Click nucleic acids (CNAs) are a new, low-cost class of xeno nucleic acid (XNA) oligonucleotides synthesized by an efficient and scalable thiol-ene polymerization. In this work, a thorough characterization of oligo(thymine) CNA-oligo(adenine) DNA ((dA)₂₀) hybridization was performed to guide the future implementation of CNAs in applications that rely on sequence-specific interactions. Microscale thermophoresis provided a convenient platform to rapidly and systematically investigate the effects of several factors (i.e., sequence, length, and salt concentration) on the CNA-DNA dissociation constant (K_app). Because CNAs have limited water solubility, all studies were performed in aqueous-DMSO mixtures. CNA-DNA hybrids between oligo(thymine) CNA (average length of 16 bases) and (dA)₂₀ DNA have good stability despite the high organic content, a favorable attribute for many emerging applications of XNAs. In particular, the K_app of CNA-DNA hybrids in 65 vol % DMSO with 10 mM sodium chloride (NaCl) was 0.74 ± 0.1 μM, whereas the K_app for (dT)₂₀-(dA)₂₀ DNA-DNA was found to be 45 ± 2 μM in a buffer without DMSO but at the same NaCl concentration. CNA hybridized with DNA following Watson-Crick base pairing with excellent sequence specificity, discriminating even a single-base-pair mismatch, with K_app values of 0.74 ± 0.1 and 3.7 ± 0.6 μM for complementary and single-base-pair mismatch sequences, respectively. As with dsDNA, increasing CNA length led to more stable hybrids as a result of increased base pairing, where K_app decreased from 5.6 ± 0.8 to 0.27 ± 0.1 μM as the CNA average length increased from 7 to 21 bases. However, unlike DNA-DNA duplexes, which are largely unstable at low salt concentrations, the CNA-DNA stability does not depend on salt concentration, with K_app remaining consistent between 1.0 and1.9 μM over a NaCl concentration range of 1.25-30 mM.

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