From the
We report two strategies for accelerating the hybridization of
oligonucleotides to DNA. We demonstrate that oligodeoxyribonucleotides
and peptide nucleic acid oligomers hybridize to inverted repeats within
duplex DNA by D-loop formation. Oligonucleotides and duplex template
form an active complex, which can be recognized by T7 DNA polymerase to
prime polymerization. Quantitation of polymerization products allowed
the rate of hybridization to be estimated, and peptide nucleic acid
oligomers and oligonucleotide-protein adducts anneal with association
constants 500- and 12,000-fold greater, respectively, than the
analogous unmodified oligonucleotides. Together, these results indicate
that sequences within duplex DNA can be targeted by Watson-Crick base
pairing and that chemical modifications can dramatically enhance the
rate of strand association. These findings should facilitate targeting
of oligomers for priming DNA polymerization, the detection of
diagnostic sequences, and the disruption of gene expression. The
observed acceleration of hybridization may offer a new perspective on
the ability of RecA or other proteins to accelerate strand invasion.
Recognition of duplex DNA by single-stranded polynucleotides
requires that the single strand come into close proximity with the
target sequence and then initiate base pairing. Acceleration of either
of these events would increase the rate at which oligonucleotides
hybridize with complementary sequences. Such increased hybridization
rates would facilitate the recognition of short-lived single-stranded
regions by oligomers and reduce the probe concentrations or incubation
periods required for anti-gene transcriptional inhibition, polymerase
priming, diagnostic probing, or structural mapping. Increased rates for
hybridization may also afford a new perspective on the ability of RecA
and other proteins to promote strand exchange during recombination.
Sequence recognition can occur by Watson-Crick base pairing through
D-loop formation(1) . Unlike triple helix formation via
Hoogstein base pairing(2, 3, 4) , which is
restricted to target sequences that are primarily
polypurine-polypyrimidine, D-loop formation is possible at any sequence
that is transiently single stranded. Such sequences include inverted
repeats(5, 6) , H-form
DNA(7, 8, 9) , Z-DNA(10) , AT-rich
unwinding elements(11, 12) , matrix attachment
regions(13) , and RNA polymerase open complexes(14) , all
of which are destabilized relative to native B-form DNA and occur in vitro and in
vivo(15, 16, 17, 18, 19) .
Hybridization at these sites might block the selective recognition by
DNA binding proteins, initiate DNA or RNA polymerization, or provide
structural probes for open regions in DNA.
D-loop formation by long
single strands has been previously described (1), but the ability of
short (<100 base) unmodified oligonucleotides to hybridize by strand
displacement has not. Here, we demonstrate that short
oligodeoxyribonucleotides and peptide nucleic acids (PNAs)
The association rate constant for the PNA with
duplex DNA was calculated by multiplying the factor at which the
concentrations of oligonucleotide and PNA differ, 20, by the relative
difference in time required for a maximal effect on annealing, 2.5 versus 60 min. This estimate is clearly inexact and probably
reflects the minimum estimate for acceleration of PNA hybridization.
Oligonucleotides were annealed to supercoiled duplex
DNA at 37 °C. Hybridization was assayed by monitoring strand
elongation upon addition of modified T7 DNA polymerase,
deoxy/dideoxynucleotide mixes, and
radiolabeling(29, 30) . Upon separation of the
elongation products by gel electrophoresis, we observed that
polymerization was occurring (Fig. 1A). Interpretation
of the resultant DNA sequence confirmed that priming was occurring at
the sites predicted from complementary hybridization by the
oligonucleotides. Phosphorimager analysis of the radiolabeled
elongation products was done to quantitate the relative efficiency of
polymerization at the varied sequences (Fig. 1B).
Elongation was most efficient when primers were directed to inverted
repeats (Fig. 1A, lanes1 and 3) but also occurred at sequences that were near to inverted
repeats (Fig. 1A, lanes2 and 4). We also attempted to prime polymerization with several
oligonucleotides that were 200-500 bases away from any inverted
repeats in pUC 19 (i.e. V) but did not observe strand
elongation (Fig. 1A, lane5).
