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Updated: May 9, 2026

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
Published on: March 31, 2010
Modeling stopped-flow data for nucleic acid duplex formation reactions: the importance of off-path intermediates
Jacqueline R Sikora1, Brittany Rauzan, Rachel Stegemann
1Department of Chemistry, Allegheny College, 520 North Main Street, Meadville, Pennsylvania 16335, USA.
Abstract:
Evidence for unexpected off-path intermediates to DNA duplex formation is presented. These off-path intermediates are shown to involve unimolecular and, in one case, bimolecular structure in one of the single strands of complementary DNA. Three models are developed to account for the observed single-stranded structures that are formed in parallel with duplex formation. These models are applied to the analysis of stopped-flow data for eight different nonself-complementary duplex formation reactions in order to extract the elementary rate constant for formation of the duplex from the complementary random coil single-stranded DNA. The free energy of activation (at 25 °C) for the denaturation of each duplex is calculated from these data and is shown to have a linear correlation to the overall standard free energy for duplex formation (also at 25 °C). Duplexes that contain mismatches obey a parallel linear free-energy (LFE) relationship with a y-intercept that is greater than that of duplexes without mismatches. Slopes near unity for the LFE relationships indicate that all duplexes go through an early, unstructured transition state.
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