Sticky DNA: Effect of the Polypurine·Polypyrimidine Sequence*

The polypurine·polypyrimidine sequence requirements for the formation of sticky DNA were evaluated inEscherichia coli plasmid systems to determine the potential occurrence of this conformation throughout biological systems. A mirror repeat, dinucleotide tract of (GA·TC)37, which is ubiquitous in eukaryotes, formed sticky DNA, but shorter sequences of 10 or 20 repeats were inert. (GGA·TCC) n inserts (where n = 126, 159, and 222 bp) also formed sticky DNA. As shown previously, the control sequence (GAA·TTC)150 (450 bp) readily adopted the X-shaped sticky structure; however, this structure has never been found for the nonpathogenic (GAAGGA·TCCTTC)65 of the same approximate length (390 bp). A sequence that is replete with polypurine·polypyrimidine tracts that can form triplexes and slipped structures but lacks long repeating motifs (the 2.5-kbp intron 21 sequence from the polycystic kidney disease gene 1) was also inert. Interestingly, tracts of (GAA·TTC) n (wheren = 176 or 80) readily formed sticky DNA with (GAAGGA·TCCTTC)65 cloned into the same plasmid when the pair of inserts was in the direct, but not in the indirect (inverted), orientation. The stabilities of the triple base (Watson-Crick and Hoogsteen) interactions in the DNA/DNA associated triplex region of the sticky conformations account for these observations. Our results have significant chemical and biological implications for the structure and function of this unusual DNA conformation in Friedreich's ataxia.

The salient clinical and molecular biological features of Friedreich's ataxia (FRDA) 1 as well as the properties of DNA threestranded structures (triplexes) and sticky DNA were reviewed in the companion paper (1). Because this novel DNA structure was discovered (2) in the long GAA⅐TTC mutation in intron 1 of the frataxin gene, which is responsible for most cases of FRDA (3)(4)(5)(6)(7), we wished to determine the sequence requirements for its stabilization. Sticky DNA is a polypurine⅐polypurine⅐ polypyrimidine (R⅐R⅐Y) triplex; hence, the features must include a mirror repeat sequence. However, the role of the distribution of purines and pyrimidines on the complementary strands has not been explored. Also, because a (GAA-GGA⅐TCCTTC) 65 tract is present in the same intron, the significance of the DNA sequence on triplex and sticky DNA formation may have further relevance. At present, the function of this unusual repeating hexanucleotide tract is unclear, especially because it does not track through family pedigrees with the disease or inhibit transcription (2, 8 -10).
The effect of GGA⅐TCC-interrupted triplets in long GAA⅐TTC repeat tracts was investigated (10) to determine some of the sequence requirements for sticky DNA and to evaluate further the veracity of its long GAA⅐GAA⅐TTC triplex structure. Studies were conducted on a family of seven periodically substituted inserts (all ϳ130 repeats in length) which contain 0, 4, 7, 8, 11, 20, or 50% substitution of GAA⅐TTC with GGA⅐TCC triplets. A relatively small amount of substitution (less than 11%) caused no inhibitory effects. However, higher levels of GGA⅐TCC interruptions reduced the formation of sticky DNA, alleviated transcription inhibition, and reduced genetic instabilities. We wished to further our studies with long DNA tracts with uniformly repeating polypurine⅐pyrimidine sequence motifs such as GA⅐TC, GGA⅐TCC, and GAAGGA⅐TCCTTC.
Herein, we have evaluated the capacity of related types of long repeating R⅐Y sequences to form the sticky DNA structure, including GA⅐TC, GGA⅐TCC, and GAAGGA⅐TCCTTC. Also, the ability of the FRDA GAA⅐TTC repeat to interact with each of these three repeat sequences was determined. These results provide important confirmatory evidence on the R⅐R⅐Y conformation of sticky DNA and give insights into possible DNA loop interactions in FRDA chromosomes.
A companion article (1) demonstrates that sticky DNA is only formed intramolecularly between a pair of GAA⅐TTC tracts in one DNA molecule.

