization of a complex between the antiprion RNA aptamer R12 and the N-terminal region of PrPC. A dimer of R12 [pink, r(GGAGGAGGAGGA)] associates with two N-terminal PrPC peptides (green, Gly-Gln-Trp-Asn-Lys-Pro-Ser-Lys-Pro-Lys-Thr-Asn) providing a 1:1 stoichiometric ratio. The illustration was created using PyMOL from PDB ID: 2RU7.

      Computer-assisted development and structure prediction of aptamers

      Selection and clustering of aptamers by high-throughput sequencing

      Despite the large potential of aptamers as therapeutic agents, biosensors, and research tools, technical issues can require major time and money investments, limiting development and progress. One such technical issue is the use of classic Sanger sequencing of the selected oligonucleotides. Although the selection process removes the vast majority of the sequences, the final pool still may contain thousands of distinct oligonucleotide sequences, making sequencing of this pool and identifying the sequences possessing the best affinity and/or specificity labor-intensive and time-consuming.
      In silico approaches, previously reviewed by Hamada (
      • Hamada M.
      In silico approaches to RNA aptamer design.
      ), have been useful for aptamer design. Artificial intelligence (AI) coupled with machine learning algorithms assists in identifying potential aptamer candidates from the selected sequences expeditiously, leading to improvement compared with classical prediction tools (
      • Chen Z.
      • Hu L.
      • Zhang B.T.
      • Lu A.
      • Wang Y.
      • Yu Y.
      • Zhang G.
      Artificial intelligence in aptamer-target binding prediction.
      ). As only the oligonucleotide pool of the last round of selection typically is cloned and sequenced in conventional SELEX, superior aptamers may be overlooked. In this section, we discuss the role of bioinformatics in aptamer development, including in sequencing, secondary-structure prediction, and simulation of aptamer–target interaction, and highlight the use of these advances in selection of aptamers against amyloidogenic proteins.
      With the advent of new sequencing technologies, such as Next-Generation sequencing, exhaustive parallel sequencing of all the selection rounds has facilitated high-throughput SELEX (HT-SELEX) (
      • Cho M.
      • Xiao Y.
      • Nie J.
      • Stewart R.
      • Csordas A.T.
      • Oh S.S.
      • Thomson J.A.
      • Soh H.T.
      Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing.
      ,
      • Jolma A.
      • Kivioja T.
      • Toivonen J.
      • Cheng L.
      • Wei G.
      • Enge M.
      • Taipale M.
      • Vaquerizas J.M.
      • Yan J.
      • Sillanpaa M.J.
      • Bonke M.
      • Palin K.
      • Talukder S.
      • Hughes T.R.
      • Luscombe N.M.
      • et al.
      Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities.
      ,
      • Kupakuwana G.V.
      • Crill 2nd, J.E.
      • McPike M.P.
      • Borer P.N.
      Acyclic identification of aptamers for human α-thrombin using over-represented libraries and deep sequencing.
      ). In the first report of this technique, a KD in the picomolar range was achieved after only three rounds of selection for aptamers against the BB subunits of platelet-derived growth factor (
      • Cho M.
      • Xiao Y.
      • Nie J.
      • Stewart R.
      • Csordas A.T.
      • Oh S.S.
      • Thomson J.A.
      • Soh H.T.
      Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing.
      ). The abundance of sequences read in all the SELEX pools can be ranked by various parameters in HT-SELEX, e.g., sequence enrichment (
      • Hoinka J.
      • Berezhnoy A.
      • Dao P.
      • Sauna Z.E.
      • Gilboa E.
      • Przytycka T.M.
      Large scale analysis of the mutational landscape in HT-SELEX improves aptamer discovery.
      ), and meta-Z-score (
      • Jiang P.
      • Meyer S.
      • Hou Z.
      • Propson N.E.
      • Soh H.T.
      • Thomson J.A.
      • Stewart R.
      MPBind: A meta-motif-based statistical framework and pipeline to predict binding potential of SELEX-derived aptamers.
      ). The meta-Z-score is a statistical scoring method for high prediction accuracy of binding potential. This method allows gleaning insight into whether sequence enrichment occurs, rather than whether the affinity for the target is enhanced at substantially earlier stages of the aptamer-selection process, resulting in considerable time saving. Additionally, a decreased number of selection rounds helps reduce PCR bias (nonspecific amplification) caused by over selection or excessive cycle numbers in PCR (
      • Nguyen Quang N.
      • Perret G.
      • Duconge F.
      Applications of high-throughput sequencing for in vitro selection and characterization of aptamers.
      ). Comprehensive analysis of very large sequence datasets by such bioinformatics methods has enabled not only accurate characterization of aptamers, data alignment, and clustering, but also improved prediction of aptamer structure and aptamer–target interaction mode.
      After collecting and ranking datasets of sequences, data clustering typically is a next step, which can be achieved using free software tools, such as AptaCluster (
      • Hoinka J.
      • Berezhnoy A.
      • Sauna Z.E.
      • Gilboa E.
      • Przytycka T.M.
      AptaCluster - a method to cluster HT-SELEX aptamer pools and lessons from its application.
      ) (https://www.ncbi.nlm.nih.gov/CBBresearch/Przytycka/index.cgi#aptatools) and FASTAptamer (
      • Alam K.K.
      • Chang J.L.
      • Burke D.H.
      FASTAptamer: A bioinformatic toolkit for high-throughput sequence analysis of combinatorial selections.
      ) (https://burkelab.missouri.edu/fastaptamer.html), both of which use RNA sequence, but not structural information. Ikebukuro and colleagues proposed a genetic methodology named evolution-mimicking algorithm, to identify optimized aptamer sequences in rugged sequence spaces. Using this methodology, by combining several in vitro assays, e.g., binding affinity, inhibitory activity, specificity, and 3D structure, in silico maturation that included selection and duplication, recombination, and point mutation has facilitated identifying several aptamers against PrP and αSyn (
      • Ikebukuro K.
      • Okumura Y.
      • Sumikura K.
      • Karube I.
      A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm.
      ,
      • Noma T.
      • Ikebukuro K.
      Aptamer selection based on inhibitory activity using an evolution-mimicking algorithm.
      ,
      • Hasegawa H.
      • Savory N.
      • Abe K.
      • Ikebukuro K.
      Methods for improving aptamer binding affinity.
      ).

      Analysis of secondary and tertiary structure of aptamers by motif finding and structure optimization

