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.
