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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Am Chem Soc. Author manuscript; available in PMC 2010 September 2.
Published in final edited form as:
PMCID: PMC2742410

Strand-Invasion of Extended, Mixed-Sequence B-DNA by γPNAs


In this Communication we show that peptide nucleic acids (PNAs), 15 to 20nt in length, when preorganized into a right-handed helix, can invade mixed-sequence double helical B-form DNA (B-DNA). Strand-invasion occurs in a highly sequence-specific manner through direct Watson-Crick base-pairing. Unlike the previously developed double-duplex invasion strategy that requires simultaneous binding of two strands of pseudocomplementary PNAs to DNA, only a single strand of γPNA is required for invasion in this case, and no nucleobase substitution is needed.

Peptide nucleic acids (PNAs) are a promising class of nucleic acid mimics, developed in the last two decades, in which the naturally occurring sugar phosphodiester backbone has been replaced by achiral N-(2-aminoethyl) glycine units (Scheme 1A).1 In addition to their ability to hybridize to complementary DNA or RNA strands, PNAs can invade double-stranded DNA.2 Strand-invasion occurs predominantly in two modes, through Watson-Crick base-pairing to form a 1:13 or in combination with Hoogsteen base-pairing to form a 2:1 (PNA:DNA) complex,4 depending on the target sequence. The simplicity and generality of their recognition, along with the ease and flexibility of their synthesis make PNAs an attractive class of antigene reagents. However, with the current design, not every sequence can be accessed by PNAs—in particular, those that are comprised of all four nucleobases. Mixed-sequence PNAs have been shown to be capable of invading supercoiled plasmid DNA5-7 and certain regions of genomic DNA;8 but they are not capable of invading intact, linear double helical B-form DNA (B-DNA). Recently, two approaches, “tail-clamp”9,10 and “double duplex invasion,”11 have been developed, enabling mixed-sequence PNAs to invade B-DNA. But, they are not without limitations. The first approach still requires a stretch of homopurine target for anchoring triplex binding while the second, although more relaxed in sequence selection, requires an elaborate nucleobase substitution and the need to use two strands of PNAs to invade B-DNA—which complicates their design and greatly limits their utility.12 In this Communication we show that mixed-sequence PNAs, 15 to 20 nucleotides in length, when preorganized into a right-handed helix, can invade double helical B-DNA. Strand-invasion occurs in a sequence-specific manner through direct Watson-Crick base-pairing. In this case only a single strand of γPNA is required for invasion, and no nucleobase substitution is needed.

Scheme 1
(A) Chemical structure of PNA and γPNA, and (B) sequence of γPNAs and a selected region of the DNA target.

Recently, we showed that PNAs, which, as individual strands do not have well-defined conformations, can be preorganized into a right-handed helix by installing an (S)-Me stereogenic center at the γ-backbone position (Scheme 1A).13 These helical γPNAs exhibit strong binding affinity for complementary DNA and RNA strands. As decamers, mixed-sequence γPNAs are unable to invade B-DNA, but strand-invasion can be rescued by appending an acridine moiety (DNA-intercalator) to one of the termini13 or by replacing a cytosine nucleobase with an energetically more favorable synthetic analog.14 These results suggest that the binding free energies of the decameric γPNAs are already near the invasion threshold and that not much more is needed to invade B-DNA. On the basis of these findings, we speculated that alternative to the aformentioned strategies the required binding free energy may also be attained by extending the size of the γPNA oligomers since a γPNA-DNA is generally thermodynamically more stable than a DNA-DNA duplex. If it can be achieved, this design strategy would not only circumvent the need to attach ancillary agents to PNAs, which may compromise their recognition specificity and complicate their synthesis, but also allow γPNAs to recognize extended DNA targets, which has been a goal of antigene design.2,15,16 To test this hypothesis, we synthesized a series of γPNA oligomers with varied sizes (Scheme 1B) that are designed to bind to both the top and bottom strands of the DNA target, and assessed their invasion capability using a combination of gel-shift and enzymatic and chemical probing assays.