EXPERIMENTAL PROCEDURES
Preparation of Dimeric and Monomeric Plasmids-Plasmid preparations after isolation from Escherichia coli contain monomeric as well as dimeric (and higher oligomeric) isoforms of DNA. The typical biological dimer studied herein is shown in Fig. 1. Plasmids containing a single GA⅐TC tract (which are pUC9 derivatives) and plasmids containing one GGA⅐TCC tract (which are pUC19 derivatives) used in these studies are shown in Table I and have been described previously (11,12). pBS4.0, which is a pBluescript KS derivative harboring the 2.5-kb R⅐Y tract from intron 21 of the PKD1 gene, has also been described previously (13,14). Plasmids with two R⅐Y tracts, which are pBR322 derivatives, are shown in Fig. 2. The constructions of these DNAs are described below.
Mixtures of supercoiled dimeric and higher oligomeric forms of the pUC9 and pUC19 derivatives were isolated after overnight growth in E. coli SURE strain, as described (1).
Cloning of a Pair of R⅐Y Tracts into pBR322-Fragments containing the (GAA⅐TTC) 176 tract and the (GAAGGA⅐TCCTTC) 65 tract were prepared from pRW3808 (15,16) and pMP193 (2), respectively, by BssHII and HaeIII digestion (New England Biolabs, Inc.) followed by filling in the recessed BssHII 3Ј termini with 0.1 unit of the Klenow fragment of E. coli DNA polymerase I (U. S. Biochemical Corp.) and dCTP plus dGTP (0.1 mM each) (17). The repeating tracts are flanked by 34 and 54 bp of the human FRDA gene (3). The digested DNA was electrophoresed in a 7% polyacrylamide gel, stained with EtBr, and the band containing the triplet repeat fragment was excised. The DNA was eluted from the excised band, purified by phenol-chloroform extraction, and precipitated with ethanol (15). Linearized pBR322 was ligated with the (GAA⅐TTC) 176 -containing insert. The ligation and all subsequent cloning steps were performed as described earlier (17). Clones that included trinucleotide repeat sequence tracts, cloned into PvuII site, were subsequently digested by EcoRI/HindIII, followed by filling in the recessed 3Ј termini. Subsequent blunt end ligation of these DNAs with the insert harboring the (GAAGGA⅐TCCTTC) 65 tract enabled the construction of a set of plasmids (pRW5000 -pRW5005) harboring the (GAAGGA⅐ TCCTTC) 65 tract in the EcoRI site of pBR322 in both orientations and the (GAA⅐TTC) 176 tract in the PvuII site; the two inserts were oriented as direct repeats or inverted repeats (Fig. 2). pRW5001 and pRW5003, harboring (GAA⅐TTC) 80 , were isolated as deletion mutants of pRW5000 and pRW5002, respectively. From the other side, subsequent blunt end ligation of the above mentioned DNA, harboring the (GAA⅐TTC) 176 tract in orientation I, with the insert, harboring the (GAA⅐TTC) 176 tract, enabled the construction of a pair of plasmids (pRW4886 -pRW4887), harboring a pair of the (GAA⅐TTC) 176 tracts both in direct and inverted orientations (Fig. 2).
For the preparation of the plasmid harboring two contiguous inserts, where two directly oriented (GAA⅐TTC) 60 are separated by 88 bp of human flanking sequence, a number of clones from (GAA⅐TTC) 60 blunt end ligation into the PvuII site of pBR322 (1) were studied.
All plasmids were fully characterized by restriction mapping (to determine the orientation and length of the cloned trinucleotide repeat sequence) and dideoxy sequencing of both strands with Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (U. S. Biochemical Corp.).
Detection of Retarded Band (RB) Formation-The presence of sticky DNA in a DNA preparation is determined routinely by the detection of a substantially RB on agarose gel electrophoresis after plasmid linearization as described (1).

RESULTS
Sticky DNA Formation by R⅐Y Sequences-Prior investigations (2,9,10) demonstrated that long tracts of GAA⅐TTC repeats readily form a new type of complex triplex called sticky DNA. However, a closely related repeating hexanucleotide sequence, GAAGGA⅐TCCTTC, does not form sticky DNA under identical conditions (2, 8 -10). Furthermore, studies on a family  Table I. B, 1% agarose gel analyses of RB formation after HindIII cleavage of dimeric forms of pUC9 derivatives harboring (GA⅐TC) n . C, 1% agarose gel analyses of RB formation after EcoRI cleavage of dimeric forms of pUC19 derivatives harboring (GAA⅐TTC) n . D, 0.7% agarose gel analyses of RB formation after EcoRI cleavage of dimeric forms of pSPL3 derivatives harboring (GAAGGA⅐TCCTTC) 65 (left lane) and (GAA⅐TTC) 150 (right lane), used as a point of reference for 0 and 100% RB formation.

