Abstract
DNA carries genetic information in its sequence of bases. Synthetic oligonucleotides that can sequence-specifically recognize a target gene sequence are a useful tool for regulating gene expression or detecting target genes. Among the many synthetic oligonucleotides, tail-clamp peptide nucleic acid (TC-PNA) offers advantages since it has two homopyrimidine PNA strands connected via a flexible ethylene glycol-type linker that can recognize complementary homopurine sequences via Watson-Crick and Hoogsteen base pairings and form thermally-stable PNA/PNA/DNA triplex structures. Here, we synthesized a series of TC-PNAs that can possess different lengths of azobenzene-containing linkers and studied their binding behaviours to homopurine single-stranded DNA. Introduction of azobenzene at the N-terminus amine of PNA increased the thermal stability of PNA-DNA duplexes. Further extension of the homopyrimidine PNA strand at the N-terminus of PNA-AZO further increased the binding stability of the PNA/DNA/PNA triplex to the target homopurine sequence; however, it induced TC-PNA/DNA/TC-PNA complex formation. Among these TC-PNAs, 9W5H-C4-AZO consisting of nine Watson-Crick bases and five Hoogsteen bases tethered with a beta-alanine conjugated azobenzene linker gave a stable 1:1 TC-PNA/ssDNA complex and exhibited good mismatch recognition. Our design for TC-PNA-AZO can be utilized for detecting homopurine sequences in various genes.
Highlights
IntroductionSequence-specific detection of target genes using oligonucleotides is a key technology for detecting pathogens and disease-related genes
Introduction of azobenzene at theN-terminus amine of Peptide nucleic acid (PNA) increased the thermal stability of PNA-DNA duplexes
Tail-clamp PNA (TC-PNA), which is composed of two homopyrimidine PNA strands connected via a linker molecule, 2-aminoethoxy2-ethoxy acetic acid (AEEA, Figure 1B), can form a stable complex with its target homopurine DNA
Summary
Sequence-specific detection of target genes using oligonucleotides is a key technology for detecting pathogens and disease-related genes. Various types of chemically modified nucleic acids have been developed to improve binding affinity and sequence specificity. Tail-clamp PNA (TC-PNA), which is composed of two homopyrimidine PNA strands connected via a linker molecule, 2-aminoethoxy2-ethoxy acetic acid (AEEA, Figure 1B), can form a stable complex with its target homopurine DNA sequence consisting partly of a duplex via Watson-Crick base pairing and partly of a triplex via both. Schematic image azobenzene amino acid linker (C3-, C4-, C5- and C6-AZO (n = 1, 2, 3 and 4)); (E) Schematic image of of triplex formation between ssDNA. Homopyrimidine bases azobenzene linker (Figure 1C,D, TC-PNA AZO) and different numbers of homopyrimidine. 1:1 complex with complementary homopurine ssDNA with homopurine high sequence binding affinity and This goodresult sequence specificity.
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