      When conducting an aptamer discovery campaign, the three-dimensional structures of the selected aptamers and aptamer–target complexes are important considerations. The relatively simpler conformational space of nucleic acids compared with proteins makes computer-aided calculation and modeling of secondary and tertiary structures particularly useful for aptamers. The secondary structure of oligonucleic acids is determined by canonical Watson–Crick base-paring interactions or noncanonical Hoogsteen base pairs in single or double strands (Fig. 6). Due to the additional hydroxyl group in ribose compared with deoxyribose, RNA single strands are believed to form more complex and diverse structures than DNA, such as stem loops and pseudoknots.
      Figure thumbnail gr6
      Figure 6Canonical and noncanonical stabilization of natural base-pairs. Canonical, Watson–Crick interactions have three hydrogen bonds between guanine and cytosine (O6–N4, N1–N3, and N2–O2), and two hydrogen bonds between adenine and thymine (N6–O4 and N1–N3). In noncanonical Hoogsteen interactions, the adenine or guanine is rotated 180° around the glycosidic bond, resulting in alternative hydrogen base pairs—two hydrogen bonds between guanine and cytosine (O6–N4 and N7–N3), and two hydrogen bonds between adenine and thymine (N6–O4 and N7–N3). Hoogsteen interactions are minor compared with the Watson–Crick structures but may contribute to formation of unique DNA or RNA structures.
      The program Mfold, originally developed by Zuker (
      • Zuker M.
      On finding all suboptimal foldings of an RNA molecule.
      ,
      • Zuker M.
      Mfold web server for nucleic acid folding and hybridization prediction.
      ) and later modified by incorporating into the University of Wisconsin Genetics Computer Group software suites (
      • Devereux J.
      • Haeberli P.
      • Smithies O.
      A comprehensive set of sequence analysis programs for the VAX.
      ), uses minimal free energy optimization and is one of the most popular tools for determination of nucleic acids’ secondary structure. Typically used after Mfold, MEMERIS is a general motif-finding algorithm for integration of secondary structures (
      • Hiller M.
      • Pudimat R.
      • Busch A.
      • Backofen R.
      Using RNA secondary structures to guide sequence motif finding towards single-stranded regions.
      ). AptaMotif is a program designed for identifying binding motifs in aptamers identified through SELEX and is based on structural processing, including suboptimal secondary structures, for prediction of sequence motifs in the loop regions (
      • Hoinka J.
      • Zotenko E.
      • Friedman A.
      • Sauna Z.E.
      • Przytycka T.M.
      Identification of sequence-structure RNA binding motifs for SELEX-derived aptamers.
      ). The tertiary structures of aptamers can be predicted based on the calculated information of secondary structures. Online programs and servers are available for this purpose, including RNAComposer (
      • Biesiada M.
      • Purzycka K.J.
      • Szachniuk M.
      • Blazewicz J.
      • Adamiak R.W.
      Automated RNA 3D structure prediction with RNAComposer.
      ) and SimRNAweb (
      • Magnus M.
      • Boniecki M.J.
      • Dawson W.
      • Bujnicki J.M.
      SimRNAweb: A web server for RNA 3D structure modeling with optional restraints.
      ). A putative flow from an identified sequence to computer-aided structural optimization of aptamers is shown in Figure 7. To allow convenient selection of the appropriate tool for each task, Rtools (http://rtools.cbrc.jp/) was developed as an integrated web-server hosting existing prediction algorithms for RNA secondary structure analysis (
      • Hamada M.
      • Ono Y.
      • Kiryu H.
      • Sato K.
      • Kato Y.
      • Fukunaga T.
      • Mori R.
      • Asai K.
      Rtools: A web server for various secondary structural analyses on single RNA sequences.
      ).
      Figure thumbnail gr7
      Figure 7A recommended workflow for in silico aptamer design, analysis, and optimization. Candidates of DNA or RNA aptamers are subjected to clustering by high-throughput sequence analysis programs, such as meta-Z-score or AptaCluster. To predict secondary structures and motifs, the resulting clusters are processed by secondary-structure and motif-finding software tools such as Mfold, MEMERIS, and/or QGRS Mapper. Next, prediction of the tertiary structure of the aptamers is performed by RNAComposer. Then, the binding site(s) may be deciphered by molecular docking of the aptamers with their target, e.g., an amyloid protein, using machine learning tools, such as FTDock. Finally, the resulting complex structure is further tested by SMART-Aptamer, followed by AptaMut analysis for considering point mutations that may improve the aptamer’s affinity and/or specificity.
      Structural prediction programs have begun being applied in the amyloid field. Mfold likely is most used program in the field due to its free availability. Mfold analysis of the anti-Aβ40 DNA aptamer RNV95 suggested a stem-loop structure, which was validated by CD spectroscopy measurements (
      • Chakravarthy M.
      • AlShamaileh H.
      • Huang H.
      • Tannenberg R.K.
      • Chen S.
      • Worrall S.
      • Dodd P.R.
      • Veedu R.N.
      Development of DNA aptamers targeting low-molecular-weight amyloid-β peptide aggregates in vitro.
      ). Bunka et al. (
      • Bunka D.H.
      • Mantle B.J.
      • Morten I.J.
      • Tennent G.A.
      • Radford S.E.
      • Stockley P.G.
      Production and characterization of RNA aptamers specific for amyloid fibril epitopes.
      ) utilized Mfold for the prediction of RNA aptamer structure against β2-microglobulin, a protein whose deposition as amyloid in joints is associated with symptoms of dialysis-related amyloidosis, to reveal an enzyme cleavage site in a stem-loop structure, facilitating the identification of the aptamer’s aptatope.
      G-quadruplex is a noncanonical structure in both RNA and DNA aptamers, which, as mentioned in previous sections, has been found in several cases to play key roles in aptamer binding to amyloid–protein targets. The G-quadruplex structure includes a stable planar core comprising four guanine bases in the same plane forming G-tetrads (Fig. 4A) stabilized by π–π interactions (
      • Davis J.T.
      G-quartets 40 years later: From 5'-GMP to molecular biology and supramolecular chemistry.
      ,
      • Gatto B.
      • Palumbo M.
      • Sissi C.
      Nucleic acid aptamers based on the G-quadruplex structure: Therapeutic and diagnostic potential.
      ). The structure can be stabilized further by metal ions (
      • Wu Y.
      • Shi Y.
      • Deng S.
      • Wu C.
      • Deng R.
      • He G.
      • Zhou M.
      • Zhong K.
      • Gao H.
      Metal-induced G-quadruplex polymorphism for ratiometric and label-free detection of lead pollution in tea.
      ) or by flat molecule-induced chelation (Fig. 4B) (
      • Tsukakoshi K.
      • Ikuta Y.
      • Abe K.
      • Yoshida W.
      • Iida K.
      • Ma Y.
      • Nagasawa K.
      • Sode K.
      • Ikebukuro K.
      Structural regulation by a G-quadruplex ligand increases binding abilities of G-quadruplex-forming aptamers.
      ). However, prediction of G-quadruplex motifs is particularly difficult because of the need to bring together four noncontiguous guanines that may be far apart from each other in the sequence (
      • Kwok C.K.
      • Merrick C.J.
      G-quadruplexes: Prediction, characterization, and biological application.
      ).
      Several software tools have been developed to address this challenge, such as QGRS (Quadruplex forming G-Rich Sequences) Mapper (
      • Kikin O.
      • D'Antonio L.
      • Bagga P.S.
      QGRS mapper: A web-based server for predicting G-quadruplexes in nucleotide sequences.
      ), GRSdb (
      • Kikin O.
      • Zappala Z.
      • D'Antonio L.
      • Bagga P.S.
      GRSDB2 and GRS_UTRdb: Databases of quadruplex forming G-rich sequences in pre-mRNAs and mRNAs.
      ), and QuadBase2 (
      • Dhapola P.
      • Chowdhury S.
      QuadBase2: Web server for multiplexed guanine quadruplex mining and visualization.
      ), which are specific to prediction of G-quadruplexes in RNA and DNA sequences. For example, the formation of G-quadruplex in an RNA aptamer against Aβ42 protofibrils predicted by QGRS Mapper was validated by detection of a negative peak at ∼240 nm and a positive peak at ∼265 nm in the CD spectrum (
      • Burge S.
      • Parkinson G.N.
      • Hazel P.
      • Todd A.K.
      • Neidle S.
      Quadruplex DNA: Sequence, topology and structure.
      ), and by observation of a 1650 cm−1 absorbance peak in the Attenuated Total Reflection-FTIR spectrum (
      • Andrushchenko V.
      • Tsankov D.
      • Krasteva M.
      • Wieser H.
      • Bour P.
      Spectroscopic detection of DNA quadruplexes by vibrational circular dichroism.
      ), both of which are unique to guanine carbonyl groups in a G-quadruplex (
      • Murakami K.
      • Obata Y.
      • Sekikawa A.
      • Ueda H.
      • Izuo N.
      • Awano T.
      • Takabe K.
      • Shimizu T.
      • Irie K.
      An RNA aptamer with potent affinity for a toxic dimer of amyloid β42 has potential utility for histochemical studies of Alzheimer's disease.
      ,
      • Obata Y.
      • Murakami K.
      • Kawase T.
      • Hirose K.
      • Izuo N.
      • Shimizu T.
      • Irie K.
      Detection of amyloid β oligomers with RNA aptamers in AppNL-G-F/NL-G-F mice: A model of Arctic Alzheimer's disease.
      ). Similarly, a DNA aptamer (T-SO508) against αSyn oligomers was predicted to contain a G-quadruplex by QGRS Mapper, and the prediction was confirmed by CD spectroscopy (
      • Tsukakoshi K.
      • Ikuta Y.
      • Abe K.
      • Yoshida W.
      • Iida K.
      • Ma Y.
      • Nagasawa K.
      • Sode K.
      • Ikebukuro K.
      Structural regulation by a G-quadruplex ligand increases binding abilities of G-quadruplex-forming aptamers.
      ).
      In the course of sequence optimization after structure prediction, to increase the affinity for the targets, the program AptaMut (
      • Hoinka J.
      • Berezhnoy A.
      • Dao P.
      • Sauna Z.E.
      • Gilboa E.
      • Przytycka T.M.
      Large scale analysis of the mutational landscape in HT-SELEX improves aptamer discovery.
      ) can provide clues for the potential effects of point mutations on affinity and structure. To enhance the resistance of RNA aptamers to chemical and enzymatic degradation, inclusion of modified nucleotides, such as 2′-O-methyl, 2′-fluoro, or other unnatural nucleotides (
      • Duffy K.
      • Arangundy-Franklin S.
      • Holliger P.
      Modified nucleic acids: Replication, evolution, and next-generation therapeutics.
      ,
      • Hirao I.
      • Kimoto M.
      • Yamashige R.
      Natural versus artificial creation of base pairs in DNA: Origin of nucleobases from the perspectives of unnatural base pair studies.
      ) in place of the natural 2′-OH ribose are common approaches. A step forward was introduced independently by Hirao and colleagues (
      • Kimoto M.
      • Yamashige R.
      • Matsunaga K.
      • Yokoyama S.
      • Hirao I.
      Generation of high-affinity DNA aptamers using an expanded genetic αbet.
      ,
      • Kimoto M.
      • Hirao I.
      Genetic alphabet expansion technology by creating unnatural base pairs.
      ), and Tan, Benner and coworkers (
      • Sefah K.
      • Yang Z.
      • Bradley K.M.
      • Hoshika S.
      • Jimenez E.
      • Zhang L.
      • Zhu G.
      • Shanker S.
      • Yu F.
      • Turek D.
      • Tan W.
      • Benner S.A.
      In vitro selection with artificial expanded genetic information systems.
      ), who incorporated modified nucleotides originally designed to form unnatural base pairs for synthetic xenobiology, into aptamers, increasing the resistance of the oligonucleotides to degradation (Fig. 8).
      Figure thumbnail gr8
      Figure 8Noncanonical artificial base pairs used in aptamers. Artificial base pairs amenable to PCR amplification and aptamer development together with the natural ones: P, 2-amino-8-(1′-β-D-2-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)one; Z, 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone; Ds, 7-(2-thienyl)-imidazo[4,5-b]pyridine; and Px, 2-nitro-4-propynylpyrrole. Hydrogen-bond stabilization for the Ds-Px pair has not been described.
      Among the challenges in translating aptamers against amyloidogenic proteins into therapeutic applications is also their potential toxicity, similar to other nucleic-acid-based drugs. For example, in the development of antisense therapy, acute hepatotoxicity of 2′-fluoro-modified antisense oligonucleotides was found in 6- to 8-week-old wild-type mice, in which the oligonucleotides were administered subcutaneously at 20 mg/kg three times a week (
      • Shen W.
      • De Hoyos C.L.
      • Sun H.
      • Vickers T.A.
      • Liang X.H.
      • Crooke S.T.
      Acute hepatotoxicity of 2' fluoro-modified 5-10-5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins.
      ). In contrast, in other studies, a 2′-fluoro nucleotide-stabilized siRNA was administered subcutaneously as a single, 30-mg/kg bolus for 2 years in a rat or at daily 7.5-mg/kg injections for 5 days in healthy volunteers without apparent toxicity (
      • Janas M.M.
      • Zlatev I.
      • Liu J.
      • Jiang Y.
      • Barros S.A.
      • Sutherland J.E.
      • Davis W.P.
      • Liu J.
      • Brown C.R.
      • Liu X.
      • Schlegel M.K.
      • Blair L.
      • Zhang X.
      • Das B.
      • Tran C.
      • et al.
      Safety evaluation of 2'-deoxy-2'-fluoro nucleotides in GalNAc-siRNA conjugates.
      ). Similarly, antisense oligonucleotides containing 2′-O-methyl nucleotides were administered to cynomolgus macaques (Macaca fascicularis) at 20 mg/kg by subcutaneous injections for 9 months and to patients with hereditary transthyretin amyloidosis in a phase 2/3 clinical study at 300 mg for 15 months without cytotoxic effects (
      • Yu R.Z.
      • Wang Y.
      • Norris D.A.
      • Kim T.W.
      • Narayanan P.
      • Geary R.S.
      • Monia B.P.
      • Henry S.P.
      Immunogenicity assessment of inotersen, a 2'-O-(2-methoxyethyl) antisense oligonucleotide in animals and humans: Effect on pharmacokinetics, pharmacodynamics, and safety.
      ). Another concern is that as is the case with antibodies (see, e.g., an interesting report by Hatami et al. (
      • Hatami A.
      • Monjazeb S.
      • Glabe C.
      The anti-amyloid-β monoclonal antibody 4G8 recognizes a generic sequence-independent epitope associated with α-synuclein and islet amyloid polypeptide amyloid fibrils.
      )), aptamers may cross-react with off-targets. In particular, aptamers against amyloid proteins might cross-react with functional human amyloids (
      • Fowler D.M.
      • Koulov A.V.
      • Balch W.E.
      • Kelly J.W.
      Functional amyloid--from bacteria to humans.
      ,
      • Maji S.K.
      • Perrin M.H.
      • Sawaya M.R.
      • Jessberger S.
      • Vadodaria K.
      • Rissman R.A.
      • Singru P.S.
      • Nilsson K.P.
      • Simon R.
      • Schubert D.
      • Eisenberg D.
      • Rivier J.
      • Sawchenko P.
      • Vale W.
      • Riek R.
      Functional amyloids as natural storage of peptide hormones in pituitary secretory granules.
      ) and disrupt their function, leading to cytotoxicity. Thus, in addition to characterization of their binding and efficacy, aptamers intended for in vivo use must be evaluated for their safety and be applied only if they have a sufficiently large therapeutic index. In this context, an advantage of aptamers as chemotypes for drug development is the ease with which they can be modified and subjected to structure–activity relationship studies compared with small molecules or protein biologics.