PNAs are DNA analogs in
which the phosphate backbone has been replaced by amide linkages (Fig. 2A). Base pairing of the PNA to DNA can
occur(31, 32) , but no chain elongation would be
expected because of the absence of a 3`-hydroxyl. As noted,
oligonucleotide I primed efficient DNA synthesis (Fig. 2B, lane1). Addition of PNA VI,
by contrast, blocked elongation when added prior to I (Fig. 2B, lane3) but had no effect
when added after (Fig. 2B, lane2).
The ability of the PNA to block polymerization when added before the
analogous oligonucleotide indicated that PNAs can hybridize to inverted
repeats. Conversely, the failure of the PNA to block DNA synthesis when
added after the analogous oligonucleotide indicated that the
oligonucleotide must have hybridized before the addition of the PNA.
Since polymerase is added after addition of either PNA or DNA, their
hybridization must be independent of protein addition. Surprisingly, an
excess of the PNA strand does not block priming by the analogous
pre-hybridized DNA oligomer after a 30-min co-incubation at 37 °C.
Since the PNA would be expected to rapidly hybridize to any
complementary sequence vacated by the DNA oligomer, this result
suggests that the off rate for the DNA oligomer is very slow.
Most previous studies of modified oligomers have focused on
the strength of strand association as measured by the melting
temperature. Measurement of association rates has received less
attention but may have equally important implications. For example, the
ability to accelerate hybridization would lower the concentration of
oligomer or template required to yield a given level of hybridization.
Acceleration of hybridization may be particularly critical for
oligomers, which are directed to duplex DNA and structured RNA, where
the single-stranded regions needed to initiate base pairing may have
short lifetimes.
To approach the target sequence, an oligonucleotide
must first overcome the electrostatic repulsion between its phosphate
backbone and the phosphate backbones of the base-paired duplex. Once an
oligonucleotide is near the duplex, strand invasion will be further
hindered by native base pairing at the target. Hybridization must then
rely on the formation of transiently single-stranded regions to permit
the initiation of base pairing between target and the invading strand.
These disadvantages, however, must be balanced with the potential of
Watson-Crick base pairing to direct oligomers to any sequence. If such
hybridization could be made efficient, a wider range of sequences could
be targeted to disrupt key interactions in transcription,
recombination, and replication.
We evaluated the ability of short
oligonucleotides to hybridize with supercoiled DNA by Watson-Crick
pairing. For base pairing to occur, one strand of the target sequence
must be displaced, an entropically disfavored event. To overcome the
obstacle to hybridization posed by pre-existing base pairing, we chose
to target oligomers to duplex sequences within supercoiled DNA that
have the potential for alternate secondary structure. The existence of
non B-form DNA structure would increase the likelihood for transient
formation of the single-stranded regions that are necessary to initiate
pairing. Supercoiling destabilizes the native B-form duplex and has
been demonstrated to promote the formation of cruciform structures at
sequences that are inverted repeats(5, 6) . We chose,
therefore, to target oligonucleotides to sequences near to or within
inverted repeats.
We monitored hybridization by assaying the ability
of annealed oligonucleotides to act as primers for Sanger dideoxy
sequencing with modified T7 DNA polymerase. This method has many
advantages as an assay for strand association: 1) interpretation of the
resultant dideoxy sequencing unambiguously locates the site of
hybridization; 2) there is no requirement for washing or purification
steps, procedures which might remove bound oligonucleotide and prevent
accurate evaluation of the concentration of the template-primer
complex; 3) the amount of radiolabel incorporated during elongation
affords a quantitative measure related to hybridization efficiency; and
4) the complex between primer and duplex template is relevant to
enzymological studies because the three-stranded complex closely mimics
intermediate structures that occur naturally(34) . We found that
the sequencing-based assay was superior to measurement by gel shift
assay, S1 nuclease nicking, or filter binding, as none of these
alternate methods conferred the advantages noted above.
