Krishna N. Ganesh

Peptide nucleic acids are neutral analogues of DNA with an aminoethyl glycine polyamide backbone, which renders a higher binding affinity to complementary DNA but reduces cell permeability due to increased hydrophobicity. We have conjugated aeg-PNA with the cationic amino acids lysine (lys), arginine (arg), and the dipeptides lys-lys and arg-arg at the N/C-terminus and introduced cationic S-aminomethylene (S-am) and S-methylene guanidine (S-mgd) side chains at Cα in 8-mer PNAs. The thermal stability of cationic PNA:DNA duplexes was enhanced due to electrostatic interactions between the cationic PNA substitutions and the anionic DNA in the range of 6–11 °C and in the following order: PNA 12-S-am2 >PNA 9-S-mgd >PNA 10-lys2 >PNA 5-S-mgd >PNA 4 S-am)∼PNA 8-arg>PNA 11-arg2, and the rest were < 3.0 °C. The side-chain-modified substitutions were generally better than the end-conjugated amino acids. The single-stranded C-terminus cationic PNAs showed 1.4–1.7-fold higher cell uptake than aeg-PNA in SH-SY5Y cells, in the following order: S-am2>S-mgd >arg2>lys> lys2>arg>S-am>aeg-PNA. FRET experiments of cationic PNA hybridized with fluorescently labeled DNA within cells exhibited increased cellular uptake of PNA:DNA duplexes, with the CF-PNA 12-S-am2 up by 12.7-fold, suggesting significantly improved cell uptake of cationic PNA:DNA duplexes. FRET imaging indicated the uptake of intact cationic PNA:DNA duplexes into the cells, suggesting their application potential for transporting DNA into cells.

Collagen peptide-PNA(GGG)₃ covalent conjugate with complementary DNA shows pH induced reversible switching from triplex–duplex supramolecular complex at neutral pH 7.2 to triplex:i-motif composite at acidic pH 4.5.

Nucleic acids (DNA and RNA) can form diverse secondary structures ranging from hairpins to duplex, triplex, G4-tetraplex and C4-i-motifs. Many of the DNA analogues designed as antisense oligonucleotides (ASO) are also adept at embracing such folded structures, although to different extents with altered stabilities. One such analogue, peptide nucleic acid (PNA), which is uncharged and achiral, forms hybrids with complementary DNA/RNA with greater stability and specificity than DNA:DNA/RNA hybrids. Like DNAs, these single-stranded PNAs can form PNA:DNA/RNA duplexes, PNA:DNA:PNA triplexes, PNA-G4 tetraplexes and PNA-C4-i-motifs. We have recently designed Janus-like bimodal PNAs endowed with two different nucleobase sequences on either side of a single aminoethylglycyl (aeg) PNA backbone and shown that these can simultaneously bind to two complementary DNA sequences from both faces of PNA. This leads to the formation of supramolecular polyplexes such as double duplexes, triple duplexes and triplexes of double duplexes with appropriate complementary DNA/RNA. Herein, we demonstrate that Janus/bimodal PNA with a poly G-sequence on the triazole side of the PNA backbone and mixed bases on the t-amide side, templates the initial formation of a (PNA-G5)₄ tetraplex (triazole side), followed by the formation of a PNA:DNA duplex (t-amide side). Such a polyplex shows synergistic overall stabilisation compared to the isolated duplexes/quadruplex. The assembly of polyplexes with a shared backbone for duplexes and tetraplexes is programmable and may have potential applications in the self-assembly of nucleic acid nano- and origami structures. It is also shown that Janus PNAs enter the cells better than the standard aeg-PNA oligomers, and hence have implications for in vivo applications as well.

Peptide nucleic acids (PNAs) consist of an aminoethylglycine (aeg) backbone to which the nucleobases are linked through a tertiary amide group and bind to complementary DNA/RNA in a sequence-specific manner. The flexible aeg backbone has been the target for several chemical modifications of the PNA to improve its properties such as specificity, solubility, etc. PNA monomers exhibit a mixture of two rotamers (Z/E) arising from the restricted rotation around the tertiary amide N-CO bond. We have recently demonstrated that achiral gemdimethyl substitution at the α, β, and γ sites on the aeg backbone induces exclusive Z (α-gdm)- or E-rotamer (β-gdm) selectivity at the monomer level. It is now shown that γ/β-gdm-PNA:DNA parallel duplexes are more stable than the analogous antiparallel duplexes, while γ/β-gdm-PNA:RNA antiparallel duplexes are more stable than parallel duplexes. Furthermore, the γ/β-gdm-PNA:RNA duplexes are more stable than the γ/β-gdm-PNA:DNA duplexes. These results with γ/β-gdm-PNA are the reverse of those previously seen with α-gdm-PNA oligomers that stabilized antiparallel α-gdm-PNA:DNA duplexes compared to α-gdm-PNA:RNA duplexes. The stability of antiparallel/parallel PNA:DNA/RNA duplexes is correlated with the preference for Z/E-rotamer selectivity in α/β-gdm-PNA monomers, with Z-rotamers (α-gdm) leading to antiparallel duplexes and E-rotamers (β/γ-gdm) leading to parallel duplexes. The results highlight the role and importance of Z- and E-rotamers in controlling the structural preferences of PNA:DNA/RNA duplexes.

The replacement of alpha(CH2) by NH in monomers of standard aeg PNA and its homologue beta-ala PNA leads to respective aza-PNA monomers (1 and 2) in which the N alpha H can form either an 8-membered H-bonded ring with folding of the backbone (DMSO and water) or a 5-membered N alpha H-alpha CO (water) to stabilize E-type rotamers. Such aza-PNA oligomers with exclusive E rotamers and intraresidue backbone H-bonding can modulate its DNA/RNA binding and assembling properties.