An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase-DNA complexes to surfaces.
Single molecule analysis of individual enzymes can require oriented immobilization of the subject molecules on a detection surface. As part of a technology development project for single molecule DNA sequencing, we faced the multiple challenges of immobilizing both a DNA polymerase and its DNA template together in an active, stable complex capable of highly processive DNA synthesis on a nonstick surface. Here, we report the genetic modification of the archaeal DNA polymerase 9 degrees N in which two biotinylated peptide 'legs' are inserted at positions flanking the DNA-binding cleft. Streptavidin binding on either side of the cleft both traps the DNA template in the polymerase and orients the complex on a biotinylated surface. We present evidence that purified polymerase-DNA-streptavidin complexes are active both in solution and immobilized on a surface. Processivity is improved from <20 nt in the unmodified polymerase to several thousand nucleotides in the engineered complexes. High-molecular weight DNA synthesized by immobilized complexes is observed moving above the surface even as it remains tethered to the polymerase. Pre-formed polymerase-DNA-streptavidin complexes can be stored frozen and subsequently thawed without dissociation or loss of activity, making them convenient for use in single molecule analysis.
Williams
John G K
JG
Advanced Research & Development, LI-COR Biosciences, Lincoln, NE, USA. john.williams@licor.com
Steffens
David L
DL
Anderson
Jon P
JP
Urlacher
Teresa M
TM
Lamb
Donald T
DT
Grone
Daniel L
DL
Egelhoff
Jolene C
JC
D019921:Biotinylation; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D005615:Freezing; D007700:Kinetics; D015202:Protein Engineering; D017422:Sequence Analysis, DNA; D019809:Streptavidin / Q000737:chemistry*; D013696:Temperature; D013698:Templates, Genetic
D004247:DNA; D019809:Streptavidin; D004259:DNA-Directed DNA Polymerase