©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Accelerated Hybridization of Oligonucleotides to Duplex DNA (*)

Mridula Iyer (§) , James C. Norton (§) , David R. Corey (¶)

From the (1)Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)()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.


MATERIALS AND METHODS

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.

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.


RESULTS

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.

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).


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.

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.


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 for Hybridization of Oligonucleotides to Duplex DNA

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.


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.




DISCUSSION

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 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.

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 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.

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.


FOOTNOTES

*
This work was supported by Grant I-1244 from the Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Both authors contributed equally to this work.

An Assistant Investigator with the Howard Hughes Medical Institute. To whom all correspondence should be addressed: The Howard Hughes Medical Institute, Dept. of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX, 75235. E-mail: Corey@howie.swmed.edu.

The abbreviation used is: PNA, peptide nucleic acid.


ACKNOWLEDGEMENTS

We thank John Waggenspack, Elana Varnum, Debbie Munoz-Medellin, and Dana Jones for technical assistance.


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