Strand Invasion of Mixed-Sequence, Double-Helical B-DNA by γ-Peptide Nucleic Acids Containing G-Clamp Nucleobases under Physiological Conditions

Abstract

Peptide nucleic acids (PNAs) are the only class of nucleic acid mimics developed to date that has been shown to be capable of invading double helical B-form DNA. Recently, we showed that sequence limitation associated with PNA recognition can be relaxed by utilizing conformationally-preorganized γ-peptide nucleic acids (γPNAs). However, like all the previous studies, with the exception of triplex-binding, DNA strand invasion was performed in relatively low salt concentrations. When subjected to physiological ionic strengths, little to no binding was observed. On the basis of this finding it was not clear whether the lack of binding is due to the lack of base-pair opening, or to the lack of binding free energy—either of which would result in no productive binding. In this Article, we show that it is the latter. Under simulated physiological conditions DNA double helix is sufficiently dynamic to permit strand invasion by the designer oligonucleotide molecules provided that the required binding free energy can be met. This finding has important implication for the design oligonucleotides for recognition of B-DNA via direct Watson-Crick base-pairing.

Cooperative hybridization of γPNA miniprobes to a repeating sequence motif and application to telomere analysis

“…18, 19 Ly and coworkers reported that incorporation of guanidino G-clamp residues into γPNA significantly enhances strand invasion into duplex DNA. 20 As shown in Scheme 1, hybridization of the 12mer or 6mer γPNA miniprobes to a complementary DNA of a given length will result in 1.5 or 3 times as many dyes being delivered to the target nucleic acid as the conventional PNA probe.…”

Design of Bivalent Nucleic Acid Ligands for Recognition of RNA-Repeated Expansion Associated with Huntington’s Disease

“…In contrast, we did not observe any evidence of ligand binding taking place at a physiologically relevant ionic strength (Figure S21), indicating that the RNA hairpin was not able to mediate template-directed ligand oligomerization under such a condition. This outcome is unlikely due to the inability of ligand to access the H-bond donors and acceptors of RNA nucleobases, as we have demonstrated in the invasion of the DNA double helix by MP γ PNA, 80 but rather due to the lack of binding free energy. There are several ways in which the binding affinity of ligand can be improved.…”

Single-Stranded γPNAs for In Vivo Site-Specific Genome Editing via Watson-Crick Recognition

“…Rapireddy et al have demonstrated that incorporation of an appropriate chiral substituent at the position in the PNA backbone induces right-handed helical pre-organization in the γPNA oligomers, enabling them to bind to double-stranded DNA targets via strand invasion with substantially increased affinity relative to standard PNAs [38]. In keeping with this, UV-melting analyses revealed that for both the MP γPNA1/DNA and MP γPNA2/DNA duplexes, the thermal stabilities were much higher than that of the PNA3/DNA (Supplemental Data, Table 1 ).…”

“…The gamma substitution creates chirality and provides helical pre-organization to the PNA oligomer, yielding substantially increased binding affinity to the target DNA [38]. The lack of editing by the mismatched γPNA, as well as the deep-sequencing results showing almost no effect at a partially homologous site, further demonstrates the specificity of binding by γPNAs.…”

“…Further the gels were stained with SYBR-Gold (Catalog #S11494, Invitrogen) for 10 min, washed twice with TBE buffer (1X) and imaged using a gel documentation system (BioDoc-It System) [37, 38]. The images were inverted using Adobe Photoshop 6.0 and the amounts of fraction-bound and-unbound DNA were quantified by using ImageJ software [37, 38]. …”