TABLE I Amount of RB formed from plasmid dimers as a function of the sequence and length of the repeating tract
To compare the yield of RB formed from plasmid dimers containing different lengths of (GA⅐TC) n , (GGA⅐TCC) n , and (GAAGGA⅐ TCCTTC) 65 , we studied purified dimers of each plasmid (see "Experimental Procedures"). Yields of RB (in percent) were compared with the amount observed from purified dimers of pRW3822 (1, 2, 9, 10) under the same conditions (HindIII cleavage for pUC9 derivatives, EcoRI cleavage for pUC19 and pSPL3 derivatives, and SacI cleavage for the pBluescript KS Ϫ derivative). All plasmids had the superhelical density as isolated from E. coli (Ϫ ϭ 0.05) (25)(26)(27). In parallel studies, the isolated monomeric forms of all 11 plasmids were studied; no RB was found in any case. The actual sequence of the insert in pRW3892 is ((AGG) 58 CCTGG(AGG) 16 ⅐(CCT) 16 CCAGG(CCT) 58 ), as described earlier (12), but it is designated as (GGA⅐TCC) 74 for simplicity. of mutated GAA⅐TTC repeat tracts in plasmids containing various amount of GGA⅐TCC interruptions (ranging from 4 to 50%) revealed that GAA⅐TTC repeats with less than 20% interruptions form triplexes and/or sticky DNA similar to the uninterrupted repeat sequence (10).
To investigate further the sequence requirements for the formation of sticky DNA, we constructed and characterized families of recombinant plasmids containing all purines on one strand and all pyrimidines on the complementary strands of the inserts. (GA⅐TC) n (where n ϭ 10, 20, and 37) as well as (GGA⅐TCC) n (where n ϭ 16, 30, 42, 53, and 74) were prepared. Investigations were also conducted on a plasmid containing a 2.5-kbp R⅐Y tract that is present in intron 21 of the polycystic kidney disease 1 (PKD1) gene (14). This sequence contains 23 perfect mirror repeats that can form DNA triplexes with stems of at least 10 bp and are clustered into three distinct regions of the 2.5-kbp tract. More than a thousand perfect tandem repeats, which will form slipped structures (18), are present. The dinucleotides TC and CT are the most common; however, they are excluded from the 5Ј-end where the mirror repeats predominate. The trinucleotide repeats are mostly CCT and are localized within one end of the tract. Pentanucleotide repeats include CTCCC, CTCCT, and CCCAT. These pentanucleotide repeats are also clustered within the R⅐Y tract. In summary, this PKD1 sequence shows the presence of many mirror and direct repeats that are localized within the sequence. This tract contains the highest density of unorthodox simple sequence repeat features (mirror, direct repeats, and R⅐Y strand bias) of any known sequence of this length (14).
To analyze the sequence requirements of the R⅐Y region for sticky DNA formation in the dimeric form, we isolated dimers from plasmid preparations grown in E. coli SURE cells. The accompanying paper (1) demonstrates that only dimeric and higher oligomeric, but not monomeric, forms of the plasmids will form sticky DNA. Thus, it was concluded (1) that two long tracts of R⅐Y must be present in one plasmid to generate sticky DNA. The plasmid monomeric forms of uninterrupted as well as periodically interrupted long GAA⅐TTC repeats will not form sticky DNA (1). Also, we would have liked to investigate the behaviors of long homopolymeric tracts of A⅐T as well as G⅐C. However, these investigations are precluded by the extreme genetic instability of these simple sequences (19,20).
Studies were conducted to evaluate the capacity of the sequences described above and listed in Table I to form sticky DNA. Fig. 1A shows the generic structure of a biological dimer, obviously containing two identical tracts of the repeating se- quences. The repeats were dinucleotide or trinucleotide or hexanucleotide or 2.5-kb R⅐Y tracts. Fig. 1B shows the gel electrophoretic analyses for the GA⅐TC tract; Fig. 1C shows the electrophoretic analysis for the GGA⅐TCC tracts, and D presents the control data for (GAA⅐TTC) 150 and a similar length of the repeating hexanucleotide sequence (pMP193).
