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J Biol Chem, Vol. 274, Issue 31, 21763-21768, July 30, 1999
From the Department of Molecular Biology, Princeton University,
Princeton, New Jersey 08544
A DNA third strand with a 3'-psoralen substituent
was designed to form a triplex with the sequence downstream of the
T·A mutant base pair of the human sickle cell Nucleic acid third strand binding provides an effective and
specific means of directing reagents to unique sites in a complex duplex genome (1-4). When the reagent linked to the third strand allows covalent attachment to a mutated base pair, the resultant adduct
may be recognized as a defect to be corrected by some DNA repair
mechanism. Error-prone repair in such a situation can lead to
correction of the mutation. This approach has been utilized to effect
specific base pair changes following psoralen photoadduct formation
site-directed by specific sequence third strands (5-8). In particular,
the use of irradiation times and wavelength favorable for monoadduct
formation has been shown to induce the transversion T·A To make such a third strand-mediated strategy for site-specific base
pair change viable, it is necessary to overcome two primary barriers.
One stems from the fact that ideal sites for spontaneous third strand
binding, i.e. perfect homopurine·homopyrimidine target sequences 15-25 base-pairs long occur with limited frequency. The
second barrier is that in the face of imperfect targets, third strand
binding energy is often insufficient to permit stable triplex formation.
We have exploited the triplex-based approach to develop a strategy for
correcting the mutation that underlies human sickle cell anemia, which
is due to an A·T Deoxyoligonucleotides--
These were synthesized by automated
phosphoramidite chemistry, purified to homogeneity by denaturing PAGE,
and recovered from gel slices via the "modified crush-and soak"
method (11). Final purification was by acetonitrile/water (50/50)
elution from C18 Sep-Pak reverse phase columns (Millipore), followed by
spin evaporation to dryness. Oligomer concentrations were adjusted
spectrophotometrically in Milli-Q-purified water. Psoralen was attached
to the 3'-end of designated oligomers by incorporating the
phosphoramidite of psoralen CPG (containing a C-16 linker between
psoralen C-3 and DMT) (Chemgenes Corp., Waltham, Massachusets) during
oligomer synthesis. Oligomer homogeneity was ascertained by
32P-5'-end-labeling and denaturing PAGE. Oligomers were
32P-5'-end-labeled preparatively by incubating
approximately 4 pmol of oligomer with 2 mCi [ Buffers--
All experiments, unless otherwise noted, were
performed in 0.1 M NaOAc, pH 5.0, 0.01 M
Mg(OAc)2 (standard buffer).
Triplexes--
Duplexes were formed by mixing equimolar amounts
of each strand, heating to 80 °C and slowly annealing to room
temperature in the standard buffer. Various ratios of third strand were
added to pre-formed duplex, and resulting mixtures were incubated for 1 h at room temperature, and then at 4 °C overnight.
Electrophoresis--
Denaturing PAGE was performed on slabs
25 × 45-cm using 8 M urea, TBE, 16% polyacrylamide
gels (1:37 bisacrylamide/acrylamide). Samples were dissolved in
denaturing loading buffer (12) and heated to 80 °C before loading.
Denaturing gels were run for 2-6 h at 1.5 kV at room temperature.
Nondenaturing PAGE was conducted on a 20 × 20-cm slab of 15%
polyacrylamide in the standard buffer (see above) and run at 4 °C
for 12 h at 150 V. Denaturing gels were soaked in standard fixing
solution (12), then in 100% methanol to remove water (13), and dried
onto Whatman filter paper on a Bio-Rad gel dryer. Gels were visualized
using either or both NEN Life Science Products x-ray film and an
ImageQuest PhosphorImager, which was also used to quantitate gel bands.
DNase I Footprinting--
Triplexes with 32P on the
5'-end of the D-2 strand (0.5 µM duplex) were
mixed with 0.6 units of DNase I (U. S. Biochemical Corp.) in a final
volume of 8 µl and incubated at 8 °C in the standard buffer. At
designated times, 2 µl aliquots were removed to 2 µl of denaturing
gel-loading buffer, frozen in a dry ice/ethanol bath, and analyzed by
denaturing PAGE. After fixing and drying on filter paper, gels were
imaged and quantitated. Band assignments of DNase I digests were
determined from sequencing ladders (see below) run in lanes adjacent to
lanes with DNase I-treated samples.
