Environment-sensitive nucleotide analogs serve as good substrates and probes to study the activity of nucleic acid processing enzymes

Abstract

Enzymatic functionalization of nucleic acids with biophysical tools and probes is used in many biotechnological applications as well as in fundamental research of nucleic acid structure and function. This process mainly depends on native and engineered DNA and RNA polymerases, transferases and their intrinsic capability to incorporate the modified nucleotides into oligonucleotides. However, the mechanism by which polymerases accept unnatural nucleotides as substrates and incorporate them into the nascent nucleic acid chains is not yet fully understood, so prediction of substrate tolerance is often not possible. Here, we demonstrate the utility of environment-sensitive heterocycle-modified fluorescent nucleotide substrates in probing the incorporation mechanism of template-dependent and template-independent DNA polymerases in real-time and at the atomic level. A novel platform is envisaged, capitalizing on these fluorescent nucleotides and X-ray crystallography and NMR spectroscopy to explore enzymatic incorporation. In the first study, it is observed that the nucleotide analogs containing a selenophene, benzofuran and benzothiophene moiety at the C5 position of 2′-deoxyuridine are incorporated into oligonucleotides (ONs) with varying efficiency, which depends on the size of the heterocycle modification and the DNA polymerase sequence family used. Notably, benzofuran-modified nucleotide (BFdUTP) serves as an excellent reporter by providing real time fluorescence readouts to monitor enzyme activity and estimate the binding events in the catalytic cycle. Further, a direct comparison of the incorporation profiles, fluorescence data, and crystal structure of a ternary complex of KlenTaq DNA polymerase with BFdUTP poised for catalysis provides a detailed understanding of the mechanism of incorporation of heterocycle-modified nucleotides. In the second study, the incorporation of 5-fluoro benzofuran-modified 2′-dUTP, which functions as a responsive fluorescent and 19F NMR probe by DNA polymerases, was studied. It shows differences in the incorporation efficiency for KlenTaq and KOD DNA polymerases. Importantly, it opens up a channel to monitor the binding events and incorporation by fluorescence and 19F NMR. Lastly, we have studied the incorporation of these modified nucleotides by TdT (terminal deoxynucleotidyl transferase enzyme), which belongs to the family X DNA polymerases. This enzyme has a unique ability to incorporate nucleotides at the 3'-end of a single-stranded DNA (ssDNA) in a template independent manner. Herein, our modified nucleotide analogs demonstrate high substrate efficacy for enzymatic incorporation by TdT as compared to thymidine. Subsequently, the utility of BFdUTP is expanded, wherein it assists in providing insights into the catalytic cycle and can facilitate the development of discovery platforms to identify TdT inhibitors.