Progress toward Plug-and-Play Polymer Strings for Optical Tweezers Experiments: Concatenation of DNA Using Streptavidin Linkers

Nimisha Mohandas; Lisa M Kent; Allan Raudsepp; Geoffrey B Jameson; Martin A K Williams

doi:10.1021/acsomega.2c00198

Progress toward Plug-and-Play Polymer Strings for Optical Tweezers Experiments: Concatenation of DNA Using Streptavidin Linkers

ACS Omega. 2022 Feb 10;7(7):6427-6435. doi: 10.1021/acsomega.2c00198. eCollection 2022 Feb 22.

Authors

Nimisha Mohandas¹, Lisa M Kent¹, Allan Raudsepp¹, Geoffrey B Jameson^{1

2

3}, Martin A K Williams^{1

2

3}

Affiliations

¹ School of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
² MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington 6012, New Zealand.
³ Riddet Institute, Massey University, Palmerston North 4442, New Zealand.

Abstract

Streptavidin is a tetrameric protein that is renowned for its strong binding to biotin. The robustness and strength of this noncovalent coupling has led to multitudinous applications of the pairing. Within the streptavidin tetramer, each protein monomer has the potential to specifically bind one biotin-bearing moiety. Herein, by separating various streptavidin species that have had differing numbers of their four potential binding sites blocked, several different types of "linking hub" were obtained, each with a different valency. The identification of these species and the study of the plugging process used to block sites during their preparation were carried out using capillary electrophoresis. Subsequently, a specific species, namely, a trans-divalent linker, in which the two open biotin-binding pockets are approximately opposite one another, was used to concatenate two ∼5 kb pieces of biotin-terminated double-stranded DNA. Following the incubation of this DNA with the prepared linker, a fraction of ∼10 kb strings was identified using gel electrophoresis. Finally, these concatenated DNA strings were stretched in an optical tweezer experiment, demonstrating the potential of the methodology for coupling and extending molecules for use in single-molecule biophysical experiments.