UV Irradiation--
Triplexes in 10-µl droplets in appropriate
buffer were placed on a parafilm-covered glass plate lying on ice and
positioned 10 cm under a UV light source ("Blac Ray", 15 mW/cm2 at 365 nm). Thermocouple readings showed that sample
temperature was maintained at ~8 °C. During irradiation, the ice
container was placed on a rotating turntable to ensure even exposure of all samples to UV light. At indicated times, 2-µl aliquots were added
to 2 µl of denaturing gel-loading buffer, and after mixing and
heating to 80 °C were analyzed by denaturing PAGE.
Sequenase Primer Extension--
UV-irradiated complexes were
fractionated by denaturing PAGE. After extraction from gel slices,
photoproducts were purified on C-18 columns, and utilized as templates
for primer extension by Sequenase 2.0 (U. S. Biochemical Corp.).
Gel-purified 32P-5'-end-labeled 8-nt primers complementary
to the 3'-end of D-1 were annealed to the template by heating to
80 °C and slowly cooling to 8 °C. Primer extension reactions were
carried out according to Sequenase 2.0 kit instructions (14), and
reaction products were analyzed by denaturing PAGE. Gels were fixed,
dried, and analyzed by autoradiography and phosphorimaging.
Plasmid Binding--
A plasmid pSCe was constructed by inserting
a 610-base pair sickle cell Experimental Plan--
Fig. 1,
a and b, show the 38- and 35-base pair linear
target fragments, DL-1·DL-2 and D-1·D-2,
respectively, of the
The strategy ultimately employed to achieve more effective third strand
binding to the target in a linear duplex fragment is depicted in Fig.
1b. Additional binding energy was designed into the system
by truncating the 3'-end of the DL-1 target strand (resulting in the D-1 strand) to produce a 6-nt single-stranded "sticky" 5'-end of D-2. The 5'-end of PsT-1 (third strand) was then
elongated in complementary fashion (via a linker of four T residues) to
produce a duplex-forming hook to bind to the D-2 sticky end (Fig.
1b), resulting in third strand PsT-2. The combined strategy
of using stronger binding modified residues, crossing-over of the
third strand, and a duplex-forming hook proved successful in forming
the structure in Fig. 1b. PsT-3, a scrambled sequence containing the 5'-duplex binding hook and 3'-psoralen, was used as a negative control (Fig. 1c).
Third Strand Binding--
Band-shift assays at 4 °C evaluated
by native PAGE were used to assess third strand binding. Fig.
2 shows that both PsT-2 and PsT-3 induce
a band-shift of 32P-labeled target duplex D-1·D-2,
whereas PsT-1, the hookless third strand, does not with
DL-1·DL-2. This indicates that the binding energy between the sticky end of D-2 and the 6-nt hook of either PsT-2
or PsT-3 is sufficient to form a stable complex with apparent triplex
stoichiometry under the electrophoretic conditions, whereas that
between the triplex-forming domain alone and the duplex target is not.
However, the results with PsT-2 and PsT-3 do not discriminate between
true triplexes and complexes in which the third strand is merely bound
by the hook. Note also that the mobility of the PsT-3-containing
complex is somewhat lower, which may be a consequence of the
"dangling" third strand making the complex much less compact than a
(presumably) true triplex formed by PsT-2. Furthermore, electrophoretic
analysis at 25 °C shows no band-shift for PsT-3, whereas PsT-2 does
form a complex (data not shown). This is consistent with melting by
that temperature of the hook from the complex that is not a
triplex.
DNase I protection experiments (18) were performed to discriminate
between triplexes, which should be relatively protected, and complexes
formed merely by the third strand hook, which should be sensitive to
the enzyme. Fig. 3a shows the
results of denaturing PAGE analysis of such digests. Complexes formed
with 10:1 (0.5 µM duplex, 5 µM PsT-2) and
100:1 (0.5 µM duplex, 50 µM PsT-2, data not
shown) ratios of PsT-2:duplex display reduced sensitivity to DNase I
along D-2 target segments B and C, but not along the unprotected
segment A (cf. Fig. 1a). In contrast, complexes
formed with similar ratios of the negative control strand PsT-3 do not display reduced DNase I sensitivity along any segment of D-2.
