DNA Balance for Native Characterization of Chemically Modified DNA

Chemical modification is a powerful approach to expand the chemical diversity and functionality of natural DNA. However, when chemically modified oligonucleotides are employed in DNA-based reactions or structures, it becomes quite difficult to predict, understand, and control their kinetics and thermodynamics. To address this challenge, we introduce a rationally designed DNA balance capable of measuring critical thermodynamic and kinetic properties of chemically modified DNA in their native environment. Our DNA balance is operated using the principle of toehold-exchange, where a panel of weight probes were designed by tuning the lengths of forward and reverse toeholds. Once placed on the DNA balance, the chemical modification will be interrogated using the weight probes to determine changes in both Gibbs free energy and hybridization rate constant. Using cyclic-azobenzene (cAB)-modified DNA as a model system, we demonstrated that our DNA balance could not only measure stable chemical modifications, but also solve more challenging issues where unstable chemical modifications and transient isomerization reactions were involved. We anticipate that our DNA balance will find wide uses for measuring important thermodynamic and kinetic parameters for DNA carrying various chemical modifications, as well as for probing transient chemical changes in DNA.

Automated summary

Chemical modification is a powerful method to expand the diversity and functionality of natural DNA, but predicting and controlling chemically modified DNA in reactions is challenging. A DNA balance has been introduced to measure critical properties of chemically modified DNA, showing the ability to measure stable modifications as well as more challenging issues.