Gel-shift assay was performed by incubating a 291bp-PCR fragment with perfect-match (PM) binding site with different concentrations of γPNA1 through 3 in 10 mM sodium phosphate (NaPi) buffer at 37 °C for 16 hrs, followed by gel-separation and SYBR-Gold staining. Consistent with our earlier result,13 we did not observe binding for γPNA1, a decamer, at γPNA:DNA ratios as high as 20:1 (Figure 1A, lanes 3 & 4). However, in the case of γPNA2 (15mer) and γPNA3 (20mer), we noticed the appearance of a shifted band (lanes 5, 6, 8 & 9). The intensity of the new band, relative to that of the free DNA, gradually increased with increasing γPNA concentrations, and the mobility decreased with increasing oligomer lengths. Formation of this complex appeared to be more efficient with γPNA3 than with γPNA2, as reflected in the ratio of the bound-to-unbound DNA (compare lanes 8 with 5 and lanes 9 with 6). This is expected; since γPNA3 is longer in length it would form a thermodynamically more stable complex with DNA. Binding appeared to be unique to γPNAs and occurred in a sequence-specific manner—neither incubation of DNA with PM binding site with PNAs of identical nucleobase sequence (PNA2, lane 7; PNA3, lane 10) nor incubation of DNA with single-base mismatch in the middle (MM1) or toward one end (MM2) of the binding site with γPNAs resulted in formation of any shifted band (Figure 1B, lanes 4-9). Similar findings were also observed with γPNA4 (15mer) and γPNA5 (20mer), which were designed to bind to the top strand of the DNA target (Figure 2). Formation of the retarded band was only observed with PM (lanes 1-8) and none with MM1 (lane 9) or MM2 (lane 10). However, when an equimolar ratio of γPNA3 and γPNA5 was added, no retarded band was observed (lane 8). This was expected since the two γPNA strands are complementary to one another; they would hybridize to each other rather than invade the DNA double helix because a γPNA-γPNA is thermodynamically more stable than a γPNA-DNA duplex. A time-course study revealed that formation of this complex reached equilibrium within ~ 8 hrs of incubation and followed a pseudo-first order kinetic (Figure 3).

Figure 1
Gel-shift assay following incubation of 0.1 μM 291bp-DNA fragment with (A) PM binding site with different concentrations of γPNAs (lanes 3-6, 8 & 9) and PNAs (lanes 7 and 10), and (B) PM (lanes 1-3), MM1 (lanes 4-6) and MM2 (7-9) ...
Figure 2
Gel-shift assay following incubation of 0.1 μM 291bp-PCR fragment with PM (lanes 1-8), MM1 (lane 9) and MM2 (lane 10) binding site with different concentrations of γPNAs (γPNA4, lanes 2 & 3; γPNA5, lanes 5, 6, 9 ...
Figure 3
Time-dependent strand-invasion of DNA by γPNA2 and γPNA3. 0.1 μM of a 291bp-PCR fragment with PM binding site was incubated with 1.0 μM of each γPNA oligomer in 10 mM NaPi at 37 °C for the indicated periods. ...

