Abstract
The switchSENSE technology is a recent approach based on surface sensor chips for the analysis of interactions of macromolecules. The technology relies on electro-switchable DNA nanolevers tethered at one end on a gold surface via a sulfur linker and labeled with a Cy3 dye on the other end. The switchSENSE approach is effective in the determination of a large panel of biophysical parameters such as binding kinetics, dissociation constant, hydrodynamic radius, or melting temperature. In addition, it can also give access to some enzymatic data such as nuclease or polymerase activity. Here we describe a DNA polymerase assay that allows retrieving, in a single experimental set, association and dissociation rates, as well as the catalytic rate of the enzyme.
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References
Rich RL, Myszka DG (2011) Survey of the 2009 commercial optical biosensor literature. J Mol Recognit 24:892–914
Knezevic J, Langer A, Hampel PA et al (2012) Quantitation of affinity, avidity, and binding kinetics of protein analytes with a dynamically switchable biosurface. J Am Chem Soc 134:15225–15228
Langer A, Hampel PA, Kaiser W et al (2013) Protein analysis by time-resolved measurements with an electro-switchable DNA chip. Nat Commun 4:2099
Alt A, Dang HQ, Wells OS et al (2017) Specialized interfaces of Smc5/6 control hinge stability and DNA association. Nat Commun 8:14011
Blocquel D, Li S, Wei N et al (2017) Alternative stable conformation capable of protein misinteraction links tRNA synthetase to peripheral neuropathy. Nucleic Acids Res 45:8091–8104
Denichenko P, Mogilevsky M, Clery A et al (2019) Specific inhibition of splicing factor activity by decoy RNA oligonucleotides. Nat Commun 10:1590
Nemoz C, Ropars V, Frit P et al (2018) XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining. Nat Struct Mol Biol 25:971–980
Ploschik D, Ronicke F, Beike H et al (2018) DNA primer extension with cyclopropenylated 7-Deaza-2′-deoxyadenosine and efficient bioorthogonal labeling in vitro and in living cells. Chembiochem 19:1949–1953
Rueda FO, Bista M, Newton MD et al (2017) Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease. Nat Commun 8:1610
Webster MW, Chen YH, Stowell JAW et al (2018) mRNA deadenylation is coupled to translation rates by the differential activities of Ccr4-not nucleases. Mol Cell 70:1089–1100.e8
Webster MW, Stowell JA, Passmore LA (2019) RNA-binding proteins distinguish between similar sequence motifs to promote targeted deadenylation by Ccr4-Not. elife 8:e40670
Kroener F, Heerwig A, Kaiser W et al (2017) Electrical actuation of a DNA origami nanolever on an electrode. J Am Chem Soc 139:16510–16513
Clery A, Sohier TJM, Welte T et al (2017) switchSENSE: a new technology to study protein-RNA interactions. Methods 118-119:137–145
Langer A, Schraml M, Strasser R et al (2015) Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces. Sci Rep 5:12066
Freisz S, Bec G, Radi M et al (2010) Crystal structure of HIV-1 reverse transcriptase bound to a non-nucleoside inhibitor with a novel mechanism of action. Angew Chem Int Ed Engl 49:1805–1808
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Bec, G., Ennifar, E. (2021). switchSENSE Technology for Analysis of DNA Polymerase Kinetics. In: Poterszman, A. (eds) Multiprotein Complexes. Methods in Molecular Biology, vol 2247. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1126-5_8
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DOI: https://doi.org/10.1007/978-1-0716-1126-5_8
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