We observed
that both PNAs and unmodified oligodeoxyribonucleotides hybridize to
plasmid DNA by strand invasion at a complementary sequence. This
hybridization is spontaneous and does not require addition of protein
to facilitate strand uptake. Hybridization of the unmodified
oligonucleotide is relatively slow, with a k
As noted, accelerated hybridization by the PNA
oligomer with plasmid DNA may be due to the absence of repulsive
electrostatic interactions during strand association. Another strategy
to mitigate repulsion between approaching strands is to couple the
oligonucleotide to a positively charged protein domain capable of
independently associating with DNA. We reasoned that the protein domain
would act to hold the oligonucleotide in proximity with the DNA
template, thereby substantially increasing its effective concentration
near its target sequence. Presumably, this might mimic some of the
functions of RecA or other proteins involved in strand exchange or the
ability of DNA binding proteins to find target sequences at rates that
are faster than the diffusion-controlled limit by reducing a
three-dimensional search to one
dimension(35, 36, 37) . We utilized
staphylococcal nuclease because we had previously observed that
coupling of the nuclease had a powerful effect on the stability of
hybridization(25) . We observed that the
oligonucleotide-staphylococcal nuclease conjugate possessed a k
Staphylococcal nuclease has a
net surface positive charge of 12(39) , and this may facilitate
the approach of the conjugate to the target sequence and subsequent
hybridization through interactions with the phosphodiester backbone. In
addition, to perform its physiological function, staphylococcal
nuclease must insert a nucleotide base into its active site, an outcome
that demands the unpairing of substrate (39). Therefore, an alternative
explanation for the rate enhancement may be that staphylococcal
nuclease actively facilitates or stabilizes the unwinding of DNA,
thereby increasing the opportunity for the initiation of base pairing.
Again, these potential contributions to hybridization are not mutually
exclusive. High concentrations of proteins with no known role in
recombination, such as bovine serum albumin(40) , as well as
cationic detergents (37) have been shown to accelerate
hybridization and strand exchange, so it is not surprising that a
tethered protein, which is held in a high effective concentration
relative to the oligonucleotide, is also able to promote annealing.
Various short peptides have been reported to associate with micromolar
affinity to duplex DNA(41, 42) . This property may
enable them to accelerate hybridization of attached oligonucleotides,
and an understanding of the effect on strand association of attachment
of various positively charged peptides and small molecules to
oligonucleotides may help determine the relative contributions of
charge and three-dimensional structure.
These experiments afford new
insights into the origin of some of the remarkable properties of PNAs.
For example, PNA oligomers have shown the ability to displace one
strand of duplex DNA and form PNA-PNA-DNA triplexes at non-supercoiled
polypurine polypyrimidine sites(31, 32, 44) .
This observation is surprising since the analogous DNA oligomers have
not been reported to hybridize in this manner. Our results are
consistent with a mechanism in which the acceleration of hybridization
by PNAs facilitates initial strand invasion at transiently
single-stranded regions by the first PNA strand. Continued
hybridization, rather than reformation of the parent duplex, might then
be stabilized by similarly accelerated Hoogstein base pairing by the
second PNA to form a stable triple-stranded structure. If true, this
would suggest that PNAs and other chemically modified oligomers may be
relatively well suited for hybridization to target sequences that are
not readily accessible to unmodified oligomers.
In conclusion, we
find that a PNA and an oligonucleotide-protein conjugate achieve
greatly accelerated hybridization to duplex DNA relative to the
analogous unmodified oligonucleotide. This implies that the
electrostatic interactions involved in the approach and recognition of
complementary strands can be manipulated through chemical modification
to increase the rate of association. PNAs are uncharged, whereas the
staphylococcal nuclease domain of the conjugate has a net charge of
+12, a fundamental difference which indicates that different
mechanisms may be involved to accelerate hybridization for the two
different species. The ability to readily manipulate hybridization
suggests that tailored oligomers can be optimized kinetically for
particular in vivo or in vitro applications. Such
oligomers should facilitate the rapid recognition of rare target
sequences by low concentrations of oligonucleotides or oligonucleotide
analogues.