      Prediction of aptamer–protein interaction by machine learning and molecular docking

      After determination of the secondary and tertiary structures of new aptamers, deciphering the aptamer–protein complex structure often is the next goal. Molecular docking of aptamers and target proteins on each other is a forecasting method that typically consists of searching all the potential binding modes between the pair of molecules in silico and scoring each complex based on its thermodynamic stability. As an example, a docking simulation of multiple aptamer–protein pairs was carried out to optimize the aptamer sequence using different proteins in combination either with RNA aptamers or with ribosomal RNA by Zhang et al. (
      • Zhang Z.
      • Lu L.
      • Zhang Y.
      • Hua Li C.
      • Wang C.X.
      • Zhang X.Y.
      • Tan J.J.
      A combinatorial scoring function for protein-RNA docking.
      ) using FTDock. The report was primarily methodological, but we expect that this methodology will be useful for future drug development.
      Recently, a new Sequential Multidimensional Analysis algoRiThm for aptamer discovery (SMART-Aptamer) using HT-SELEX based on machine learning has been described (
      • Song J.
      • Zheng Y.
      • Huang M.
      • Wu L.
      • Wang W.
      • Zhu Z.
      • Song Y.
      • Yang C.
      A sequential multidimensional analysis algorithm for aptamer identification based on structure analysis and machine learning.
      ). This system of molecular docking allowed obtaining high-affinity aptamers with low false-positive and false-negative rates. This new system coupled with modeling of the tertiary structure has high promise for optimization of the interaction mode between aptamers and amyloid–protein targets. However, although these approaches have helped optimizing aptamers in different cases, such as for human embryonic stem cells, epithelial cell adhesion molecule, and cell-surface vimentin (
      • Song J.
      • Zheng Y.
      • Huang M.
      • Wu L.
      • Wang W.
      • Zhu Z.
      • Song Y.
      • Yang C.
      A sequential multidimensional analysis algorithm for aptamer identification based on structure analysis and machine learning.
      ), they have not yet been used in the amyloid field.

