A simple cytosine to G-clamp nucleobase substitution enables chiral gamma-PNAs to invade mixed-sequence double-helical B-form DNA.

Nature uses Watson–Crick base pairings as a means to store and transmit genetic information because of their high fidelity. These specific A–T (or A–U) and G–C nucleobase interactions, in turn, provide chemists and biologists with a general paradigm for designing molecules to bind to DNA and RNA. With knowledge of the sequence information, one can design oligonucleotides to bind to just about any part of these biopolymeric targets simply by choosing the corresponding nucleobase sequence according to these digital base-pairing rules. Although conceptually simple, such principles in general can only be applied to the recognition of single-stranded DNA or RNA, but not the double-stranded form. The reason is that in double-helical DNA (or RNA) not only are the Watson–Crick faces of the nucleobases already occupied, they are buried within the double helix. Such molecular encapsulation imposes a steep energetic barrier on the designer molecules. To establish binding, not only would they need to be able to gain access to the designated nucleobase targets, which are blocked by the existing base pairs, they would also need to be able to compete with the complementary DNA strand to prevent it from re-annealing with its partner—a task that has rarely been accomplished by any class of molecules. To circumvent this challenge, most of the research effort to date has been focused on establishing principles for recognizing chemical groups in the minor and major groove instead because they are more readily accessible and energetically less demanding. While impressive progress has been made on this front, especially in the development of triplex-forming oligonucleotides, polyamides, and zinc-finger-binding peptides, the issues of sequence selection, specificity and/or target length still remain unresolved for many of these approaches. In the last decade, however, several studies have shown that peptide nucleic acid (PNA), a particular class of nucleic acid mimics that are comprised of pseudopeptide backbone (Scheme 1), could invade double-stranded DNA (dsDNA). This finding is significant because it demonstrates that the same Watson–Crick base-pairing principles that guide the recognition of single-stranded DNA and RNA can also be applied to dsDNA. Aside from the simplicity, this recognition strategy is general and could potentially be applied to any sequence or target length, just as in the recognition of single-stranded DNA or RNA. The downside to this approach, however, is that PNA can only recognize homopurine and homopyrimidine targets. Mixed-sequence PNAs do not have sufficient binding free energy to invade double-helical B-DNA. Though a double-duplex invasion strategy has been developed to try to overcome this energetic barrier, the issue of sequence selection still remains due to the unresolved issue with self-quenching. In this Communication, we show that a simple nucleobase substitution, that is, replacing cytosine with a G-clamp (Scheme 1) provides the necessary energetics for chiral g-PNAs to invade mixed-sequence B-DNA. Unlike the double-duplex invasion strategy, which requires two strands of PNAs, only a single strand of g-PNA is required for binding to B-DNA. Recently, we showed that randomly folded, single-stranded PNAs can be preorganized into a right-handed helix simply by installing an l-alanine-derived, S chiral center at the g-position of the N-(2-aminoethyl)glycine backbone unit. These helical g-PNAs exhibit strong binding affinity and sequence selectivity for DNA and RNA and are capable of invading mixed-sequence Scheme 1. Chemical structures of PNA, g-PNA, C–G and X–G base pairs along with the sequences of the oligomers used in this study. Bold letters indicate g-backbone modifications.