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.
Highlights
DNA is a semi-flexible polymer composed of deoxyribonucleotide triphosphates that are joined together by phosphodiester bonds
Methods of stretching and immobilizing DNA molecules have been explored extensively over last decade in an attempt to develop DNA templates that meet the following requirements: (1) the stretched DNA molecules should preserve the base stacking structure of unstretched DNA, allowing normal DNA–protein interactions; (2) the DNA should be immobilized in such way that it is firmly held to withstand the hydrodynamic flow while providing ample space for proteins to move freely along the DNA; (3) the DNA should remain stretched and immobilized at physiological pH and salt concentrations
Yin et al [24] measured the transcriptional velocity of E. coli RNA polymerase (RNAP) against an applied force on a DNA by directly monitoring the displacement of a DNA molecule held by an optical trap as it is transcribed by an immobilized RNAP (Fig. 2A)
Summary
DNA is a semi-flexible polymer composed of deoxyribonucleotide triphosphates (dNTPs) that are joined together by phosphodiester bonds. Flow can produce two kinds of forces that can stretch DNA molecules, namely, the viscous drag produced by the bulk flow surrounding the DNA, and the meniscus force created by an air–solvent interface moving along the DNA The latter method, more commonly known as ‘‘molecular combing’’ [16, 17], often produces highly overstretched DNA molecules in which the bases are unstacked into a flat parallel ladder, and will be discussed in more detail in section ‘‘Stretching by a moving interface.’’ There are many ways in which flow fields can be generated to stretch and immobilize DNA molecules. A novel method that we call protein-assisted DNA immobilization uses a flow field containing DNA-binding proteins to stretch and immobilize DNA molecules in a microfluidic device [19]. We review the aforementioned methods of DNA stretching and immobilization and how these DNA molecules are used to investigate interactions with proteins at the single-molecule level
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