An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase–DNA complexes to surfaces

Abstract

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°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.

Fluorescent Structural DNA Nanoballs Functionalized with Phosphate-Linked Nucleotide Triphosphates

“…Gel extension assay results showing the successful incorporation of nucleotide triphosphates that were attached to DNA nanoballs by a mutant DNA polymerase 6. A positive control (+) containing natural dNTP’s, and negative controls (−) containing no dNTP’s are shown, along with reactions performed exclusively with nucleotide triphosphate functionalized nanoballs, using either a mutant polymerase or the wild-type (WT) version of Therminator DNA polymerase (Invitrogen).…”

“…We classify this method of single-molecule sequencing as Field-Switch Sequencing6, where a surface bound polymerase containing a primed single-stranded DNA template is provided with labeled nanoballs containing attached dNTP’s. In this method the nanoballs would be moved near the polymerase using an electric field, allowing the binding and eventual incorporation of the corresponding nucleotide.…”

Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase

“…To date, a large number of studies pursuing different biotechnological goals have documented in vitro evolution of DNA polymerases. Accordingly, DNA polymerases with expanded recognition of natural ( 8 – 11 ) or unnatural ( 12 – 19 ) substrates, increased processivity ( 20 – 22 ), altered fidelity ( 23 – 26 ), enhanced resistance to inhibitors ( 27 , 28 ) or thermostability ( 29 ) have been engineered by different methods. In most of these cases, researches have taken advantage of the thermostability or processivity traits of polymerases, allowing the use of exponential amplification or directed selection techniques to find desired polymerase variants ( 30 ).…”

Gene Regulatory Scenarios of Primary 1,25-Dihydroxyvitamin D3 Target Genes in a Human Myeloid Leukemia Cell Line

“…At a lower rate, the VDR is able to contact genomic DNA already in the absence of 1,25(OH) 2 D 3 and then preferentially forms complexes with co-repressor proteins [15,16] and chromatin modifying enzymes, such as histone deactylases (HDACs) [17,18]. However, the binding of 1,25(OH) 2 D 3 to VDR’s ligand-binding domain induces a conformational change to the latter, so that VDR changes its interaction partners, a few of which have chromatin modifying activity like histone acetylation [19].…”