The gel from Fig. 3a was quantitated, and the photodensity
of each D-2 segment was determined. These results, expressed in Table
I as the fraction of the total strand
cuts within each D-2 segment, confirm that complexes formed with PsT-2
display much reduced DNase I sensitivity along the D-2 target segments B and C, but not at unprotected segment A. This protection is very
strong for segment C, but less evident for segment B (Table I), which
contains the strand-switching triplex domain. Further, in Fig.
3b, where DNase I sensitivity is shown relative to that of
naked duplex, the data clearly indicate protection along the D-2 target
segment in the presence of PsT-2, but a lack of protection in the
presence of PsT-3. Thus, triplex formation occurs only with PsT-2; so
PsT-3 must be bound to duplex only by the hook.
UV Irradiation of Complexes--
The complexes formed with PsT-1,
PsT-2, and PsT-3 were UV-irradiated and the products analyzed by
denaturing PAGE. Based upon the expected triplex structure, a psoralen
on the 3'-end of PsT-1 or PsT-2 should principally form monoadducts
(19) to residue T11 on the D-1/DL-1 strand,
resulting in covalent attachment of the third strand. The long length
of the linker (C16) between the psoralen moiety and the
third strand makes possible additional monoadducts to T9 of
D-1/DL-1 and to various pyrimidine residues along
D-2/DL-2, the complementary duplex strand. Interstrand
psoralen crosslinks (19) are also possible between pyrimidine residues of D-1/DL-1 and D-2/DL-2.
Fig. 4 shows denaturing PAGE analysis of
UV-irradiated complexes containing 32P-end-label on the D-1
or DL-1 strand that contained either 100 nM or
40 µM of the appropriate third strand. Photoproducts are observed in the presence of PsT-2, and also in the presence of the
hookless PsT-1 at 40 µM; neither duplex alone nor any
complex of PsT-3 and duplex produce higher molecular weight bands,
notwithstanding the psoralen moiety tethered to the 3'-end of PsT-3
(Fig. 4).
Third strand binding affinity of PsT-1 and PsT-2 was evaluated by the
dependence of photoproduct formation on third strand concentration.
Varying concentrations of third strand were annealed to target duplex,
irradiated, and analyzed by denaturing PAGE analysis (Fig.
5a). Bands were quantitated
(Fig. 5b), giving apparent Kd values of
<3 nM for PsT-2 and ~3,000 nM for PsT-1. The
1,000-fold difference in Kd values between PsT-1 and
PsT-2 clearly demonstrates the advantage of the third strand with the
duplex-forming hook, and all further work was performed using
PsT-2.
Photoproduct Identification--
To investigate the interaction of
psoralen-linked third strand with the coding and noncoding target
strands, irradiation experiments were performed using duplex with
either 32P-D-1 or 32P-D-2. Triplexes with D-1
labeled show a major photoproduct and two minor ones, one slightly
faster moving than the major one, and one much slower-moving. The
D-2-labeled triplexes (Fig.
6a) also show one major
photoproduct, but three faster moving minor products, and one much
slower moving one. The slower moving band has the same mobility in both
cases and it is also visible when PsT-2 is labeled (not shown). Hence,
this slower moving photoproduct contains D1, D2, and PsT-2; it is
therefore identified as a crosslink between PsT-2 and D-1·D-2. Based
upon the sequence of the triplex, previous observations (17), the
yield, and primer extension results described below, the major
D-1-labeled photoproduct is identified as a monoadduct between PsT-2
and D-1 at residue T11, whereas the faster running minor
product is probably the monoadduct formed at residue T9 of
D-1. Similar considerations, including primer extension results (not
shown), identify the major D-2-labeled photoproduct as a monoadduct
between PsT-2 and residue T22 of D-2 and the three faster
moving minor products as monoadducts formed with residues
C21, C23, and C24,
respectively.
