We hypothesize that programmable hybridization to noncanonical nucleic acid motifs may be achieved by macromolecular display of binders to individual noncanonical pairs (NCPs). As each recognition element may individually have weak binding to an NCP, we developed a semi‐rational approach to detect low affinity interactions between selected nitrogenous bases and noncanonical sites in duplex DNA and RNA. A set of fluorogenic probes was synthesized by coupling abiotic (triazines, pyrimidines) and native RNA bases to thiazole orange (TO) dye. This probe library was screened against duplex nucleic acid substrates bearing single abasic, single NCP, and tandem NCP sites. Probe engagement with NCP sites was reported by 100–1000× fluorescence enhancement over background. Binding is strongly context‐dependent, reflective of both molecular recognition and stability: less stable motifs are more likely to bind a synthetic probe. Further, DNA and RNA substrates exhibit entirely different abasic and single NCP binding profiles. While probe binding in the abasic and single NCP screens was monotonous, much richer binding profiles were observed with the screen of tandem NCP sites in RNA, in part due to increased steric accessibility. In addition to known binding interactions between the triazine melamine (M) and T/U sites, the NCP screens identified new targeting elements for pyrimidine‐rich motifs in single NCPs and 2×2 internal bulges. We anticipate that semi‐rational approaches of this type will lead to programmable noncanonical hybridization strategies at the macromolecular level.
We report herein a study on the impact of bifacial peptide nucleic acid (bPNA) amino acid composition and backbone modification on DNA binding. A series of bPNA backbone variants with identical net charge were synthesized to display either 4 or 6 melamine (M) bases. These bases form thymine‐melamine‐thymine (TMT) base‐triples, resulting in triplex hybrid stem structures with T‐rich DNAs. Analyses of 6 M bPNA‐DNA hybrids suggested that hybrid stability was linked to amino acid secondary structure propensities, prompting a more detailed study in shorter 4 M bPNAs. We synthesized 4 M bPNAs predisposed to adopt helical secondary structure via helix‐turn nucleation in 7‐residue bPNAs using double‐click covalent stapling. Generally, hybrid stability improved upon stapling, but amino acid composition had a more significant effect. We also pursued an alternative strategy for bPNA structural preorganization by incorporation of residues with strong backbone amide conformational preferences such as 4
- NSF-PAR ID:
- 10366693
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ChemBioChem
- Volume:
- 23
- Issue:
- 8
- ISSN:
- 1439-4227
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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