Single-molecule force spectroscopy can precisely probe the biomechanical interactions of proteins that unwind duplex DNA and bind to and wrap around single-stranded (ss)DNA. Yet assembly of the required substrates, which often contain a ssDNA segment embedded within a larger double-stranded (ds)DNA construct, can be time-consuming and inefficient, particularly when using a standard three-way hybridization protocol. In this chapter, we detail how to construct a variety of force-activated DNA substrates more efficiently. To do so, we engineered a dsDNA molecule with a designed sequence of specified GC content positioned between two enzymatically induced, site-specific nicks. Partially pulling this substrate into the overstretching transition of DNA (~65 pN) using an optical trap led to controlled dissociation of the ssDNA segment delineated by the two nicks. Here, we describe protocols for generating ssDNA of up to 1000 nucleotides as well as more complex structures, such as a 120-base-pair DNA hairpin positioned next to a 33-nucleotide ssDNA segment. The utility of the hairpin substrate was demonstrated by measuring the motion of E. coli. RecQ, a 3'-to-5' DNA helicase.
BT - Methods in Biology DA - 2022-12 DO - 10.1007/978-1-0716-2229-2_10 N2 -Single-molecule force spectroscopy can precisely probe the biomechanical interactions of proteins that unwind duplex DNA and bind to and wrap around single-stranded (ss)DNA. Yet assembly of the required substrates, which often contain a ssDNA segment embedded within a larger double-stranded (ds)DNA construct, can be time-consuming and inefficient, particularly when using a standard three-way hybridization protocol. In this chapter, we detail how to construct a variety of force-activated DNA substrates more efficiently. To do so, we engineered a dsDNA molecule with a designed sequence of specified GC content positioned between two enzymatically induced, site-specific nicks. Partially pulling this substrate into the overstretching transition of DNA (~65 pN) using an optical trap led to controlled dissociation of the ssDNA segment delineated by the two nicks. Here, we describe protocols for generating ssDNA of up to 1000 nucleotides as well as more complex structures, such as a 120-base-pair DNA hairpin positioned next to a 33-nucleotide ssDNA segment. The utility of the hairpin substrate was demonstrated by measuring the motion of E. coli. RecQ, a 3'-to-5' DNA helicase.
PB - Springer US PY - 2022 SN - 9781071622285/1064-3745, 1940-6029 SP - 273 EP - 312 T2 - Methods in Biology TI - Force-Activated DNA Substrates for In Situ Generation of ssDNA and Designed ssDNA/dsDNA Structures in an Optical-Trapping Assay ER -