Kinetics of Photoproduct Formation and Yields--
The
major D-1 monoadduct, which amounts to more than 50% of
the total photoproduct, represents psoralen interaction with the mutated T nucleotide of the human sickle cell hemoglobin gene. As
indicated in Fig. 6b, ~ 40% of the D-1 strand is
converted to the major photoproduct by 5 min of irradiation. In
contrast, the photocrosslinked product forms at much lower yield
(<3%) and does not level off over the timecourse studied, which is
consistent with previous observations (5, 19). The kinetics of major photoproduct formation is similar for the D-1- and D-2-labeled complexes (Fig. 6b). The maximum yield is attained by 5 min
of irradiation and levels off. The yield is significantly lower for the
major D-2 monoadduct (15 versus 40%). This is consistent
with the more distant location of the target residue in that strand and
the less favorable orientation of the intercalated psoralen moiety
(18).
Fig. 7 shows the dependence of
photoproduct yield on pH. The yield decreases significantly as pH is
increased from 5 to 7. Additional modifications of the third strand
sequence that use C analogs that need not be protonated should improve
binding at physiological pH (22, 23).
Sequenase Primer Extension--
To confirm the photoaddition site
of the major D-1 photoproduct, it was eluted from PAGE gel slices and
used as a DNA template in primer extension reactions (20, 21). Fig.
8 shows these results along with a mixed
ddNTP sequencing ladder obtained from nonirradiated D-1 control
strands. It can be seen that synthesis continues only up to residue
T11. Apparently, the polymerase is unable to synthesize
past the PsT-2 photoattachment site. This observation identifies
residue T11, the site of the A·T Specific Third Strand Binding and Photoaddition to Plasmids
Containing the Target--
Band-shift assays (Fig.
9) indicate that whereas PsT-3, the
scrambled third strand, does not bind to the plasmid with the With respect to our long term goal, this study indicates that the
efficiency of photochemical modification at the desired target site is
relatively high; 40% of the duplex target is converted to monoadduct
at the pyrimidine residue of the mutated base pair. Another 2% at this
site are crosslinked, whereas a minor photoproduct on this strand
accounts for ~8% of the duplex. The C16 psoralen linker
additionally permits substantial photoproduct formation of the opposite
target strand. Model building suggests that shortening the linker
should substantially reduce or even eliminate those reactions, in which
case the yield and specificity of the desired psoralen photoaddition
can be raised. In fact, preliminary experiments demonstrate that use of
a C6 psoralen linker markedly reduces photoproduct
formation with the noncoding strand.
It would appear that early steps of a strategy for triplex-mediated
repair of the sickle cell mutation are now in place. A strand has been
designed with suitable affinity for third strand binding, which forms a
psoralen monoadduct to the mutated base pair in high yield. DNA under
superhelical stress unwinds to relieve this stress, thereby creating
dynamic unwound single-stranded patches no less accessible than the
sticky end in the linear target. In fact, the preliminary results
suggest that this third strand with the hook is capable of binding to a
supercoiled plasmid containing the target sequence, indicative of a
strand-invading mechanism (28). This opens the possibility of
exploiting various DNA repair mechanisms that have been shown to effect
site-specific base pair changes. For example, T·A Chimeric structures with linked duplex and triplex elements have been
exploited for other purposes (24-28). In this study, the
duplex-forming hook represents a novel approach for achieving effective
third strand binding to a relatively poor DNA duplex target, coupled to
a potential for strand invasion. Direct comparison of binding affinity
to the same target (by photoproduct formation) of a third strand with a
hook versus a hookless third strand demonstrates at least
1,000-fold difference in Kd values. Moreover, the
hookless third strand does not induce a band-shift on native PAGE even
at high third strand concentrations (Fig. 2). This indicates that the
complex is transient and is trapped only by formation of covalent
photoproducts under UV-irradiation. In contrast, the addition of the
duplex-forming hook to the third strand makes the complex stable even
without irradiation. The approach developed in the present work should
find wider applicability to other unfavorable target sequences.
We are grateful to Peter Glazer for helpful
discussions. We thank Lydia Lin, Neelesh Prakash, and Katherine Lee for
valuable assistance and Dmitry Klimov for help with preparation of the figures.
*
This work was supported in part by fellowships from Codon
Pharmaceutical, Inc. and Oncor, Inc. (to O. A. and N. G. D., respectively), and by Grant DE-FG02-96-ER62202.A001 from the
Department of Energy.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Present address: Dept. of Chemistry, Lomonosov Moscow State
University, Moscow, Russia.