We thank John Waggenspack, Elana Varnum, Debbie
Munoz-Medellin, and Dana Jones for technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)can hybridize to duplex DNA by D-loop formation
independently of added proteins. Hybridized oligonucleotides act as
primers for modified T7 polymerase, affording a method for monitoring
hybridization rate and efficiency. We have measured the relative rates
of hybridization by oligonucleotides, oligonucleotide-protein adducts,
and PNAs and find that recognition by the latter two species is greatly
accelerated. The ability to tailor hybridization rates by chemical
modification should facilitate the optimization of oligonucleotide
probes and therapeutics and afford insights into the molecular
mechanisms which facilitate sequence recognition, strand invasion, and
recombination.
Preparation of Oligonucleotides and
Plasmids
Supercoiled pUC 19 DNA (20) (
= 0.54 ± 0.02) was prepared by mild, non-denaturing lysis
followed by two successive cesium chloride gradient centrifugations to
minimize the likelihood of contamination by denatured duplex
DNA(21) . Oligonucleotides were synthesized on an Applied
Biosystems 451 DNA synthesizer (Foster City, CA). Sequences of
oligonucleotides, the PNA, and the oligonucleotide-nuclease conjugate
used in these studies and the location of hybridization in pUC19 are as
follows: I, 5`-GGATCTTCACCTAGATCCT-3`(1546-1565); II,
5`-ATATACTTTAGATTGATTT-3`(1587-1606); III,
5`-GAGCGGATACATATTTGAATGT-3`(2540-2561); IV,
5`-GGGGTTCCGCGCACATTTCCCCG-3`(2582-2605);and V,
5`-AGTAGTTCGCCAGTTAATAG-3`(1898-1917). Sequences of
oligonucleotides, the PNA, and the oligonucleotide-nuclease conjugate
used in these studies and the location of hybridization in pUC19 are as
follows: I, 5`-GGATCTTCACCTAGATCCT-3`(1546-1565); II, 5`-ATATACTTTAGATTGATTT-3`(1587-1606); III,
5`-GAGCGGATACATATTTGAATGT-3`(2540-2561); IV,
5`-GGGGTTCCGCGCACATTTCCCCG-3`(2582-2605); V,
5`-AGTAGTTCGCCAGTTAATAG-3` (1898-1917); VI, PNA sequence
Gly-GGATCTTCACCTAGATCCT-Lys(1546-1565); VII,
Staphylococcal nuclease-cysteine-S-5`-GGATCTTCACCTAGATCCT-3`
(1546-1565). Additional oligonucleotides were synthesized to be
complementary to sites lacking obvious potential for secondary
structure. Melting temperatures of PNA VI and oligonucleotide I were
determined using a Hewlett Packard 8452 diode array UV
spectrophotometer and a Peltier temperature controller in 10 mM Tris-HCl, pH 7.5. PNA VI was synthesized manually according to
published procedures (22, 43) using PNA monomers
obtained from Millipore. The PNA was purified by reverse phase high
pressure liquid chromatography as described (22) and was
predominantly one peak. PNA purity was evaluated by mass spectral
analysis. The calculated and observed molecular weights for PNA VI were
5532.96 and 5533.13 ± 0.98.
Synthesis of Oligonucleotide-Staphylococcal Nuclease
Conjugate VII
Oligonucleotides containing an introduced
thiol group were synthesized by standard methods using C-6
Thiolmodifier reagent (Clontech)(23) . The oligonucleotide was
cleaved from the resin and deprotected as described to generate the
reduced oligomer in a dithiothreitol-containing solution. This solution
was desalted by size exclusion chromatography (BioSpin 6, Bio-Rad) and
collected directly into a 20 mM solution of
2,2`-dithiodipyridine in 100 µl of acetonitrile. This solution was
incubated for 15 min at room temperature to allow formation of the
5`-S-thiopyridyl adduct. Unreacted 2,2`-dithiodipyridine was
removed by extraction with diethyl ether (3 0.5 ml). The
solution was frozen, concentrated by vacuum centrifugation briefly to
remove residual diethyl ether, and desalted by size exclusion
chromatography (BioSpin 6) to yield the 5`-S-thiopyridyl
oligonucleotide adduct. The presence of the attached thiopyridyl group
was confirmed by monitoring the release of thiopyridyl anion at 343 nm
(
= 7060 M
) upon addition of
dithiothreitol. Modified oligonucleotides were cross-linked to
K116C-containing staphylococcal nuclease by disulfide exchange,
purified, and analyzed as
described(24, 25, 26) . An attenuated variant of
staphylococcal nuclease with reduced catalytic activity, Y113A, K116C
(27, 28), or the specific calcium chelator EGTA (Sigma) was used for
some experiments to prevent DNA nicking during incubations.