      Brain-targeting delivery of aptamers by exosomes and nanoliposomes

      Oligonucleic acids are too large and negatively charged to pass through the blood–brain barrier (BBB). To exert the functions of amyloid-targeting aptamers in the CNS, the development of effective delivery systems is an important challenge. Since bare nucleic acid delivered systemically also would be subject to enzymatic degradation and may not even reach the BBB in sufficient amounts (
      • Reichmuth A.M.
      • Oberli M.A.
      • Jaklenec A.
      • Langer R.
      • Blankschtein D.
      mRNA vaccine delivery using lipid nanoparticles.
      ), innovative engineering of CNS-targeting delivery methods of aptamers using nanocarriers, such as exosomes or nanoliposome, has been a major focus in recent years.
      Exosomes are nanovesicles, 30 to 200 nm in diameter, secreted by virtually all cell types and are thought to mediate intercellular and interorgan communication, as well as serve a mechanism for removal of cellular stressors (
      • van Niel G.
      • D'Angelo G.
      • Raposo G.
      Shedding light on the cell biology of extracellular vesicles.
      ,
      • Hill A.F.
      Extracellular vesicles and neurodegenerative diseases.
      ,
      • Vassileff N.
      • Cheng L.
      • Hill A.F.
      Extracellular vesicles - propagators of neuropathology and sources of potential biomarkers and therapeutics for neurodegenerative diseases.
      ). They can be used as a delivery mechanism when the cargo is expressed in cultured cells and the exosomes containing the cargo are isolated from the cell-culture medium.
      A follow-up study on the DNA aptamer F5R1 (
      • Zheng Y.
      • Qu J.
      • Xue F.
      • Zheng Y.
      • Yang B.
      • Chang Y.
      • Yang H.
      • Zhang J.
      Novel DNA aptamers for Parkinson's disease treatment inhibit α-synuclein aggregation and facilitate its degradation.
      ) mentioned above revealed that it bound preferably to fibrillar, rather than monomeric αSyn (
      • Ren X.
      • Zhao Y.
      • Xue F.
      • Zheng Y.
      • Huang H.
      • Wang W.
      • Chang Y.
      • Yang H.
      • Zhang J.
      Exosomal DNA aptamer targeting α-synuclein aggregates reduced neuropathological deficits in a mouse Parkinson's disease model.
      ). To investigate a therapeutic potential of F5R1, it was encapsuled, following polyethylenimine-assisted transfection, in exosomes isolated from the culture medium of HEK293 cells. It was then delivered into the brain of wild-type mice, which received intrastriatal injection of preformed recombinant αSyn fibrils prepared from recombinant αSyn, by taking advantage of a virus-transmission system (Fig. 9A) (
      • Ren X.
      • Zhao Y.
      • Xue F.
      • Zheng Y.
      • Huang H.
      • Wang W.
      • Chang Y.
      • Yang H.
      • Zhang J.
      Exosomal DNA aptamer targeting α-synuclein aggregates reduced neuropathological deficits in a mouse Parkinson's disease model.
      ). The cells also expressed rabies virus glycoprotein (RVG) fused with lamp2B, which positions the RVG on the outer membrane of exosomes secreted by these cells. Intraperitoneal administration of the RVG-decorated exosomes containing F5R1 led to retrograde transport and transsynaptic transmission into the CNS through the axons and synapses of peripheral neurons, circumventing the BBB. The treatment decreased αSyn aggregation in the substantia nigra and ameliorated motor dysfunction in the treated mice (
      • Ren X.
      • Zhao Y.
      • Xue F.
      • Zheng Y.
      • Huang H.
      • Wang W.
      • Chang Y.
      • Yang H.
      • Zhang J.
      Exosomal DNA aptamer targeting α-synuclein aggregates reduced neuropathological deficits in a mouse Parkinson's disease model.
      ). This pioneering work provided proof of concept for application of DNA–aptamer therapy delivered into the CNS using an innovative delivery system that bypasses the BBB, resulting in reduction of the aggregation, transmission, and toxicity of αSyn in a preclinical model of synucleinopathy.
      Figure thumbnail gr9
      Figure 9Strategies for delivering aptamers into the CNS by encapsulation in nanovesicles. A, aptamers encapsulated in exosome expressing rabies virus glycoprotein (RVG) on their surface are retrogradely and transsynaptically transported from the peripheral nervous system into the brain, circumventing the BBB. B, aptamers included in nanoliposome carrying ligands of transferrin receptor (TfR) are transported from the blood to the brain parenchyma by receptor-mediated transcytosis.
      A different aptamer-delivery method into the CNS using nanoliposomes was reported by McConnell et al. (Fig. 9B) (
      • McConnell E.M.
      • Ventura K.
      • Dwyer Z.
      • Hunt V.
      • Koudrina A.
      • Holahan M.R.
      • DeRosa M.C.
      In vivo use of a multi-DNA aptamer-based payload/targeting system to study dopamine dysregulation in the central nervous system.
      ). Like exosomes, nanoliposomes are nanovesicles, yet they are prepared synthetically, as opposed to the natural origin of exosomes, allowing a greater degree of control over their composition and cargo. Similar to exosomes, the nanoliposomes are enclosed by a lipid bilayer surrounding trapped water in which hydrophilic compounds can be transported. McConnell et al. encapsuled a dopamine-specific DNA aptamer, Da20m, into nanoliposomes conjugated on their surface by a different aptamer, specific for the transferrin receptor (TfR). TfR is expressed on endothelial cells in the luminal surface of the BBB. Binding of the anti-TfR aptamer to TfR followed by clathrin-mediated transcytosis of TfR allowed delivery of the nanoliposomes across the BBB and release of the antidopamine aptamer cargo on the CNS-parenchymal side. Compared with unencapsulated Da20m, which had little or no effect, systemic administration of Da20m via the functionalized nanoliposomes potently attenuated abnormal behavior of wild-type mice triggered by aberrantly increased extracellular dopamine following exposure to cocaine (
      • McConnell E.M.
      • Ventura K.
      • Dwyer Z.
      • Hunt V.
      • Koudrina A.
      • Holahan M.R.
      • DeRosa M.C.
      In vivo use of a multi-DNA aptamer-based payload/targeting system to study dopamine dysregulation in the central nervous system.
      ). This system appears to be highly promising, yet to our knowledge it has not been used yet for delivery of aptamers against amyloidogenic proteins.
      To date, over 20 liposomal products have been approved by the FDA, including anticancer drugs and vaccine formulations against SARS-CoV-2 containing RNA molecules (
      • Sahin U.
      • Muik A.
      • Derhovanessian E.
      • Vogler I.
      • Kranz L.M.
      • Vormehr M.
      • Baum A.
      • Pascal K.
      • Quandt J.
      • Maurus D.
      • Brachtendorf S.
      • Lorks V.
      • Sikorski J.
      • Hilker R.
      • Becker D.
      • et al.
      COVID-19 vaccine BNT162b1 elicits human antibody and TH1 T cell responses.
      ,
      • Baden L.R.
      • El Sahly H.M.
      • Essink B.
      • Kotloff K.
      • Frey S.
      • Novak R.
      • Diemert D.
      • Spector S.A.
      • Rouphael N.
      • Creech C.B.
      • McGettigan J.
      • Khetan S.
      • Segall N.
      • Solis J.
      • Brosz A.
      • et al.
      Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine.
      ). Encapsulation of RNA (and DNA) in liposomes promotes the biosafety and stability of the exogenous nucleic acids inside the body. Both exosomes and nanoliposomes are attractive delivery systems for aptamers. Exosome cargo and surface markers are easier to manipulate genetically, compared with those of nanoliposomes, yet nanoliposomes can be produced more easily on large scales (
      • Raemdonck K.
      • Braeckmans K.
      • Demeester J.
      • De Smedt S.C.
      Merging the best of both worlds: Hybrid lipid-enveloped matrix nanocomposites in drug delivery.
      ). In addition, because exosomes are produced in cells and may contain unintended biological material, it is crucial to establish ways of controlling and monitoring their clinical safety if they are going to be approved for therapeutic applications (
      • Zhu X.
      • Badawi M.
      • Pomeroy S.
      • Sutaria D.S.
      • Xie Z.
      • Baek A.
      • Jiang J.
      • Elgamal O.A.
      • Mo X.
      • Perle K.
      • Chalmers J.
      • Schmittgen T.D.
      • Phelps M.A.
      Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells.
      ). Although exosomes are less immunogenic than cell-based therapies, a concern in using them as drug carriers is that they may be recognized as nonself by the immune system and induce an immune response, potentially causing damaging inflammation. Should this become a problem, it could be overcome by extracting the patient’s own cells and preparing the exosomes using the patient-derived cells. Though more time-consuming and potentially expensive, such a procedure could be justified for life-saving treatments.

