Stretching and immobilization of DNA for studies of protein–DNA interactions at the single-molecule level

Abstract

Single-molecule studies of the interactions of DNA and proteins are important in a variety of biological or biotechnology processes ranging from the protein's search for its DNA target site, DNA replication, transcription, or repair, and genome sequencing. A critical requirement for single-molecule studies is the stretching and immobilization of otherwise randomly coiled DNA molecules. Several methods for doing so have been developed over the last two decades, including the use of forces derived from light, magnetic and electric fields, and hydrodynamic flow. Here we review the immobilization and stretching mechanisms for several of these techniques along with examples of single-molecule DNA-protein interaction assays that can be performed with each of them.

Synthesis and application of streptavidin functionalized organosilica microparticles

“…Although SAs modified microspheres were often exploited in single molecule manipulation using optical or magnetic tweezers, [10][11][12][13][14] it was a time-consuming process to obtain particles carrying only one DNA molecule. 53 According to Poisson distribution, one can calculate the proportion of one DNA-one particle complexes by controlling the DNA-to-particle ratio, which depends on the size of the particles and the radius of gyration of (R g ) of the DNA.…”

“…8,9 Microparticles and nanoparticles, such as gold, magnetic, latex, polystyrene, and silica particles, are also used as carriers for single molecular manipulation with optical [10][11][12][13] or magnetic tweezers 14 in combination with microfluidics. These systems have been used in single-molecule DNA stretching 14,15 and behavior observation, [16][17][18] speed control and electrophoretic dynamics of DNA translocation through nanopore, 19,20 and DNA-protein interactions. [10][11][12][13][14]21,22 Majority of these experiments rely on attachment of DNA molecules onto carrier microparticles.…”

“…These systems have been used in single-molecule DNA stretching 14,15 and behavior observation, [16][17][18] speed control and electrophoretic dynamics of DNA translocation through nanopore, 19,20 and DNA-protein interactions. [10][11][12][13][14]21,22 Majority of these experiments rely on attachment of DNA molecules onto carrier microparticles. There are many approaches to attach DNA onto particles.…”

Bioanalytical separations using electric field gradient techniques

“…After creating DNA fragments, AC DEP was used to stretch fragments ranging in length from 3 to 60 kbp in the microdevice. For more information, a review article addressing different methods of stretching and immobilizing DNA was published by Kim et al [133]. The review investigated various means of manipulating DNA, including using light, magnets, electric fields, hydrodynamic flow, and DEP forces.…”

Physical manipulation of single-molecule DNA using microbead and its application to analysis of DNA–protein interaction

“…Because the form of DNA molecules always changes in solution by Brownian motion, a ''physical form control'' technique is required to measure the length of individual fluorescent-stained DNA and position of fluorescentlabeled proteins. DNA manipulation methods using a flow solution or electrostatic force are widely used [1,2]. Optical or magnetic trapping for a microbead is also a powerful technique in singlemolecule studies [1][2][3].…”