Base-Functionalized Nucleotide Analogs: From Chemical Synthesis to Enzymatic Processing and Applications in Nucleic Acid Labeling

Abstract

The landscape of nucleic acid modification technologies is rapidly evolving with enzymatic and post-enzymatic labeling strategies emerging as very important tools for accessing functionalized oligonucleotides (ONs). These approaches rely heavily on enzyme promiscuity to accommodate and process modified nucleotide analogs, offering a robust and versatile alternative to conventional solid-phase synthesis (SPS). Various nucleic acid–processing enzymes, such as DNA and RNA polymerases, nucleotide transferases, and viral reverse transcriptases, play central roles in genome replication and maintenance in all living organisms. These processes utilize the cell’s natural machinery to incorporate native nucleotides into a growing ON sequence. Inspired by their precision and efficiency, such enzymatic systems have been adapted to incorporate unnatural, chemically modified nucleotide analogs, thereby expanding the functional repertoire of labeled nucleic acids. In particular, the ability of both native and engineered enzymes to incorporate modified nucleotides into ONs has found wide applications, not only in accessing functionalized ONs for diagnostic and therapeutic purposes, but also in new-generation sequencing technologies and selection protocols for identifying robust aptamers. Biochemical studies over the years have revealed that C5-modified pyrimidines and C7-modified purines are better tolerated by enzymes, enabling access to base-modified ONs equipped with fluorescent, EPR-, redox-, and NMR-active labels or probes for various applications.However, site-specific labeling of ONs is not straightforward and importantly, enzymes don’t effectively incorporate all types of modifications as they may not be accommodated well in their active site. These shortcomings led to the development of chemoenzymatic nucleic acid labeling technologies that harness the ability of enzymes to incorporate small chemo-selective reactive handles that are compatible for post-enzymatic chemical functionalization in chemo-selective manner. These approaches typically rely on chemo-selective reactions involving nucleophiles (e.g., amines and thiols) or bioorthogonal handles (e.g., azides and alkynes) with suitable reaction partners. Although, several of the nucleic acid processing enzymes accept and incorporate a variety of structurally diverse substrates, the structural basis by which the enzymes accommodate such modifications in the reaction site, and how the interplay between the enzyme and substrate dictate the catalytic efficiency of incorporation of modified nucleotides are not fully understood. Further, development of new and complementing chemoenzymatic labeling methods will greatly aid in incorporating multiple functional tags (e.g., probes, drug molecules) site-specifically onto ONs for advanced applications. In this context, the theme of this doctoral work is to expand the functionalized ON toolbox to understand enzyme-substrate (nucleotide) plasticity, design enzyme-compatible nucleotide probes, and develop versatile and novel post-synthetic ON modification strategies. The nucleotide analogs and nucleic acid functionalization strategies presented in this thesis provide valuable insights into how polymerases incorporate modified nucleotides into DNA oligomers. This effort will not only deepen our understanding of polymerase mechanisms but also aid in the rational design of nucleotide probes that are appropriate for enzymatic functionalization of ONs.