      Conclusions and future perspectives

      In 2004, the first aptamer drug, the RNA aptamer Macugen (pegaptanib sodium), which acts as an antagonist of vascular endothelial growth factor, was approved by FDA for treatment of age-related macular degeneration. Several additional aptamers have been tested in clinical trials for cancer, cardiovascular disease, and eye diseases, yet to date, despite the growing number of reports on antiamyloid aptamers and the advances in their design and delivery discussed above, aptamers have not yet advanced to clinical trials in the neurodegenerative disease field.
      To advance the development of aptamers targeting amyloidogenic protein for biomedical applications, several issues should be addressed. First, the conformational metastability and heterogeneity of the targets, particularly oligomers of amyloidogenic proteins, are major impediments for aptamer selection. This challenge can be addressed by covalent tethering of the protein monomers to create a more stable aptagen that mimics the metastable oligomers, as was demonstrated in the case of Aβ42 (
      • Murakami K.
      • Obata Y.
      • Sekikawa A.
      • Ueda H.
      • Izuo N.
      • Awano T.
      • Takabe K.
      • Shimizu T.
      • Irie K.
      An RNA aptamer with potent affinity for a toxic dimer of amyloid β42 has potential utility for histochemical studies of Alzheimer's disease.
      ), by identification of conformations or sequences responsible for pathology, as was done for prion (
      • Rhie A.
      • Kirby L.
      • Sayer N.
      • Wellesley R.
      • Disterer P.
      • Sylvester I.
      • Gill A.
      • Hope J.
      • James W.
      • Tahiri-Alaoui A.
      Characterization of 2'-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion.
      ) or tau (
      • Kim J.H.
      • Kim E.
      • Choi W.H.
      • Lee J.
      • Lee J.H.
      • Lee H.
      • Kim D.E.
      • Suh Y.H.
      • Lee M.J.
      Inhibitory RNA aptamers of tau oligomerization and their neuroprotective roles against proteotoxic stress.
      ), and by competitive selection of target assemblies in combination with counterselection of other species, as was successfully carried out for αSyn (
      • Tsukakoshi K.
      • Abe K.
      • Sode K.
      • Ikebukuro K.
      Selection of DNA aptamers that recognize α-synuclein oligomers using a competitive screening method.
      ).
      Second, isolating aptamers with high affinity and specificity for the desired target is a labor-intensive and time-consuming process that is always accompanied by a level of uncertainty. Next-Generation sequencing and structural prediction programs coupled with AI and machine learning algorithms are promising approaches for increasing both the speed of the process and the likelihood of success. Application of these modern techniques has been limited in the amyloid field but is beginning to catch up. Additional advances are expected when detailed structures of aptamers and their complexes with their respective targets become available and support structure prediction algorithms. To our knowledge, to date, only two X-ray crystallography studies reporting such structures have been published (
      • Nomura Y.
      • Sugiyama S.
      • Sakamoto T.
      • Miyakawa S.
      • Adachi H.
      • Takano K.
      • Murakami S.
      • Inoue T.
      • Mori Y.
      • Nakamura Y.
      • Matsumura H.
      Conformational plasticity of RNA for target recognition as revealed by the 2.15 angstrom (Å) crystal structure of a human IgG-aptamer complex.
      ,
      • Kato K.
      • Ikeda H.
      • Miyakawa S.
      • Futakawa S.
      • Nonaka Y.
      • Fujiwara M.
      • Okudaira S.
      • Kano K.
      • Aoki J.
      • Morita J.
      • Ishitani R.
      • Nishimasu H.
      • Nakamura Y.
      • Nureki O.
      Structural basis for specific inhibition of Autotaxin by a DNA aptamer.
      ). However, the increasing popularity of high-resolution structural determination by cryo-electron microscopy suggests that additional data will be forthcoming. High-resolution structures and improved structural prediction technology not only will facilitate obtaining improved aptamers, but also will help clarify the molecular basis of target recognition by aptamers, including in the amyloid field.
      Finally, the inability of aptamers to cross the BBB on their own can now be addressed by at least two delivery systems, including exosomes and nanoliposomes, as discussed above. Each approach has its advantages and limitations, yet we expect that future research will address the current shortcomings and lead to successful application of these techniques in delivering aptamers targeting amyloidogenic proteins into the CNS, as has been demonstrated recently for aptamer F5R1 in a mouse model of synucleinopathy (
      • Ren X.
      • Zhao Y.
      • Xue F.
      • Zheng Y.
      • Huang H.
      • Wang W.
      • Chang Y.
      • Yang H.
      • Zhang J.
      Exosomal DNA aptamer targeting α-synuclein aggregates reduced neuropathological deficits in a mouse Parkinson's disease model.
      ). Recent advances in both computational and experimental approaches suggest that using aptamers as antiamyloid therapeutics is achievable in the near future.

      Conflicts of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Author contributions

      K. M. and G. B. conceptualization; K. M. supervision; K. M. and N. I. writing—original draft; G. B. writing—review and editing.

      Funding and additional information

      This study was supported in part by the JSPS KAKENHI, grant number 20KK0126 to K. M. and N. I, and by NIH / NIA grant RF1AG054000 , The Alzheimer's Association , The Michael J. Fox Foundation , Weston Brain Institute , and Alzheimer’s Research UK Biomarkers Across Neurodegenerative Diseases (BAND 3) grant 17990 , and CurePSP grant 665-2019-07 to G. B.