The dimers of pUC9-derived plasmids harboring GA⅐TC tracts revealed the formation of sticky DNA for pGA37 which contains 37 dimeric repeats; however, little or no sticky DNA was observed for the plasmids containing the two shorter dinucleotide repeat tracts (10 and 20 units in length) ( Fig. 1 and Table I). Unfortunately, longer repeat tracts of GA⅐TC were not available for investigation. Thus, repeating dinucleotide sequences containing all purines on one strand and all pyrimidines on the complementary strand can also form sticky DNA.
Studies were also conducted on the repeating trinucleotide sequence, GGA⅐TCC, containing 16 -74 triplet repeats. This sequence contains 66% GC, whereas the FRDA mutation contains only 33% GC. Interestingly, the three plasmids (pUC19 derivatives) which contain 42-74 repeats readily formed sticky DNA, whereas the two shorter inserts did not have this capability ( Fig. 1 and Table I). Hence, the threshold length for sticky DNA formation is between 40 and 74 bp for GA⅐TC, 90 and 126 bp for GGA⅐TCC, and 99 and 177 bp for GAA⅐TTC. No hereditary neurological diseases have been identified yet where GGA⅐TCC is involved in its etiology (18).
A R⅐Y sequence containing an irregular distribution of A and G nucleotides in the polypurine strain was investigated to evaluate the necessity of a repetitive sequence. The 2.5-kbp tract from intron 21 of the PKD1 gene was employed because of its known sequence features (for review, see Ref. 14) as well as its interesting biological characteristics. This 2.5-kbp sequence contains four P1 nuclease-sensitive regions (18), which apparently form triplexes (14,18). One of these regions contains a 404-bp noninterrupted R⅐Y tract (13). Because this length is approximately twice the known threshold (described above) for the formation of sticky DNA for GAA⅐TTC as well as GGA⅐TCC, we felt that it might be possible for this PKD1 insert to adopt a sticky DNA structure. However, as revealed in Table I, no RB was found. Because this 404-bp R⅐Y tract contains no mirror repeats with at least 80% matching triple base interactions (13,14) that are longer than ϳ200 bp, this result is not unexpected.
Further gel electrophoretic analyses revealed that the RB found with GA⅐TC as well as with GGA⅐TCC inserts were, in fact, sticky DNA, by evaluating the lengths of the arms in the X-shaped structures formed after cleavage at different unique restriction sites. For the pUC19 derivatives, the DNAs were linearized with Eco0109I, EcoRI or NdeI and for pUC9 derivatives, the plasmids were cleaved with EcoRI or HindIII. In all cases, the correlation described earlier (2) between the extent of retardation of the RB and the distance between the restriction enzyme cleavage site and the R⅐Y tract was observed (data not shown).
Sticky DNA Formation by Two Nonhomologous Sequences in One Plasmid-Further investigations on the role of DNA sequence on the capacity of two tracts of different types of R⅐Y sequences to form sticky DNA were conducted. Plasmids containing one tract of (GAAGGA⅐TCCTTC) 65 and another block of (GAA⅐TTC) 176 were prepared and characterized (Fig. 2). Similar plasmids were also prepared in which the (GAA⅐TTC) 176 was replaced with (GAA⅐TTC) 80 . Plasmids were characterized which contained the inserts in the direct repeat orientations as well as in the inverted repeat orientations (Fig. 2). Prior investigations (1) revealed that only plasmids containing inserts in the direct repeat orientation could form sticky DNA, and our current work confirms this conclusion (Table II).
Table II reveals that pRW4886, which contains two tracts of 176 repeats of GAA⅐TTC in the direct repeat orientation (Fig.  2), readily forms sticky DNA (as described under "Experimental Procedures"). However, pRW4887, containing the same in-

TABLE II Amount of RB formed from plasmids harboring two inserts as a function of repeating sequence orientations and the length of the (GAA⅐TTC) n tract
To compare the yield of RB formed from plasmids containing one (GAAGGA⅐TCCTTC) 65 tract and one (GAA⅐TTC) n tract, we studied purified monomers (see "Experimental Procedures"). Yields of RB (in percent) were compared with the amount observed from purified monomers of pRW4886 (Fig. 2), harboring two (GAA⅐TTC) 176 tracts, both in orientation I, under the same conditions. All DNAs were cleaved simultaneously with EcoRI and EcoNI. All plasmids had the superhelical density as isolated from E. coli (Ϫ ϭ 0.05) (25)(26)(27). RB formation was monitored as described under "Experimental Procedures."

