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 mu M, whereas the K-app for (dT)(20)-(dA)(20) DNA-DNA was found to be 45 +/- 2 mu M 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 mu M 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 mu M 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 mu M over a NaCl concentration range of 1.25-30 mM.