¶
To whom correspondence should be addressed. Tel. 609-258-3927;
Fax: 609-258-1028; E-mail: jrfresco@princeton.edu.
2
O. Amosova and J. R. Fresco, unpublished.
The abbreviations used are:
nt, nucleotide(s);
PAGE, polyacrylamide gel electrophoresis;
D-1/DL-1, coding
strand of target duplex;
D-2/DL-2, noncoding strand of
target duplex;
PsT-1, third strand without hook;
pr5U, 5-propynyluracil;
me5C, 5-methylcytosine;
PsT-2, third
strand with hook;
PsT-3, third strand with hook and scrambled
triplex-forming sequence.
Repairing the Sickle Cell Mutation
I. SPECIFIC COVALENT BINDING OF A PHOTOREACTIVE THIRD STRAND TO
THE MUTATED BASE PAIR*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin gene.
Triplex-mediated psoralen modification of the mutant T residue was
sought as an approach to gene repair. The 24-nucleotide purine-rich
target sequence switches from one strand to the other and has four
pyrimidine interruptions. Therefore, a third strand sequence favorable
to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third
strand binding, 5-methylcytosine and 5-propynyluracil were used in the
third strand. Further, a six residue "hook" complementary to an
overhang of a linear duplex target was added to the 5'-end of the third
strand via a T4 linker. In binding to the overhang by
Watson-Crick pairing, the hook facilitates triplex formation. This
third strand also binds specifically to the target within a supercoiled
plasmid. The psoralen moiety at the 3'-end of the third strand forms
photoadducts to the targeted T with high efficiency. Such monoadducts
are known to preferentially trigger reversion of the mutation by DNA
repair enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A·T
(5).
T·A transversion in the gene for the
-globin chain located on chromosome 11 (9). This mutation occurs
immediately upstream of a
24-nt1 purine-rich sequence
that consists of two adjacent regions located on opposite strands, the
longer downstream one containing four base pair inversions. These
encumbrances make it a difficult target for third strand binding,
requiring a strand-switching "cross-over" strategy (10), the use of
modified third strand residues and, when this target is contained in a
linear duplex fragment, a duplex-forming hook (Fig. 1b) to
provide sufficient third strand binding energy to enable
triplex-mediated psoralen photoaddition in high yield precisely at the
mutant base pair.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP
(Amersham Pharmacia Biotech), and 1 unit of T-4 polynucleotide kinase
(U. S. Biochemical Corp.) at 37 °C for 1 h, purified by denaturing PAGE, and eluted from C18 Sep-Pak columns.
-globin gene fragment surrounding the
mutation site into the pBluescript vector (Stratagene).
32P-end-labeled PsT-2 and PsT-3 were incubated at 20 °C
for 1 h with 10
8 M pSCe and/or
pBluescript (vector without the insert) at a 10:1 molar ratio in the
standard buffer and then at 4 °C overnight prior to irradiation.
Irradiated plasmids were electrophoresed on agarose gel in TBE under
conditions where nonphotoattached third strand dissociates from the
target, and the gels were evaluated by ethidium bromide staining,
autoradiography, and phosphorimaging.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin gene sequence of human chromosome 11 used in this investigation. D-1·D-2 is a version of
DL-1·DL-2 shortened at the downstream end.
Residue T11 on strand DL-1/D-1 (shown in bold)
is the consequence of the A T transversion responsible for sickle
cell anemia. Residues 1-17 on strand D-2 (4-21 on DL-2)
and 12-19 on D-1/DL-1 comprise the 24-nt purine-rich third
strand binding region that was targeted for triplex formation. The one
T and three C residues within the purine-rich sequence of the D-2
binding domain significantly decrease the effectiveness of third strand
binding to the target. Our original approach was a strand-switching
third strand with an acridine intercalator added at the 5'-end (Fig.
1a). The triplex forming domain of PsT-1 was designed to
bind to the DL-2 target segment in the pyrimidine/parallel
motif, and then cross over to bind to the DL-1 segment in
the GT/antiparallel motif. This design allows the psoralen moiety at
the 3'-end of PsT-1 to be positioned directly opposite the mutant
T11 residue of DL-1. To enhance the association
to this target,
pr5U2 was
positioned opposite the three C·G inversions, G opposite the T·A
inversion (15, 16), and me5C was substituted for C in the
third strand opposite G·C pairs (17). Despite these various
modifications, binding was not sufficiently enhanced (see below).