      References

        • Iadanza M.G.
        • Jackson M.P.
        • Hewitt E.W.
        • Ranson N.A.
        • Radford S.E.
        A new era for understanding amyloid structures and disease.
        Nat. Rev. Mol. Cell Biol. 2018; 19: 755-773
        • Hartl F.U.
        Protein misfolding diseases.
        Annu. Rev. Biochem. 2017; 86: 21-26
        • Chuang E.
        • Hori A.M.
        • Hesketh C.D.
        • Shorter J.
        Amyloid assembly and disassembly.
        J. Cell Sci. 2018; 131jcs189928
        • Otzen D.
        • Riek R.
        Functional amyloids.
        Cold Spring Harb. Perspect. Biol. 2019; 11a033860
        • Zhou J.
        • Rossi J.
        Aptamers as targeted therapeutics: Current potential and challenges.
        Nat. Rev. Drug Discov. 2017; 16: 181-202
        • Ginsberg S.D.
        • Galvin J.E.
        • Chiu T.S.
        • Lee V.M.
        • Masliah E.
        • Trojanowski J.Q.
        RNA sequestration to pathological lesions of neurodegenerative diseases.
        Acta Neuropathol. 1998; 96: 487-494
        • Ginsberg S.D.
        • Crino P.B.
        • Hemby S.E.
        • Weingarten J.A.
        • Lee V.M.
        • Eberwine J.H.
        • Trojanowski J.Q.
        Predominance of neuronal mRNAs in individual Alzheimer's disease senile plaques.
        Ann. Neurol. 1999; 45: 174-181
        • Hua Q.
        • He R.Q.
        • Haque N.
        • Qu M.H.
        • del Carmen Alonso A.
        • Grundke-Iqbal I.
        • Iqbal K.
        Microtubule associated protein tau binds to double-stranded but not single-stranded DNA.
        Cell. Mol. Life Sci. 2003; 60: 413-421
        • McKeague M.
        • McConnell E.M.
        • Cruz-Toledo J.
        • Bernard E.D.
        • Pach A.
        • Mastronardi E.
        • Zhang X.
        • Beking M.
        • Francis T.
        • Giamberardino A.
        • Cabecinha A.
        • Ruscito A.
        • Aranda-Rodriguez R.
        • Dumontier M.
        • DeRosa M.C.
        Analysis of in vitro aptamer selection parameters.
        J. Mol. Evol. 2015; 81: 150-161
        • Shu Y.
        • Pi F.
        • Sharma A.
        • Rajabi M.
        • Haque F.
        • Shu D.
        • Leggas M.
        • Evers B.M.
        • Guo P.
        Stable RNA nanoparticles as potential new generation drugs for cancer therapy.
        Adv. Drug Deliv. Rev. 2014; 66: 74-89
        • Qu J.
        • Yu S.
        • Zheng Y.
        • Zheng Y.
        • Yang H.
        • Zhang J.
        Aptamer and its applications in neurodegenerative diseases.
        Cell. Mol. Life Sci. 2017; 74: 683-695
        • Bouvier-Muller A.
        • Duconge F.
        Nucleic acid aptamers for neurodegenerative diseases.
        Biochimie. 2018; 145: 73-83
        • Rahimi F.
        Aptamers selected for recognizing amyloid β-protein-a case for cautious optimism.
        Int. J. Mol. Sci. 2018; 19: 668
        • Fezoui Y.
        • Teplow D.B.
        Kinetic studies of amyloid β-protein fibril assembly. Differential effects of α-helix stabilization.
        J. Biol. Chem. 2002; 277: 36948-36954
        • Glenner G.G.
        • Wong C.W.
        Alzheimer's disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein.
        Biochem. Biophys. Res. Commun. 1984; 120: 885-890
        • Masters C.L.
        • Simms G.
        • Weinman N.A.
        • Multhaup G.
        • McDonald B.L.
        • Beyreuther K.
        Amyloid plaque core protein in Alzheimer disease and Down syndrome.
        Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249
        • Karran E.
        • Mercken M.
        • De Strooper B.
        The amyloid cascade hypothesis for Alzheimer's disease: An appraisal for the development of therapeutics.
        Nat. Rev. Drug Discov. 2011; 10: 698-712
        • Jarrett J.T.
        • Berger E.P.
        • Lansbury Jr., P.T.
        The carboxy terminus of the β amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer's disease.
        Biochemistry. 1993; 32: 4693-4697
        • Bitan G.
        • Kirkitadze M.D.
        • Lomakin A.
        • Vollers S.S.
        • Benedek G.B.
        • Teplow D.B.
        Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 330-335
        • Haass C.
        • Selkoe D.J.
        Soluble protein oligomers in neurodegeneration: Lessons from the Alzheimer's amyloid β-peptide.
        Nat. Rev. Mol. Cell Biol. 2007; 8: 101-112
        • Dahlgren K.N.
        • Manelli A.M.
        • Stine Jr., W.B.
        • Baker L.K.
        • Krafft G.A.
        • LaDu M.J.
        Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability.
        J. Biol. Chem. 2002; 277: 32046-32053
        • Roychaudhuri R.
        • Yang M.
        • Hoshi M.M.
        • Teplow D.B.
        Amyloid β-protein assembly and Alzheimer disease.
        J. Biol. Chem. 2009; 284: 4749-4753
        • Meyer-Luehmann M.
        • Spires-Jones T.L.
        • Prada C.
        • Garcia-Alloza M.
        • de Calignon A.
        • Rozkalne A.
        • Koenigsknecht-Talboo J.
        • Holtzman D.M.
        • Bacskai B.J.
        • Hyman B.T.
        Rapid appearance and local toxicity of amyloid-β plaques in a mouse model of Alzheimer's disease.
        Nature. 2008; 451: 720-724
        • Benilova I.
        • Karran E.
        • De Strooper B.
        The toxic Aβ oligomer and Alzheimer's disease: An emperor in need of clothes.
        Nat. Neurosci. 2012; 15: 349-357
        • Murakami K.
        Conformation-specific antibodies to target amyloid β oligomers and their application to immunotherapy for Alzheimer's disease.
        Biosci. Biotechnol. Biochem. 2014; 78: 1293-1305
        • Bitan G.
        • Fradinger E.A.
        • Spring S.M.
        • Teplow D.B.
        Neurotoxic protein oligomers--what you see is not always what you get.
        Amyloid. 2005; 12: 88-95
        • Lesnè S.
        • Koh M.T.
        • Kotilinek L.
        • Kayed R.
        • Glabe C.G.
        • Yang A.
        • Gallagher M.
        • Ashe K.H.
        A specific amyloid-β protein assembly in the brain impairs memory.
        Nature. 2006; 440: 352-357
        • Walsh D.M.
        • Lomakin A.
        • Benedek G.B.
        • Condron M.M.
        • Teplow D.B.
        Amyloid β-protein fibrillogenesis. Detection of a protofibrillar intermediate.
        J. Biol. Chem. 1997; 272: 22364-22372
        • Walsh D.M.
        • Hartley D.M.
        • Kusumoto Y.
        • Fezoui Y.
        • Condron M.M.
        • Lomakin A.
        • Benedek G.B.
        • Selkoe D.J.
        • Teplow D.B.
        Amyloid β-protein fibrillogenesis. Structure and biological activity of protofibrillar intermediates.
        J. Biol. Chem. 1999; 274: 25945-25952
        • Harper J.D.
        • Wong S.S.
        • Lieber C.M.
        • Lansbury P.T.
        Observation of metastable Aβ amyloid protofibrils by atomic force microscopy.
        Chem. Biol. 1997; 4: 119-125
        • Barghorn S.
        • Nimmrich V.
        • Striebinger A.
        • Krantz C.
        • Keller P.
        • Janson B.
        • Bahr M.
        • Schmidt M.
        • Bitner R.S.
        • Harlan J.
        • Barlow E.
        • Ebert U.
        • Hillen H.
        Globular amyloid β-peptide oligomer - a homogenous and stable neuropathological protein in Alzheimer's disease.
        J. Neurochem. 2005; 95: 834-847
        • Deshpande A.
        • Mina E.
        • Glabe C.
        • Busciglio J.
        Different conformations of amyloid β induce neurotoxicity by distinct mechanisms in human cortical neurons.
        J. Neurosci. 2006; 26: 6011-6018
        • Lambert M.P.
        • Barlow A.K.
        • Chromy B.A.
        • Edwards C.
        • Freed R.
        • Liosatos M.
        • Morgan T.E.
        • Rozovsky I.
        • Trommer B.
        • Viola K.L.
        • Wals P.
        • Zhang C.
        • Finch C.E.
        • Krafft G.A.
        • Klein W.L.
        Diffusible, nonfibrillar ligands derived from Aβ1-42 are potent central nervous system neurotoxins.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6448-6453
        • Caughey B.
        • Lansbury P.T.
        Protofibrils, pores, fibrils, and neurodegeneration: Separating the responsible protein aggregates from the innocent bystanders.
        Annu. Rev. Neurosci. 2003; 26: 267-298
        • Hoshi M.
        • Sato M.
        • Matsumoto S.
        • Noguchi A.
        • Yasutake K.
        • Yoshida N.
        • Sato K.
        Spherical aggregates of β-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3β.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6370-6375
        • Tucker S.
        • Moller C.
        • Tegerstedt K.
        • Lord A.
        • Laudon H.
        • Sjodahl J.
        • Soderberg L.
        • Spens E.
        • Sahlin C.
        • Waara E.R.
        • Satlin A.
        • Gellerfors P.
        • Osswald G.
        • Lannfelt L.
        The murine version of BAN2401 (mAb158) selectively reduces amyloid-β protofibrils in brain and cerebrospinal fluid of tg-ArcSwe mice.
        J. Alzheimers Dis. 2015; 43: 575-588
        • Lambert M.P.
        • Velasco P.T.
        • Chang L.
        • Viola K.L.
        • Fernandez S.
        • Lacor P.N.
        • Khuon D.
        • Gong Y.
        • Bigio E.H.
        • Shaw P.
        • De Felice F.G.
        • Krafft G.A.
        • Klein W.L.
        Monoclonal antibodies that target pathological assemblies of Aβ.
        J. Neurochem. 2007; 100: 23-35
        • Kayed R.
        • Head E.
        • Thompson J.L.
        • McIntire T.M.
        • Milton S.C.
        • Cotman C.W.
        • Glabe C.G.
        Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.
        Science. 2003; 300: 486-489
        • Hillen H.
        • Barghorn S.
        • Striebinger A.
        • Labkovsky B.
        • Muller R.
        • Nimmrich V.
        • Nolte M.W.
        • Perez-Cruz C.
