<br/>DNA encodes the primary structure of proteins in a cell, so that single-nucleotide polymorphisms (SNPs) or chemical modifications such as oxidation or methylation, can lead to changes in protein expression with links to human diseases. DNA mismatches and chemical modifications affect Watson-Crick base pairing interactions changing the stability of the DNA double helix. Our goal is to develop single-molecule fluorescence methods to examine the fundamental thermodynamics and kinetics of DNA hybridization in the presence of DNA damage, modification, or transcription errors. In this work, we immobilize a short, unlabeled probe strand to a glass coverslip that exhibits minimal non-specific adsorption of DNA that would interfere with the analysis. We then expose this surface to solution-phase fluorescently-labeled target ssDNA that is either fully-complementary with the immobilized probe or contains a single-mismatched or chemically-modified base. Using total-internal-reflection-fluorescence (TIRF) microscopy, we detect hybridization of individual target molecules at the glass surface and measure the hybridization kinetics and equilibria by tracking individual molecules in time-resolved images. Previous studies of SNP dissociation in free solution suggested that changes in association constants compared to fully complementary strands were due solely to changes in k<sub>off</sub>.  Single-molecule kinetics at a surface reveal that changes in hybridization equilibria result from changes in both association and dissociation rate constants. Furthermore, by varying the temperature at which the reactions are carried out, contributions from enthalpy and entropy terms to the stability of hybridization and the transition state energies can be resolved. The location of a base mismatch or modification along a strand significantly influences the stability of the resulting duplex.<br/>

Single-Molecule fluorescence imaging of mismatched and modified oligonucleotide hybridization

253rd National Meeting (2017)

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