Monitoring of Hybridization by DNA
Sequencing
pUC19 (0.03-0.1 µM) was mixed
with 1-100 equivalents of oligonucleotide primer in 10 mM Tris-Cl, pH 7.5, solution for varying periods at 37 °C. The
annealed primer/template mixture was cooled on ice, and
MgCl, NaCl, and Tris-Cl, pH 7.5, were added to final
concentrations of 8, 80, and 10 mM, respectively. Labeling
mix, modified T7 polymerase (Sequenase, U. S. Biochemical Corp., 1
unit/reaction), and
S-dATP were added, and sequencing was
carried out according to the manufacturer's protocol (29, 30).
Equal volumes of the elongation reactions were applied to a denaturing
6% polyacrylamide gel and separated by electrophoresis. The products
were visualized by autoradiography and quantified using a Molecular
Dynamics model 425F phosphorimager. Phosphorimager data were processed
using ImageQuant software (versions 3.1 or 3.2). Background
radioactivity from nonspecific priming or other sources was determined
using reactions to which no primer had been added and was subtracted
from all results.
Monitoring Hybridization by Agarose Gel
Electrophoresis
Separation of the plasmid/elongation
product complex was performed by electrophoresis using 1% agarose gels
and 20 mM Tris acetate, pH 8.0, buffer. DNA polymerization was
performed as described above and was terminated by rapid chilling on
ice. Heating or addition of formamide was avoided so as not to denature
the DNA and cause dissociation of the incorporated strand. Loading
buffer consisting of 10% glycerol/water was added, and the mixture was
applied to an agarose gel and separated at 100 V. Separated DNA bands,
both plasmid and plasmid-elongation product complex, were analyzed by
ethidium bromide staining to visualize total DNA and by autoradiography
to detect and locate the incorporation of radiolabeled extension
products. The relative amounts of radiolabel extension products were
quantified by phosphorimager analysis.
Estimation of Kinetic Association Constant
k
Association constants were determined
using the equation v = k(primer)(plasmid), where v is
the velocity of strand association. The initial rate of strand
association was first shown to be linearly dependent on the
concentration of both template and primer and to follow second order
kinetics. The amount of hybridized complex was estimated by quantifying
the elongation products by phosphorimager analysis and comparing this
amount to reference elongation reactions, which had been analyzed by
agarose gel electrophoresis to observe the absolute. The amount of
hybridized complex was estimated by quantifying the elongation products
by PhosphorImager analysis and comparing this amount to reference
elongation reactions which had been analyzed by agarose gel
electrophoresis to observe the absolute amount of strand elongation.
This amount was then divided by the time of annealing to generate v. Initial rates were used, and both the primer and plasmid
concentrations were assumed to be equivalent to the initial
concentrations.
Targeting Strand Invasion to Inverted
Repeats
Oligonucleotides were synthesized to be
complementary to various sequences within supercoiled plasmid pUC19
DNA. Oligonucleotides I and III were complementary to sequences
containing inverted repeats, II and IV were complementary to sequences
near inverted repeats, while other oligonucleotides were complementary
to sequences that were not near sites known to possess non-B-form
secondary structure. Plasmid DNA was prepared by mild lysis, which
avoided high temperatures or alkaline conditions, to reduce the
likelihood that hybridization might derive from small amounts of
denatured DNA.
