Design, synthesis and applications of oligonucleotide-probe and -antibody conjugates leveraging bioorthogonal chemistry

Abstract

Nucleic acids adopt diverse secondary and tertiary structures, which play critical roles in various biological processes beyond encoding genetic information. It is essential to modify nucleic acids with biophysical probes and therapeutic cargoes to harness their diagnostic and therapeutic potentials. Traditional solid-phase synthesis (SPS) and enzymatic methods, while effective for incorporating modifications into oligonucleotides (ONs), often fail for bulky or sensitive modifications. Hence, post-synthetic labeling strategy offers a versatile alternative, leveraging biorthogonal click chemistry. In this approach, a small reactive handle is introduced first into the ON via SPS or enzymatic incorporation followed by conjugation with a cognate reactive partner containing the tag of interest. Terminal nucleotidyl transferases like TUTase and TdT, are known for incorporating modified nucleotide analogs into the 3’-end of RNA and DNA, respectively. However, the inherent processivity of these enzymes often leads to multiple incorporations, which can be challenging for controlled and precise labeling required for the development of diagnostic and therapeutic tools. Hence, in the first study, we explored strategies to control the activity of SpCID1, a TUTase, by balancing the concentration of essential metal ion cofactors (Mg²⁺ and Mn²⁺) and borate which forms chelate with the cis diols of ribose, to restrict the processivity of the enzyme. In this context, C5-modified fluorescent UTP probes that are useful in analyzing non-canonical nucleic acid structures and azide- and alkyne-modified UTP analogs that are compatible for chemoenzymatic functionalization were used as study systems. Appropriate mix of Mg2+ ions, Tris buffer strength and borate concentration afforded largely single-nucleotide incorporated RNA products with both clickable and fluorescent UTPs. Further, the clickable handles were post-synthetically functionalized with fluorophores via CuAAC and SPAAC reactions, which broadens the scope of TUTase in site-specifically installing modifications onto RNA for various applications. In the second study, this approach was extended to modify G-quadruplex (GQ) forming DNA aptamers, targeting c-Met receptors on cancer cells. Using TdT enzyme and SPS, we incorporated alkyne modification into the aptamers and functionalized them with affinity tags like biotin-azide and fluorescent probe (TAMRA-azide) post-synthetically. The aptamer-probe conjugates retained their native structure, and subsequent investigations demonstrated that they bound efficiently to c-Met in vitro. Moreover, the modified aptamers exhibited comparable efficacy to the native aptamer in binding to c-Met overexpressed A549 cells, highlighting their potential as a system for specifically targeting cancer cells. In the third study, we developed antibody-ON conjugates to target HER2+ over-expressed cancer cell lines. For that, TdT and SPS were used to synthesize alkyne-modified siRNA targeting the Plk-1 gene, a key regulator for mitotic progression. This was then conjugated with azide-modified Trastuzumab using click chemistry to target HER2+ cancer cells. Taken together, our labeling techniques broadens the scope for site-specific ON modification, enabling the development of versatile nucleic acid-based probes and conjugates for targeted diagnostic and therapeutic applications in cancer and other diseased models.