(188s) Thermodynamic Characterization of Click Nucleic Acid-DNA Binding for Biosensing

Nearly 3% of the human genome is composed of tandem repeat sequences. These sequences are commonly polymorphic and changes in the length of tandem repeat sequences are often associated with disease. Currently, detection of tandem repeat sequences is achieved using either DNA or peptide nucleic acid (PNA) probes. DNA and PNA probes are made via solid-phase synthesis, in which each nucleobase addition requires four steps, making the overall process relatively inefficient. Recently, a new approach to producing synthetic nucleic acids was developed using click chemistry to efficiently polymerize repeat sequences. These “click” nucleic acids (CNAs) have the same 6-atom spacing as native DNA and PNA. In the current work, we characterize the binding properties of CNA probes to DNA oligonucleotides and investigate how binding is affected by ionic strength and organic solvent content.

Homopolymer CNA probes (e.g., poly(T)) were synthesized via a photoinitiated thiol-ene reaction. The molecular weights of the resultant CNA probes were determined by gel permeation chromatography. CNA-DNA binding specificity and affinity were first characterized using polyacrylamide gel electrophoresis (PAGE). PAGE analysis suggested that the dissociation constant (K_D) for poly(T) CNA and A₁₀ DNA was around 70 µM in a buffer containing 50% DMSO and only 2 mM Na⁺. No binding was observed for a complete mismatch sequence (T₁₀ DNA) or a single base-pair mismatch (A₅GA₄). Microscale thermophoresis (MST) was used to further study the effects of DMSO content and ionic strength on CNA-DNA interactions. CNA-DNA binding was observed in solutions containing up to 95% DMSO and less than 1 mM Na⁺. Given the unique binding properties, reduced cost, and improved efficiency of click-chemistry synthesis over solid-phase synthesis, CNA probes are expected to serve as useful tools for applications including biosensing of tandem repeat DNA or RNA sequences.