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Fig. 1.
Oligonucleotide structures.
a, triplex structure formed by association of third strand
PsT-1 and the duplex segment of -globin gene
DL-1·DL-2 at the third strand binding target.
The A·T T·A transversion mutation site on DL-1 is
indicated in bold letters and by an arrow. PsT-1
contains modified residues (mC= me5C,
pU = pr5U) in the triplex-forming region
that binds to DL-2 in the pyrimidine/parallel motif and
then crosses over to bind to DL-1 in the GT/antiparallel
motif, terminating with a 3'-psoralen moiety positioned opposite the
mutant residue T11 on DL-1. An acridine
intercalator moiety is added to the 5'-end of PsT-1 to strengthen third
strand binding. b, triplex structure formed by association
of strand PsT-2 and duplex D-1·D-2, which are shortened versions of
DL-1 and DL-2, respectively. The 3'-end of
DL-1 is truncated by 6 nt to create a target on D-2 for a
duplex-forming hook linked to PsT-1 to create third strand PsT-2. The
hook, complementary to the 5'-end of D-2, is followed by a
(T)4 linker and then the same triplex-forming sequence
containing modified residues (mC= me5C,
pU = pr5U), that binds to duplex segment
C. PsT-2 then crosses over to bind along strand D-1 of duplex segment
B, terminating with a 3'-psoralen moiety positioned opposite the mutant
residue T11 on D-1. Duplex segment A should not be
protected from nuclease digestion by third strand binding, whereas
segments B and C are potential third strand binding targets that should
be protected. c, negative control third strand PsT-3,
containing the same 6-nt duplex-forming hook and (T)4
linker as PsT-2, that is however followed by a scrambled sequence that
should not form triplex.
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Fig. 2.
Nondenaturing PAGE band-shift analysis.
Duplexes (0.5 µM) were annealed with different
concentrations of third strand, as described. Note that some type of
complex formation is observed between D-1·D-2 and both third strand
PsT-2 and negative control PsT-3, but not between
DL-1·DL-2 and third strand PsT-1.
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Fig. 3.
DNase I footprint analysis.
a, denaturing PAGE analysis of DNase I-treated structures
(0.5 µM D-1·D-2, 5 µM PsT-2 or PsT-3).
Duplex strand D-2 of D-1·D-2 is 32P-labeled on the
5'-end; thus, observed bands represent specific cuts along D-2. D-2
residue assignments were made by running ddNTP sequencing reactions
using D-2 templates on denaturing PAGE alongside DNase I-treated duplex
(data not shown). Note that D-2 strand segments B and C (but not A)
display reduced sensitivity to DNase I cleavage only in the presence of
PsT-2 (cf. lanes 3 and 4 versus 1 and 2), but not in the
presence of PsT-3 (cf. lanes 5 and 6 versus 1 and 2). Reduced sensitivity
to DNase I at the 5'-end of the duplex control is due to the
single-stranded protruding end, which is not a substrate of DNase I;
this 5'-end becomes a substrate when bound to PsT-3, forming a duplex.
b, relative DNase I sensitivity of different D-2 segments.
Quantitated PAGE results (Table I) are represented as changes in
nuclease sensitivity relative to unprotected naked duplex along
different D-2 segments. Note that in the presence of a binding third
strand (PsT-2), relative sensitivity significantly decreases along the
protected segments, and the observed cuts are "shifted" to the
unprotected segment. In contrast, no significant change in the relative
sensitivity profile is observed in the presence of a nontriplex-forming
third strand (PsT-3). Values along the ordinate represent the
difference in the fraction of total cuts (along the designated
segments) between the three-stranded complex and the same region of
unprotected duplex.
Quantitative analysis of DNase I protection results
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Fig. 4.
Photoproduct formation along D-1-labeled
complexes. Denaturing PAGE analysis of photoproduct formation.
Complexes formed at 10 nM duplex and indicated third strand
concentrations were UV-irradiated for 10 min at 4 °C. Note that no
photoproduct is observed at either third strand concentration in the
presence of the nontriplex-forming strand PsT-3, despite the psoralen
tethered to its 3'-end. In contrast, strand PsT-2 forms photoproducts
in similar yield at both concentrations. However, PsT-1 forms
photoproducts only at very high (40 µM)
concentration.