Figure 1:
A, dideoxy DNA sequencing of
non-denatured supercoiled pUC19 by primers directed to various
sequences. The order of dideoxy addition was A, G, C, and T.
Supercoiled pUC 19 (0.14 µM) and the relevant primers (2.0
µM) were annealed in 10 mM Tris, pH 7.5, at 37
°C for 15 min. Lanes1 and 3,
oligonucleotides I and III, respectively, directed to inverted repeats. Lanes2 and 4, oligonucleotides II and IV,
respectively, directed to sequences adjacent to an inverted repeat. Lane5, oligonucleotide V directed to a sequence
lacking any predicted secondary structure. The inability of V to prime
strand elongation is representative of six similarly directed primers,
which also failed to prime polymerization. B, phosphorimager
quantification of elongation products after priming with
oligonucleotides I, II, III, IV, and V.
Effect of PNA Addition on
Hybridization
The ability of oligonucleotides to prime DNA
polymerization demonstrated hybridization through strand displacement.
The obligatory presence of polymerase, however, prevented us from
drawing the general conclusion that hybridization was independent of
added protein. To examine hybridization separately from polymerization,
we assayed the inhibition of annealing of oligonucleotide I upon
addition of the analogous PNA oligomer VI.
Figure 2:
A, structure of a PNA dimer CG showing the N(2-aminoethyl)-glycine backbone with the bases linked to the
backbone by a methylene carbonyl linkage. B, assay of
protein-independent hybridization by altering the order of addition of
PNA VI and analogous unmodified oligonucleotide I. The order of dideoxy
addition was A, C, G, and T. Supercoiled pUC19 (0.14 µM)
was present in each elongation. Lane1, 0.7
µM I, without addition of VI. Lane2,
0.7 µM I annealed for 15 min at 37 °C prior to
addition of 0.7 µM VI and continued annealing for an
additional 15 min. Lane3, 0.7 µM VI
annealed for 15 min prior to addition of 0.7 µM I and
continued annealing for an additional 15 min. Lane4,
0.7 µM VI annealed for 15 min at 37
°C.
Estimation of k
Delivery of oligonucleotides to
complementary sequences is governed by the rate at which two strands
associate in an orientation conducive to base pairing. This rate was
proportional to the concentration of primer (Fig. 3). The rate
was also proportional to the annealing time. As hybridization occurred
prior to addition of polymerase, it was possible to manipulate the
annealing period to monitor the rate of formation of the
primer-template complex. Fixed concentrations of template and
oligonucleotide were mixed and annealed for increasing periods of time,
and the efficiency of elongation was monitored.
for Hybridization of
Oligonucleotides to Duplex DNA
Figure 3:
PhosphorImager quantification of
elongation products after priming with increasing concentrations of
oligonucleotide I (0.07, 0.145, 1.45, 2.9, 4.3, 7.25, 8.7, 13, and 14.5
µM) and a fixed concentration of pUC19 template (0.145
µM). Primer and template were annealed for 30 min at 37
°C. Elongation products were separated by polyacrylamide gel
electrophoresis and quantified as described under ``Materials and
Methods.
Hybridization, as
measured by phosphorimager analysis of the polymerization products,
increased with time (Fig. 4, A and C). This
allowed estimation of the initial rate for formation of the hybridized
primer-template complex. Given knowledge of the initial concentrations
of both template and oligonucleotide, this allowed estimation of k, the rate constant for association of
oligonucleotide and template to form a primer-template complex capable
of elongation. For oligonucleotide I k
was calculated to be 14 M
s
.
Figure 4:
A
and B, effect of annealing time on priming of DNA sequencing
by oligonucleotide I and oligonucleotide-nuclease conjugate VII.
