Synthesis, incorporation and photophysical characterization of base-modified fluorescent peptide nucleic acid analogues

Abstract

Fluorescent nucleoside analogue probes incorporated into oligonucleotides have provided effective biophysical systems to study nucleic acid structure and function and in devising nucleic acid-based diagnostic tools. However, due to low fluorescence efficiency exhibited by the majority fluorescent nucleoside analogues within oligonucleotides and inherent instability of oligonucleotides in nuclease environment, the utility of nucleoside-modified oligonucleotides has been limited to in vitro systems only. In this regard, we envision that a nucleic acid mimic, peptide nucleic acid (PNA), which is resistant to nucleases and base-pairs strongly with complementary DNA and RNA oligonucleotides, would be a suitable candidate for labelling with fluorescent nucleobase analogue. Here, we report the synthesis and incorporation of fluorescent 5-benzofuran- and 5-benzothiophene-conjugated uracil PNA base monomers into PNA sequences. The base analogues incorporated into PNA oligomers and hybridized to complementary DNA oligonucleotides have marginal impact on the duplex stability. Furthermore, 5-benzofuran-conjugated uracil is highly sensitive to changes in its neighbouring base environment. Importantly, it displays significant enhancement in fluorescence intensity upon hybridization with complementary oligonucleotide, a property rarely exhibited by the majority of fluorophores. The straight forward synthesis, amicability to solid-phase PNA oligomer synthesis, structurally non-perturbing nature and sensitivity to changes in its microenvironment highlight the potential of benzofuran-conjugated pyrimidine PNA base analogue as an efficient fluorescent probe for nucleic acid diagnosis.