Plasmid
Orientation of (GAAGGA⅐TCCTTC) 65 serts but in the inverted repeat orientation (Fig. 2), were devoid of this capability, as expected (1). Interestingly, pRW5000 and pRW5004 readily formed sticky DNA; these plasmids contain one tract of (GAAGGA⅐ TCCTTC) 65 and one tract of (GAA⅐TTC) 176 ; both inserts in the two plasmids are in the direct repeat orientation, but the pair of repeats are inverted in pRW5004 compared with pRW5000. Accordingly, it is apparent that the GGAGAA tract from the repeating hexanucleotide sequence is aligning and Hoogsteen pairing in an antiparallel manner with the GAAGAA sequence from the repeating triplet sequence. Thus, five of six (83%) of the base oppositions are "normal" G⅐G⅐A and A⅐A⅐T structures with only one G⅐A⅐T opposition that is less stable (10). Therefore, by our previous analysis (Table II of Ref. 1), this extent (17%) of "misoppositions" should be tolerated. However, the investigations described above (Table I) in which two tracts of the (GAAGGA⅐TCCTTC) 65 would be required to pair successfully to form sticky DNA was forbidden; this result is as expected (1) because only four of six (66%) of the correct oppositions would be present, which is insufficient to stabilize sticky DNA (1, 10).
As expected, pRW5001 also forms sticky DNA, and thus a (GAA⅐TTC) 80 tract can replace the 176-mer and still effectively pair with the repeating hexamer. Also, all plasmids with inserts in the inverted orientation (pRW4887, 5002, 5003, and 5005) were incapable of adopting the sticky structure (1).
Effect of Length of Intervening Sequence between GAA⅐TTC Tracts-The effect of the length of the pBR322 DNA sequence between two tracts of (GAA⅐TTC) 60 was investigated to evaluate further the factors involved in the stabilization of sticky DNA. For the plasmids shown in Fig. 1 as well as Fig. 2, the repeat sequences were separated by more than 2,000 bp. To evaluate whether one very long GAA⅐TTC tract could fold back on itself to form sticky DNA and/or to determine the influence of a very short intervening sequence, we prepared and characterized pRW4881 (Fig. 3), which contains 88 bp of human FRDA flanking sequence between a pair of (GAA⅐TTC) 60 sequences. The plasmid has two cleavage sites for BssHII, which are separated by a (GAA⅐TTC) 60 tract. After cleavage of pRW4881 with BssHII and electrophoretic analysis for sticky DNA (Fig. 3), RB is found. The extent of retardation is as expected because the location of the cleavage sites is close to the (GAA⅐TTC) 60 tract (2).
Thus, the stabilizing forces for sticky DNA generated by the fold-back of two GAA⅐TTC sequences that are proximal are sufficiently great to enable the formation of sticky DNA. Sharp bending of the 88-bp intervening sequence must have been caused by the stabilization and pairing of the GAA⅐TTC tracts. Because this 88 bp is shorter than the known persistence length of DNA, which is 450 Å or 130 bp (21,22), we conclude that the interactive forces created by the pair of GAA⅐TTC tracts must be substantial. DISCUSSION Sticky DNA was described (2,9,10) as an X-shaped DNA molecule found in plasmids containing long tracts of GAA⅐TTC after linearization of the plasmid molecule. The lengths of GAA⅐TTC required for its formation correspond to the tracts found in intron 1 of the frataxin gene of FRDA patients (2). Sticky DNA was stabilized by negative supercoiling as well as divalent metal ions. The long GAA⅐TTC repeats form R⅐R⅐Y triplexes at neutral pH (23). The companion article (1) demonstrates the necessity of two tracts of GAA⅐TTC in one molecule for the intramolecular formation of sticky DNA. Interestingly, a tract of (GAAGGA⅐TCCTTC) 65 is also present in intron 1 of the frataxin gene (8) but does not inhibit transcription (9,12) nor track with the disease in family pedigrees; alternatively, sticky DNA effectively inhibits transcription (9).