Elongation products resulting from dideoxyadenosine and dideoxycytidine
termination reactions are shown. A, sequencing of 0.06
µM pUC19 by 2.0 µM oligonucleotide I. Lanes 1-8, 0-, 1-, 2-, 5-, 10-, 15-, 20-, and 40-min
annealing times, respectively. B, sequencing of 0.03
µM pUC19 by 0.06 µM oligonucleotide nuclease
conjugate VII. Lanes1-5, 0-, 30-, 45-, 60-,
and 90-s annealing times, respectively. Solutions containing DNA were
maintained at 37 °C, and primer was added to commence annealing. At
specified time points, aliquots were withdrawn and chilled in an
ice/water bath to terminate annealing. Stop buffer for elongations
primed by the oligonucleotide-nuclease elongations contained 10 mM dithiothreitol to release the nuclease prior to electrophoresis. C, phosphorimager quantification of elongation products after
priming with oligonucleotide I or oligonucleotide-nuclease VII as a
function of increasing annealing times. ODN,
oligonucleotide.
Estimation of the Rate of PNA Hybridization to Duplex
DNA
PNAs lack a 3`-hydroxyl and are inherently unsuitable
for priming DNA synthesis, so we could not use strand elongation as a
direct measure of the rate for PNA annealing. Instead, we developed an
indirect assay in which we examined the ability of PNA VI to compete
with the analogous DNA oligonucleotide for occupation of the target
site. A 5-fold excess of PNA oligomer VI relative to plasmid was
annealed for varying periods prior to addition of a 100-fold excess of
analogous DNA oligonucleotide I (Fig. 5). Had the rates of PNA
and DNA hybridization been similar, the low concentration of PNA would
have had a negligible effect on the initial rate of DNA hybridization.
This result was not observed. Even after annealing PNA VI to template
for less than 2.5 min, priming by DNA I was inhibited, indicating that
prehybridization with the PNA over this short time period was
sufficient to completely block annealing of oligonucleotide. The
ability of a 5-fold excess PNA oligomer in 2 min to block hybridization
of a 100-fold excess of oligonucleotide, which would normally require
over 1 h for complete hybridization, implies that k for PNA hybridization is
500-fold greater than k
for the unmodified oligonucleotide.
Accelerated hybridization by PNA VI at 37 °C is especially
surprising since it is self-complementary and has a melting temperature
of >90 °C, suggesting that structure within the PNA is not a
serious impediment to recognition.
Figure 5:
Indirect assay of the rate of PNA
hybridization. Effect of annealing time of PNA VI on priming by
subsequent addition of the analogous oligonucleotide. Supercoiled
plasmid template (0.14 µM) was used (A and Clanes only). Lane1, 0.7 µM oligonucleotide I annealed for 15 min a 37 °C (no PNA VI
present). Lanes2-5, polymerization by a
100-fold excess of oligonucleotide I (14 µM) added
simultaneously with 0.7 µM PNA VI or 0.5, 1, or 2.5 min
after addition of 0.7 µM PNA
VI.
Acceleration of Hybridization by
Protein-Oligonucleotide Adducts
To assay the effect of a
conjugated protein on oligonucleotide hybridization, we synthesized a
protein-oligonucleotide adduct VII consisting of an oligonucleotide
analogous in sequence to I linked by a 5`-thiol moiety to an introduced
cysteine on the surface of staphylococcal nuclease. Staphylococcal
nuclease was chosen because sequence-specific cleavage by
oligonucleotide-nuclease conjugates had demonstrated hybridization to
plasmid DNA by D-loop formation(25, 33) , and that the
presence of the attached nuclease promoted hybridization (25). The
staphylococcal nuclease-oligonucleotide conjugate was annealed to
template, and upon addition of polymerase, strand elongation was
observed, confirming that the conjugate was a functional primer. In
contrast to unmodified oligonucleotides, only short annealing times and
low primer concentrations were required (Fig. 4, B and C). The association constant k was measured to be 170,000 M
s
, a 12,000-fold increase relative to the
analogous unmodified oligonucleotide.
