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Thermodynamics of single molecule force-induced DNA-ligand interactions

Nano-Science Seminar by Mark C. Williams, Northeastern University, Boston.

Abstract
When single DNA molecules are stretched, mechanical work is performed that can induce structural and thermodynamic changes that alter DNA interactions in a measurable manner. Using an optical tweezers instrument, we obtain the force required to extend the DNA molecule and to convert double-stranded DNA into single-stranded DNA in the presence of binding ligands, which we refer to as force-induced melting. Depending on the nature of the ligand binding mode, several different effects are observed. For example, intercalators such as ethidium increase the contour length of double-stranded DNA as well as the melting force, allowing for measurement of force-dependent and zero force intercalation affinity. In contrast, single-stranded DNA binding proteins, such as gp32 from Bacteriophage T4 and gp2.5 from Bacteriophage T7, strongly destabilize double-stranded DNA, resulting in a decrease in melting force. The observed decrease in melting force as a function of protein concentration allows us to quantify the protein binding free energy, providing insights into the function of these proteins during viral replication. Finally, DNA stretching allows us to characterize the mechanism by which retroviral nucleocapsid proteins facilitate rearrangements of nucleic acid secondary structure, which are required for reverse transcription and retroviral replication.