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Fig. 5.
Binding affinity of third strands PsT-1 and
PsT-2. a, denaturing PAGE analysis of irradiated
complexes formed with either 10 nM
DL-1·DL-2 and increasing concentrations of
PsT-1 (right panel) or 1 nM D-1·D-2 duplex and
increasing concentrations of PsT-2 (left panel), using
32P-end-labeled DL-1 and D-1 duplex strands,
respectively. b, binding affinity of PsT-1 (×) and PsT-2
( 
) determined from denaturing PAGE. Results are expressed as
percent of labeled duplex converted to complex with PsT-1 or PsT-2.
Higher ximum yield for PsT-1 photoproducts could be due to higher
duplex concentration.
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Fig. 6.
Kinetics of photoproduct formation and
yields. a, denaturing PAGE analysis of photoproduct
formation by PsT-2 with D-1- and D-2-labeled strands (0.5 µM D-1·D-2 duplex, 5 µM PsT-2 third
strand). Complexes were irradiated for indicated times at 4 °C. A
slowly moving low intensity band, formed with both types of complexes,
is identified as a crosslink (XL) between the PsT-2 third
strand and D-1·D-2 duplex. The faster moving photoproduct bands are
monoadducts (MA); their different mobilities result from
difference in psoralen photoaddition site. b, kinetics and
yields of photoproduct formation. ×, D-1 monoadduct; ( 
), D-2
monoadducts; (
), crosslinks.
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Fig. 7.
Dependence of photoproduct yield on pH.
Complexes (10 nM D-1*-labeled duplex, 100 nM
PsT-2 third strand) were formed in buffers of constant ionic
strength but varying pH, irradiated for 10 min and their photoproduct
yields determined by denaturing PAGE analysis.
T·A human sickle
cell transversion, as the major site of psoralen photoaddition. Similar
observations on photoproducts formed with the D-2 target strand confirm
the sites of photoattachment indicated above (data not shown).
View larger version (36K):
[in a new window]
Fig. 8.
Primer extension from D-1 templates.
Denaturing PAGE analysis of ddNTP primer extension reactions using D-1
primary monoadduct strands (MA) as template
(lane 2) alongside a sequencing ladder obtained using
nonphotomodified D-1 control strands (lane 1). Note
that arrest of synthesis occurs at the photodamaged T11
residue on D-1 (lane 2).
-globin sickle cell target, PsT-2, the third strand with the duplex-forming hook, does bind to it, though not to the vector plasmid
without the target. Moreover, the binding is very much greater to the
supercoiled than to open circular or linearized plasmid. These results
suggest high promise for our overall approach.
View larger version (35K):
[in a new window]
Fig. 9.
Third strand binding to plasmid.
Ethidium bromide-stained agarose gel of pSCE (containing the target
sequence) and pBluescript II (vector plasmid without the target
sequence) incubated with PsT-2 or PsT-3 and then UV-irradiated
(top). Open circular (OC) and supercoiled
(SC) plasmid bands are indicated by arrows. *
indicates radioactively labeled. Bottom, autoradiograph of
the same gel. Only the supercoiled form of the plasmid with the sickle
cell target and third strand PsT-2 is strongly labeled. A very low
level of nonspecific binding of PsT-2* to the control plasmid without
the target is evident in the left lane.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A·T
substitutions in the supF gene have been triggered in
vivo by third strand-directed psoralen photomodification of
plasmids transfected into monkey COS-7 cells (6-8). Using the
methodology developed, it is anticipated that the sickle cell mutation
may be similarly repaired. In that event, acceptable levels of the
correct phenotype might be achieved if psoralen monoadduct-triggered
in vivo mutation efficiencies are sufficiently high.
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of sabbatical research support from West Chester
University. Present address: Dept. of Biology, West Chester University, West Chester, PA 19383.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Broitman, S. L.,
Im, D. D.,
and Fresco, J. R.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
5120-5124 2.
Letai, A. G.,
Palladino, M. A.,
Fromm, E.,
Rizzo, V.,
and Fresco, J. R.
(1988)
Biochemistry
27,
9108-9112[CrossRef][Medline]
[Order article via Infotrieve]
3.