Analysis of Strand Elongation by Agarose Gel
Electrophoresis
We evaluated the absolute level of strand
invasion by separating the template/elongation product complex from
native template through agarose gel electrophoresis. Upon addition of
polymerase and strand elongation, plasmid was converted to material of
lower mobility. Priming by oligonucleotide conjugate VII converted most
template, demonstrating that the majority of supercoiled plasmid was
capable of hybridization (Fig. 6A, lanes1 and 2). Much longer annealing times and much higher
concentrations of oligomer were required to achieve similar conversion
of template by the analogous unmodified oligonucleotide I (Fig. 6A, lanes3 and 4). The
acceleration of hybridization caused by attachment of the nuclease is
emphasized by the observation that a 5-min incubation in the presence
of two equivalents of oligonucleotide-nuclease conjugate led to 2-fold
greater polymerization relative to polymerization during a 1-h
incubation with 100-fold excess unmodified oligonucleotide. The lower
mobility bands were strongly radiolabeled (Fig. 6B),
consistent with their incorporation of elongation products.
Figure 6:
A,
mobility shifts resulting from priming of polymerization by
oligonucleotide-nuclease conjugate VII and oligonucleotide I.
Supercoiled plasmid template (0.012 µM) was used, and the
shift was monitored by 1% agarose gel electrophoresis. Lanes1 and 2, the gel mobility shift of pUC 19 caused
by the presence of 0.024 µM oligonucleotide-nuclease
conjugate VII, annealed for 5 min with and without subsequent addition
of polymerase. Lanes3 and 4, the gel
mobility shift of pUC 19 caused by the presence of 1.2 µM oligonucleotide I annealed for 60 min, with and without addition
of polymerase. B, phosphorimager quantification of the
elongation product-template complex after priming with oligonucleotide
I or oligonucleotide-nuclease VII as a function of increasing annealing
times. The complex is the band of lower mobility in lanes1 and 3 of Fig.
6A.
of 14 M
s
.
The analogous PNA, by contrast, hybridizes with a >500-fold higher
association constant. This suggests that hybridization is not limited
by opening of the target duplex to generate a single-stranded region
for the initiation of base pairing, since the target sequence is the
same. Rather, it is likely that the PNA is better able to approach the
duplex and initiate base pairing, which leads to strand exchange. PNAs
possess uncharged backbone linkages and have been observed to possess
higher melting temperatures than DNA of the same sequence(44) .
These higher melting temperatures may lead to more stable initiation
complexes, which are more likely to lead to full incorporation of the
PNA rather than reformation of the target duplex. Alternatively, the
uncharged backbone may facilitate approach of the PNA to its target,
thereby increasing its effective concentration. These mechanisms are
not mutually exclusive and may both contribute to accelerated
hybridization. Despite the differing rates of association, DNA
oligomers remain stably hybridized when added prior to large excesses
of the PNA, demonstrating that the PNA will not readily displace the
analogous DNA. Stable hybridization of oligonucleotides is somewhat
surprising, as reformation of the parent duplex is entropically
favored. It is also interesting to note that hybridization of PNA VI
occurs despite its ability to form an intramolecular hairpin or a
bulged self-complementary duplex with a measured melting temperature of
greater than 90 °C. Even at 37 °C, the structure of the PNA
must be dynamic enough to allow it to adopt new base pairing to
recognize its complementary sequence. It appears, therefore, that
hairpin PNAs, which may have altered membrane permeabilities or in
vivo stabilities, have the potential to readily hybridize to
complementary sequences at temperatures well below their isolated
melting temperatures.
for its complementary sequence of
170,000 M
s
,
representing a 12,000-fold enhancement for the rate of association.
This k
is similar to that recently
measured for the association of complementary 16-base
oligonucleotides(38) , which was determined by fluorescence
resonance energy transfer to be 640,000 M
s
. Similar experiments reported hybridization
to single-stranded M13 mp18 as ``very slow'' in the absence
of formamide and 57,000 M
s
for binding in the presence of 25% formamide(38) .
Attachment of the nuclease appears to have largely overcome structural
or other elements in complex templates that prevent rapid hybridization
of oligonucleotides to complementary sequences, so that the k
approaches that for hybridization of
complementary short oligonucleotides.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.