Heidi R. Culver; Jasmine Sinha; Tania R. Prieto; Christopher J. Calo; Benjamin D. Fairbanks; Christopher N. Bowman Biomacromolecules, 2020, 21(10), 4205-4211 https://doi.org/10.1021/acs.biomac.0c00996
Abstract
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 future implementation of CNAs in applications that rely on sequence specific interactions. Microscale thermophoresis (MST) provided a convenient platform to rapidly and systematically test the effects of several factors (i.e., sequence, length, and salt concentration) on the CNA-DNA dissociation constant (Kapp). 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. Specifically, the Kapp of CNA-DNA hybrids in 65 vol% DMSO with 10 mM sodium chloride (NaCl) was 0.74 0.1 μM, while the Kapp for (dT)20-(dA)20 DNA-DNA was measured 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 Kapp 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 Kapp decreased from 5.6 0.8 to 0.27 0.1 μM as CNA average length increased from 7 to 21 bases. However, unlike DNA-DNA duplexes, which are largely unstable at low salt concentrations, CNA-DNA stability does not depend on salt concentration, with Kapp remaining consistent between 1.0 – 1.9 μM over a NaCl concentration range of 1.25 to 30 mM.