        • van der Auwera I.
        • van Leuven F.
        • van Gaalen M.
        • Bespalov A.Y.
        • Schoemaker H.
        • Sullivan J.P.
        • Ebert U.
        Generation and therapeutic efficacy of highly oligomer-specific β-amyloid antibodies.
        J. Neurosci. 2010; 30: 10369-10379
        • Goñi F.
        • Marta-Ariza M.
        • Peyser D.
        • Herline K.
        • Wisniewski T.
        Production of monoclonal antibodies to pathologic β-sheet oligomeric conformers in neurodegenerative diseases.
        Sci. Rep. 2017; 7: 9881
        • Kayed R.
        • Canto I.
        • Breydo L.
        • Rasool S.
        • Lukacsovich T.
        • Wu J.
        • Albay 3rd, R.
        • Pensalfini A.
        • Yeung S.
        • Head E.
        • Marsh J.L.
        • Glabe C.
        Conformation dependent monoclonal antibodies distinguish different replicating strains or conformers of prefibrillar Aβ oligomers.
        Mol. Neurodegener. 2010; 5: 57
        • Ylera F.
        • Lurz R.
        • Erdmann V.A.
        • Furste J.P.
        Selection of RNA aptamers to the Alzheimer's disease amyloid peptide.
        Biochem. Biophys. Res. Commun. 2002; 290: 1583-1588
        • Farrar C.T.
        • William C.M.
        • Hudry E.
        • Hashimoto T.
        • Hyman B.T.
        RNA aptamer probes as optical imaging agents for the detection of amyloid plaques.
        PLoS One. 2014; 9e89901
        • Jankowsky J.L.
        • Fadale D.J.
        • Anderson J.
        • Xu G.M.
        • Gonzales V.
        • Jenkins N.A.
        • Copeland N.G.
        • Lee M.K.
        • Younkin L.H.
        • Wagner S.L.
        • Younkin S.G.
        • Borchelt D.R.
        Mutant presenilins specifically elevate the levels of the 42 residue β-amyloid peptide in vivo: Evidence for augmentation of a 42-specific γ secretase.
        Hum. Mol. Genet. 2004; 13: 159-170
        • Bitan G.
        Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.
        Methods Enzymol. 2006; 413: 217-236
        • Bitan G.
        • Teplow D.B.
        Rapid photochemical cross-linking--a new tool for studies of metastable, amyloidogenic protein assemblies.
        Acc. Chem. Res. 2004; 37: 357-364
        • Maji S.K.
        • Ogorzalek Loo R.R.
        • Inayathullah M.
        • Spring S.M.
        • Vollers S.S.
        • Condron M.M.
        • Bitan G.
        • Loo J.A.
        • Teplow D.B.
        Amino acid position-specific contributions to amyloid β-protein oligomerization.
        J. Biol. Chem. 2009; 284: 23580-23591
        • Rahimi F.
        • Murakami K.
        • Summers J.L.
        • Chen C.H.
        • Bitan G.
        RNA aptamers generated against oligomeric Aβ40 recognize common amyloid aptatopes with low specificity but high sensitivity.
        PLoS One. 2009; 4e7694
        • Rahimi F.
        • Bitan G.
        Selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.
        J. Vis. Exp. 2010; 39: 1955
        • Sipe J.D.
        • Cohen A.S.
        Review: History of the amyloid fibril.
        J. Struct. Biol. 2000; 130: 88-98
        • Tsukakoshi K.
        • Abe K.
        • Sode K.
        • Ikebukuro K.
        Selection of DNA aptamers that recognize α-synuclein oligomers using a competitive screening method.
        Anal. Chem. 2012; 84: 5542-5547
        • Zhu L.
        • Zhang J.
        • Wang F.
        • Wang Y.
        • Lu L.
        • Feng C.
        • Xu Z.
        • Zhang W.
        Selective amyloid β oligomer assay based on abasic site-containing molecular beacon and enzyme-free amplification.
        Biosens. Bioelectron. 2016; 78: 206-212
        • Takahashi T.
        • Tada K.
        • Mihara H.
        RNA aptamers selected against amyloid β-peptide (Aβ) inhibit the aggregation of Aβ.
        Mol. Biosyst. 2009; 5: 986-991
        • Babu E.
        • Muthu Mareeswaran P.
        • Sathish V.
        • Singaravadivel S.
        • Rajagopal S.
        Sensing and inhibition of amyloid-β based on the simple luminescent aptamer-ruthenium complex system.
        Talanta. 2015; 134: 348-353
        • Chakravarthy M.
        • AlShamaileh H.
        • Huang H.
        • Tannenberg R.K.
        • Chen S.
        • Worrall S.
        • Dodd P.R.
        • Veedu R.N.
        Development of DNA aptamers targeting low-molecular-weight amyloid-β peptide aggregates in vitro.
        Chem. Commun. 2018; 54: 4593-4596
        • Bernstein S.L.
        • Dupuis N.F.
        • Lazo N.D.
        • Wyttenbach T.
        • Condron M.M.
        • Bitan G.
        • Teplow D.B.
        • Shea J.E.
        • Ruotolo B.T.
        • Robinson C.V.
        • Bowers M.T.
        Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer's disease.
        Nat. Chem. 2009; 1: 326-331
        • Murakami K.
        • Irie K.
        • Morimoto A.
        • Ohigashi H.
        • Shindo M.
        • Nagao M.
        • Shimizu T.
        • Shirasawa T.
        Neurotoxicity and physicochemical properties of Aβ mutant peptides from cerebral amyloid angiopathy: Implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease.
        J. Biol. Chem. 2003; 278: 46179-46187
        • Murakami K.
        • Obata Y.
        • Sekikawa A.
        • Ueda H.
        • Izuo N.
        • Awano T.
        • Takabe K.
        • Shimizu T.
        • Irie K.
        An RNA aptamer with potent affinity for a toxic dimer of amyloid β42 has potential utility for histochemical studies of Alzheimer's disease.
        J. Biol. Chem. 2020; 295: 4870-4880
        • Obata Y.
        • Murakami K.
        • Kawase T.
        • Hirose K.
        • Izuo N.
        • Shimizu T.
        • Irie K.
        Detection of amyloid β oligomers with RNA aptamers in AppNL-G-F/NL-G-F mice: A model of Arctic Alzheimer's disease.
        ACS Omega. 2020; 5: 21531-21537
        • Murakami K.
        • Tokuda M.
        • Suzuki T.
        • Irie Y.
        • Hanaki M.
        • Izuo N.
        • Monobe Y.
        • Akagi K.
        • Ishii R.
        • Tatebe H.
        • Tokuda T.
        • Maeda M.
        • Kume T.
        • Shimizu T.
        • Irie K.
        Monoclonal antibody with conformational specificity for a toxic conformer of amyloid β42 and its application toward the Alzheimer's disease diagnosis.
        Sci. Rep. 2016; 6: 29038
        • Irie K.
        New diagnostic method for Alzheimer's disease based on the toxic conformation theory of amyloid β.
        Biosci. Biotechnol. Biochem. 2020; 84: 1-16
        • Toda T.
        • Noda Y.
        • Ito G.
        • Maeda M.
        • Shimizu T.
        Presenilin-2 mutation causes early amyloid accumulation and memory impairment in a transgenic mouse model of Alzheimer's disease.
        J. Biomed. Biotechnol. 2011; 2011: 617974
        • Saito T.
        • Matsuba Y.
        • Mihira N.
        • Takano J.
        • Nilsson P.
        • Itohara S.
        • Iwata N.
        • Saido T.C.
        Single app knock-in mouse models of Alzheimer's disease.
        Nat. Neurosci. 2014; 17: 661-663
        • Zheng Y.
        • Wang P.
        • Li S.
        • Geng X.
        • Zou L.
        • Jin M.
        • Zou Q.
        • Wang Q.
        • Yang X.
        • Wang K.
        Development of DNA aptamer as a β-amyloid aggregation inhibitor.
        ACS Appl. Bio Mater. 2020; 3: 8611-8618
        • Arendt T.
        • Stieler J.T.
        • Holzer M.
        Tau and tauopathies.
        Brain Res. Bull. 2016; 126: 238-292
        • Krylova S.M.
        • Musheev M.
        • Nutiu R.
        • Li Y.
        • Lee G.
        • Krylov S.N.
        Tau protein binds single-stranded DNA sequence specifically--the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures.
        FEBS Lett. 2005; 579: 1371-1375
        • Kim S.
        • Wark A.W.
        • Lee H.J.
        Femtomolar detection of tau proteins in undiluted plasma using surface plasmon resonance.
        Anal. Chem. 2016; 88: 7793-7799
        • Berezovski M.V.
        • Musheev M.U.
        • Drabovich A.P.
        • Jitkova J.V.
        • Krylov S.N.
        Non-SELEX: Selection of aptamers without intermediate amplification of candidate oligonucleotides.
        Nat. Protoc. 2006; 1: 1359-1369
        • Lisi S.
        • Fiore E.
        • Scarano S.
        • Pascale E.
        • Boehman Y.
        • Duconge F.
        • Chierici S.
        • Minunni M.
        • Peyrin E.
        • Ravelet C.
        Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau proteins.
        Anal. Chim. Acta. 2018; 1038: 173-181
        • Kim J.H.
        • Kim E.
        • Choi W.H.
        • Lee J.
        • Lee J.H.
        • Lee H.
        • Kim D.E.
        • Suh Y.H.
        • Lee M.J.
        Inhibitory RNA aptamers of tau oligomerization and their neuroprotective roles against proteotoxic stress.
        Mol. Pharm. 2016; 13: 2039-2048
        • Bandyopadhyay B.
        • Li G.
        • Yin H.
        • Kuret J.
        Tau aggregation and toxicity in a cell culture model of tauopathy.
        J. Biol. Chem. 2007; 282: 16454-16464
        • Breydo L.
        • Wu J.W.
        • Uversky V.N.
        α-Synuclein misfolding and Parkinson's disease.
        Biochim. Biophys. Acta. 2012; 1822: 261-285
        • Tripathi T.
        A master regulator of α-synuclein aggregation.
        ACS Chem. Neurosci. 2020; 11: 1376-1378
        • Sorrentino Z.A.
        • Giasson B.I.
        The emerging role of α-synuclein truncation in aggregation and disease.
        J. Biol. Chem. 2020; 295: 10224-10244
        • Du X.Y.
        • Xie X.X.
        • Liu R.T.
        The role of α-synuclein oligomers in Parkinson's disease.
        Int. J. Mol. Sci. 2020; 21: 8645
        • Schweighauser M.
        • Shi Y.
        • Tarutani A.
        • Kametani F.
        • Murzin A.G.
        • Ghetti B.
        • Matsubara T.
        • Tomita T.
        • Ando T.