Moser, H. E.,
and Dervan, P. B.
(1987)
Science
238,
645-650 4.
Sun, J.-S.,
and Hélène, C.
(1993)
Curr. Opin. Struct. Biol.
3,
345-356
5.
Havre, P. A.,
Gunther, E. J.,
Gasparro, F. P.,
and Glazer, P. M.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
7879-7883 6.
Wang, G.,
Seidman, M. M.,
and Glazer, P. M.
(1996)
Science
271,
802-805[Abstract]
7.
Faruqi, A. F.,
Seidman, M. M.,
Segal, D. J.,
Carroll, D.,
and Glazer, P. M.
(1996)
Mol. Cell. Biol.
16,
6820-6828[Abstract]
8.
Sandor, Z.,
and Bredberg, A.
(1995)
Biochim. Biophys. Acta
1263,
235-240[Medline]
[Order article via Infotrieve]
9.
Moschonas, N.,
de Boer, E.,
and Flavell, R. A.
(1982)
Nucleic Acids Res.
10,
2109-2120 10.
Jayasena, S.,
and Johnston, B. H.
(1992)
Biochemistry
31,
320-327[CrossRef][Medline]
[Order article via Infotrieve]
11.
Chen, Z.,
and Ruffner, D. E.
(1996)
BioTechniques
21,
820-822[Medline]
[Order article via Infotrieve]
12.
Maniatis, T.,
Fritsch, E. F.,
and Sambrook, J.
(1982)
Molecular Cloning: A Laboratory Manual
, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
13.
Thomas, M.,
Abedi, H.,
and Farzaneh, F.
(1992)
BioTechniques
13,
533[Medline]
[Order article via Infotrieve]
14.
Sequenase Version 2.0 DNA Sequencing Kit, 9th Edition
(1994) Amersham Life Science, Inc.
15.
Yoon, K.,
Hobbs, C. A.,
Koch, J.,
Sardaro, M.,
Kutny, R.,
and Weis, A. L.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
3840-3844 16.
Fossella, J. A.,
Kim, Y. J.,
Shih, H.,
Richards, E. G.,
and Fresco, J. R.
(1993)
Nucleic Acids Res.
21,
4511-4515 17.
Posvic, T. J.,
and Dervan, P. B.
(1989)
J. Am. Chem. Soc.
111,
3059-3061[CrossRef]
18.
Galas, D. J.,
and Shmitz, A.
(1978)
Nucleic Acids Res.
55,
3157-3170
19.
Gasparro, F. P.,
Havre, P. A.,
Olack, G. A.,
Gunther, E. J.,
and Glazer, P. M.
(1994)
Nucleic Acids Res.
22,
2845-2852 20.
Sage, E.,
and Moustacchi, E.
(1987)
Biochemistry
26,
3307-3314[CrossRef][Medline]
[Order article via Infotrieve]
21.
Guieysse, A.-L.,
Praseuth, D.,
Grigoriev, M.,
Harel-Bellan, A.,
and Hélène, C.
(1996)
Nucleic Acids Res.
24,
4210-4216 22.
Berressem, B. R.,
and Engels, J. W.
(1995)
Nucleic Acids Res.
23,
3465-3472 23.
Rajeev, K. G.,
Jadhav, V. R.,
and Ganesh, K. N.
(1997)
Nucleic Acids Res.
25,
4187-4193 24.
François, J.-C.,
and Hélène, C.
(1995)
Biochemistry
34,
65-72[CrossRef][Medline]
[Order article via Infotrieve]
25.
Pascolo, E.,
and Toulmé, J.-J.
(1996)
J. Biol. Chem.
271,
24187-24192 26.
Brossalina, E.,
Pascolo, E.,
and Toulmé, J.-J.
(1993)
Nucleic Acids Res.
21,
5616-5622 27.
Moses, A. C.,
and Schepartz, A.
(1997)
J. Am. Chem. Soc.
119,
11591-11597[CrossRef]
28.
Gamper, H. B., Jr.,
Hou, Y.-M.,
Stamm, M. R.,
Podyminogin, M. A.,
and Meyer, R. B.
(1998)
J. Am. Chem. Soc.
120,
2182-2183[CrossRef]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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