We have evaluated some of the sequence requirements for the mirror repeat R⅐Y insert tracts to form sticky DNA. The following R⅐Y tracts were investigated: GA⅐TC, GAA⅐TTC, GGA⅐TCC, GAAGGA⅐TCCTTC, and the 2.5-kbp R⅐Y tract from intron 21 of the polycystic kidney disease gene 1. The GA⅐TC repeat sequence is distributed widely in a variety of genomes (24). However, expanded repeats of GGA⅐TCC have not been discovered to date in conjunction with a hereditary neurological disease (18). In plasmids containing a pair of tracts of the identical repeat sequences, we found that sufficiently long tracts of GA⅐TC, GGA⅐TCC, and GAA⅐TTC adopted the sticky conformation. Alternatively, neither the GAAGGA⅐TCCTTC repeating hexanucleotide sequence nor the 2.5-kbp R⅐Y tract from the PKD1 gene adopted this unusual conformation. Because the two purine strands in the R⅐R⅐Y complex must be antiparallel (23), these results are readily explained from the known types of Hoogsteen bp schemes shown in Fig. 4; T⅐A⅐A and C⅐G⅐G are quite stable and favored structures, whereas T⅐A⅐G, C⅐G⅐A, and C⅐G⅐Aϩ are less favored. Accordingly, Fig. 5I demonstrates the facile triplex formation for GA⅐TC as well as GAA⅐TTC and Fig. 5II shows the stable bp interactions for GGA⅐TCC repeats.
Alternatively, Fig. 5III shows that for GAAGGA⅐TCCTTC interacting with the identical sequence in an intramolecular reaction, only four of six (66%) of the triple base oppositions are stable, even in the optimal frame. This figure shows the potential pairing schemes in all six frames. Thus, this degree of stability is insufficient for the formation of sticky DNA (10) and provides a rational basis for the negative results shown in Table I for this sequence.
Studies were also conducted with plasmids containing GAAGGA⅐TCCTTC and GAA⅐TTC in the same plasmid in direct repeat orientations. These plasmids showed the formation of sticky DNA. Fig. 5IV shows that in the proper frames (a and d), five of six of the pairs are in stable Hoogsteen structures (Fig. 4A). This extent of pairing is sufficient to enable the formation of a stable triplex (10). Also, as expected, when two tracts of GAA⅐TTC are present in the same plasmid (pRW4886) (Fig. 2), a stable sticky DNA structure was observed. In summary, the composite results demonstrate that the formation of sticky DNA requires the formation of at least 83% of acceptable Hoogsteen and Watson-Crick base oppositions. Accordingly, these data provide strong confirmatory evidence for the purinepurine antiparallel triplex structures proposed in Figs. 4 and 5.
Furthermore, our current results are in excellent agreement with prior studies (1), which showed that sticky DNA was an R⅐R⅐Y triplex with the purine strands in the antiparallel orientation. This investigation (1) with a family of inserts in recombinant plasmid dimers with varying extents of interruptions between the extremes of pure GAA⅐TTC repeats (0% of GGA⅐TCC in the GAA⅐TTC repeats) and the repeating hexanucleotide sequence GAAGGA⅐TCCTTC (50% of GGA⅐TCC in the GAA⅐TTC repeats) revealed that more than 20% interruptions by GGA⅐TCC in the GAA⅐TTC repeat sequence abolished the formation of sticky DNA. In summary, all of these results considered together provide important confirmatory evidence for sticky DNA as an R⅐R⅐Y triplex with the base pairing schemes shown in Fig. 4A with the two purine strands in the antiparallel arrangement.
The salient features of FRDA as well as its human and molecular genetic properties were reviewed in the Introduction to the companion paper (1). In patients, the expanded GAA⅐TTC repeats (66 -1,700 or more) may form sticky DNA and thereby inhibit transcription; prior investigations (9,16) showed the efficiency of sticky DNA in transcription inhibition, which could explain the reduction of X25 mRNA in patients, which would result in a diminution of the amount of the frataxin protein, thus causing the disease pathology. The first intron of the frataxin gene also contains a (GAAGGA⅐TCCTTC) 65 tract, which is nonpathogenic (8). Unlike the GAA⅐TTC repeat, this hexamer repeat does not form an antiparallel triplex and/or sticky DNA, does not inhibit transcription, and does not associate with the FRDA disease state (8 -10). Because our investigations demonstrated the facility of formation of sticky DNA between long tracts of GAA⅐TTC and this neighboring hexanucleotide repeat, it is possible that these two sequences interact to form a potent block for transcription in FRDA patients.