        • Hasegawa K.
        • Murayama S.
        • Yoshida M.
        • Hasegawa M.
        • Scheres S.H.W.
        • Goedert M.
        Structures of α-synuclein filaments from multiple system atrophy.
        Nature. 2020; 585: 464-469
        • McGlinchey R.P.
        • Ni X.
        • Shadish J.A.
        • Jiang J.
        • Lee J.C.
        The N terminus of α-synuclein dictates fibril formation.
        Proc. Natl. Acad. Sci. U. S. A. 2021; 118e2023487118
        • Fusco G.
        • Chen S.W.
        • Williamson P.T.F.
        • Cascella R.
        • Perni M.
        • Jarvis J.A.
        • Cecchi C.
        • Vendruscolo M.
        • Chiti F.
        • Cremades N.
        • Ying L.
        • Dobson C.M.
        • De Simone A.
        Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers.
        Science. 2017; 358: 1440-1443
        • Doherty C.P.A.
        • Ulamec S.M.
        • Maya-Martinez R.
        • Good S.C.
        • Makepeace J.
        • Khan G.N.
        • van Oosten-Hawle P.
        • Radford S.E.
        • Brockwell D.J.
        A short motif in the N-terminal region of α-synuclein is critical for both aggregation and function.
        Nat. Struct. Mol. Biol. 2020; 27: 249-259
        • Tsukakoshi K.
        • Harada R.
        • Sode K.
        • Ikebukuro K.
        Screening of DNA aptamer which binds to α-synuclein.
        Biotechnol. Lett. 2010; 32: 643-648
        • Tsukakoshi K.
        • Ikuta Y.
        • Abe K.
        • Yoshida W.
        • Iida K.
        • Ma Y.
        • Nagasawa K.
        • Sode K.
        • Ikebukuro K.
        Structural regulation by a G-quadruplex ligand increases binding abilities of G-quadruplex-forming aptamers.
        Chem. Commun. 2016; 52: 12646-12649
        • Zheng Y.
        • Qu J.
        • Xue F.
        • Zheng Y.
        • Yang B.
        • Chang Y.
        • Yang H.
        • Zhang J.
        Novel DNA aptamers for Parkinson's disease treatment inhibit α-synuclein aggregation and facilitate its degradation.
        Mol. Ther. Nucleic Acids. 2018; 11: 228-242
        • Crombez L.
        • Aldrian-Herrada G.
        • Konate K.
        • Nguyen Q.N.
        • McMaster G.K.
        • Brasseur R.
        • Heitz F.
        • Divita G.
        A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells.
        Mol. Ther. 2009; 17: 95-103
        • Prusiner S.B.
        • Scott M.R.
        • DeArmond S.J.
        • Cohen F.E.
        Prion protein biology.
        Cell. 1998; 93: 337-348
        • Collinge J.
        Prion diseases of humans and animals: Their causes and molecular basis.
        Annu. Rev. Neurosci. 2001; 24: 519-550
        • Aguzzi A.
        • Polymenidou M.
        Mammalian prion biology: One century of evolving concepts.
        Cell. 2004; 116: 313-327
        • McLennan N.F.
        • Brennan P.M.
        • McNeill A.
        • Davies I.
        • Fotheringham A.
        • Rennison K.A.
        • Ritchie D.
        • Brannan F.
        • Head M.W.
        • Ironside J.W.
        • Williams A.
        • Bell J.E.
        Prion protein accumulation and neuroprotection in hypoxic brain damage.
        Am. J. Pathol. 2004; 165: 227-235
        • Spudich A.
        • Frigg R.
        • Kilic E.
        • Kilic U.
        • Oesch B.
        • Raeber A.
        • Bassetti C.L.
        • Hermann D.M.
        Aggravation of ischemic brain injury by prion protein deficiency: Role of ERK-1/-2 and STAT-1.
        Neurobiol. Dis. 2005; 20: 442-449
        • Mitteregger G.
        • Vosko M.
        • Krebs B.
        • Xiang W.
        • Kohlmannsperger V.
        • Nolting S.
        • Hamann G.F.
        • Kretzschmar H.A.
        The role of the octarepeat region in neuroprotective function of the cellular prion protein.
        Brain Pathol. 2007; 17: 174-183
        • Sengupta I.
        • Udgaonkar J.B.
        Structural mechanisms of oligomer and amyloid fibril formation by the prion protein.
        Chem. Commun. 2018; 54: 6230-6242
        • Lauren J.
        Cellular prion protein as a therapeutic target in Alzheimer's disease.
        J. Alzheimers Dis. 2014; 38: 227-244
        • Lauren J.
        • Gimbel D.A.
        • Nygaard H.B.
        • Gilbert J.W.
        • Strittmatter S.M.
        Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers.
        Nature. 2009; 457: 1128-1132
        • Weiss S.
        • Proske D.
        • Neumann M.
        • Groschup M.H.
        • Kretzschmar H.A.
        • Famulok M.
        • Winnacker E.L.
        RNA aptamers specifically interact with the prion protein PrP.
        J. Virol. 1997; 71: 8790-8797
        • Sekiya S.
        • Noda K.
        • Nishikawa F.
        • Yokoyama T.
        • Kumar P.K.
        • Nishikawa S.
        Characterization and application of a novel RNA aptamer against the mouse prion protein.
        J. Biochem. 2006; 139: 383-390
        • Mercey R.
        • Lantier I.
        • Maurel M.C.
        • Grosclaude J.
        • Lantier F.
        • Marc D.
        Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns.
        Arch. Virol. 2006; 151: 2197-2214
        • Ogasawara D.
        • Hasegawa H.
        • Kaneko K.
        • Sode K.
        • Ikebukuro K.
        Screening of DNA aptamer against mouse prion protein by competitive selection.
        Prion. 2007; 1: 248-254
        • Bibby D.F.
        • Gill A.C.
        • Kirby L.
        • Farquhar C.F.
        • Bruce M.E.
        • Garson J.A.
        Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins.
        J. Virol. Methods. 2008; 151: 107-115
        • Rhie A.
        • Kirby L.
        • Sayer N.
        • Wellesley R.
        • Disterer P.
        • Sylvester I.
        • Gill A.
        • Hope J.
        • James W.
        • Tahiri-Alaoui A.
        Characterization of 2'-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion.
        J. Biol. Chem. 2003; 278: 39697-39705
        • Sayer N.M.
        • Cubin M.
        • Rhie A.
        • Bullock M.
        • Tahiri-Alaoui A.
        • James W.
        Structural determinants of conformationally selective, prion-binding aptamers.
        J. Biol. Chem. 2004; 279: 13102-13109
        • Proske D.
        • Gilch S.
        • Wopfner F.
        • Schatzl H.M.
        • Winnacker E.L.
        • Famulok M.
        Prion-protein-specific aptamer reduces PrPSc formation.
        ChemBioChem. 2002; 3: 717-725
        • Takemura K.
        • Wang P.
        • Vorberg I.
        • Surewicz W.
        • Priola S.A.
        • Kanthasamy A.
        • Pottathil R.
        • Chen S.G.
        • Sreevatsan S.
        DNA aptamers that bind to PrP(C) and not PrP(Sc) show sequence and structure specificity.
        Exp. Biol. Med. (Maywood). 2006; 231: 204-214
        • Murakami K.
        • Nishikawa F.
        • Noda K.
        • Yokoyama T.
        • Nishikawa S.
        Anti-bovine prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its β isoform with high affinity.
        Prion. 2008; 2: 73-80
        • Mashima T.
        • Matsugami A.
        • Nishikawa F.
        • Nishikawa S.
        • Katahira M.
        Unique quadruplex structure and interaction of an RNA aptamer against bovine prion protein.
        Nucleic Acids Res. 2009; 37: 6249-6258
        • Mashima T.
        • Nishikawa F.
        • Kamatari Y.O.
        • Fujiwara H.
        • Saimura M.
        • Nagata T.
        • Kodaki T.
        • Nishikawa S.
        • Kuwata K.
        • Katahira M.
        Anti-prion activity of an RNA aptamer and its structural basis.
        Nucleic Acids Res. 2013; 41: 1355-1362
        • Hayashi T.
        • Oshima H.
        • Mashima T.
        • Nagata T.
        • Katahira M.
        • Kinoshita M.
        Binding of an RNA aptamer and a partial peptide of a prion protein: Crucial importance of water entropy in molecular recognition.
        Nucleic Acids Res. 2014; 42: 6861-6875
        • Mashima T.
        • Lee J.H.
        • Kamatari Y.O.
        • Hayashi T.
        • Nagata T.
        • Nishikawa F.
        • Nishikawa S.
        • Kinoshita M.
        • Kuwata K.
        • Katahira M.
        Development and structural determination of an anti-PrP(C) aptamer that blocks pathological conformational conversion of prion protein.
        Sci. Rep. 2020; 10: 4934
        • Hamada M.
        In silico approaches to RNA aptamer design.
        Biochimie. 2018; 145: 8-14
        • Chen Z.
        • Hu L.
        • Zhang B.T.
        • Lu A.
        • Wang Y.
        • Yu Y.
        • Zhang G.
        Artificial intelligence in aptamer-target binding prediction.
        Int. J. Mol. Sci. 2021; 22: 3605
        • Cho M.
        • Xiao Y.
        • Nie J.
        • Stewart R.
        • Csordas A.T.
        • Oh S.S.
        • Thomson J.A.
        • Soh H.T.
        Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing.
        Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 15373-15378
        • Jolma A.
        • Kivioja T.
        • Toivonen J.
        • Cheng L.
        • Wei G.
        • Enge M.
        • Taipale M.
        • Vaquerizas J.M.
        • Yan J.
        • Sillanpaa M.J.
        • Bonke M.
        • Palin K.
        • Talukder S.
        • Hughes T.R.
        • Luscombe N.M.
        • et al.
        Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities.
        Genome Res. 2010; 20: 861-873
        • Kupakuwana G.V.
        • Crill 2nd, J.E.
        • McPike M.P.
        • Borer P.N.
        Acyclic identification of aptamers for human α-thrombin using over-represented libraries and deep sequencing.
        PLoS One. 2011; 6e19395
        • Hoinka J.
        • Berezhnoy A.
        • Dao P.
        • Sauna Z.E.
        • Gilboa E.
        • Przytycka T.M.
        Large scale analysis of the mutational landscape in HT-SELEX improves aptamer discovery.
        Nucleic Acids Res. 2015; 43: 5699-5707