To confirm that these γPNAs bind to their designated site and through a strand-invasion mechanism, we performed S1-nuclease digestion with the 3′-end of the homologous DNA strand labeled with P-32. S1-nuclease was chosen because it is known to selectively cleave single-stranded or melted regions of double-stranded DNA. Invasion of γPNAs into DNA double helix is expected to result in a local displacement of the homologous DNA strand, which can be revealed in the form of strand cleavage following S1-nuclease digestion. Figure 4 reveals the digestion patterns of a 171bp-PCR fragment containing PM (lanes 1-8), MM1 (lanes 9 & 10) and MM2 (lanes 11 & 12) binding site following incubation with γPNAs and PNA. Contrary to the gel-shift assay, where we did not observe any evidence of binding for γPNA1, we observed cleavage on the homologous DNA strand directly across from its binding site (lanes 2 & 3). This result indicates that strand-invasion did take place because of the susceptibility of the homologous strand to S1-nuclease digestion, but the fact that no retarded band was observed in the gel-shift assay indicates that the complex was not sufficiently stable to withstand the prolonged electrophoresis. Similar cleavage patterns were observed for γPNA2 and γPNA3, but they were more extensive in coverage—roughly 5 bases further toward the 3′-end for γPNA2 (lanes 4 & 5) and 5-8 bases further toward both the 3′- and 5′-end for γPNA3 (lanes 6 & 7). This was expected based on their recognition coverage. No strand cleavage was observed with unmodified PNA3 (lane 8) or with γPNA3 that have mismatched targets (lanes 10 & 12). This result was further corroborated by DEPC (diethyl pyrocarbonate) chemical probing assay,17,18 which revealed selective cleavage of the homologous DNA strand at the adenine and, to a smaller extent, guanine residues across from their binding site (Figures 1S and 2S). This was only observed with PM and none with MM1 or MM2 binding site. These results are consistent with γPNA binding through a strand-invasion mechanism.

Figure 4
S1-nuclease digestion of a 171bp-linear B-DNA with PM (lanes 1-8), MM1 (lanes 9 & 10) and MM2 (lanes 11 & 12) binding site following incubation with γPNAs and PNA at the indicated ratios in 10 mM NaPi buffer at 37 °C for ...

Further, we show that γPNAs can be used to selectively inhibit restriction enzyme digestion. Incubation of a 321bp-DNA target containing PM binding site with Bgl II resulted in two DNA fragments—142 and 175bp in length (Figures 5A and B, lane 3). When the DNA target was first incubated with γPNA3 prior to adding enzyme, the digestion was progressively inhibited with increasing γPNA3 concentrations (lanes 4 & 5). This result indicates one of two possibilities: that either γPNA3 bound to its target and thereby blocked the enzyme from cleaving the DNA at its restriction site, or that γPNA3 ‘poisoned’ the enzyme through nonspecific binding. We ruled out the latter possibility based on the mismatch result. When the DNA fragments containing MM1 and MM2 binding site were used, no apparent inhibition of the restriction enzyme digestion was observed (lanes 7 & 9), indicating that the presence of γPNA3 had no effect on the activity of Bgl II. This result shows that γPNAs can be used as molecular tools to selectively manipulate the structure of B-DNA,19 and possibly other genome-sensing applications as well.20 Once formed, the invasion complex remained stable over a prolonged period, even at a relatively high ionic strength as demonstrated in this study.

Figure 5
Restriction enzyme digestion of a 321bp-PCR fragment containing PM, MM1 and MM2 binding site with γPNA3. (A) A schematic diagram showing the Bgl II restriction and γPNA3 binding site along with the size of the predicted DNA fragments following ...

In summary, we have shown that mixed-sequence PNAs, 15 to 20nt in length, when preorganized into a right-handed helix by installing an appropriate stereogenic center at the γ-backbone, can invade double helical B-DNA. We attribute the improvements in γPNAs' ability to invade B-DNA to their preorganized structure. This structure allows them to intercept the nucleobase targets more rapidly and stabilize the invasion complex more effectively than their achiral counterparts since minimal structural rearrangement would be required prior or subsequent to invasion. The results reported herein are important because they demonstrate that the same Watson-Crick base-pairing principles that guide the recognition of single-stranded DNA and RNA can also be applied to intact double helical B-DNA. Recognition, in this case, is general to all four nucleobases, and extended target sizes that may be difficult to achieve with other classes of molecules.16,21-23

Supplementary Material



Funding was provided in part by the National Institutes of Health to D. L. (GM076251).


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