A DNA polymerase with increased reactivity for ribonucleotides and C5-modified deoxyribonucleotides.

摘要: DNA polymerases find broad applications in molecular biology techniques (e.g. , polymerase chain reaction, genome sequencing, or diagnostic methods). Recently, have increasingly been employed to synthesize highly functionalized and thereby show great promise for future ranging from new sequencing technologies diagnostics, SELEX, DNA-based nanotechnology. Moreover, it was shown that nucleotide analogues with a simplified sugar backbone such as threosyl glyceryl surrogates are polymerized by polymerases. Interestingly, these reports sequence family B superior other polymerizing modified nucleotides. The A485L mutant of Thermococcus species 98N termed Therminator (Therminator pol) widely applied due its ability accept several unnatural nucleoside triphosphate substrates some extent. However, the observed low efficiency enzyme processing often still prevents further applications. Directed evolution shows improving performance Here we present generation pol variant broadened substrate scope. identified screen library generated error-prone PCR. We is able polymerize well nucleobase-modified nucleotides greater extent than parental enzyme. Nucleic acid classified either RNA their utilize 2’-deoxyribonucleotides (dNTPs) ribonucleotides (rNTPs) substrates. share common mechanism polynucleotide synthesis leads theory ancestor enzymes. All require primer initiate synthesis. Usually this short bound canonical template sequence. there few exceptions like terminal transferases use protein-primed initiation mechanism. dependent on promoter start de novo downstream at transcription start. vitro T7 polymerase) purines whereas pyrimidines poorly processed; limits space available method. 12] Thus, template-dependent could overcome bottleneck. In last years, studies focused catalyze promoter-independent RNA. Although variants significantly increased single ribonucleotide incorporation efficiencies were identified, mostly abort after ribonucleotides. same holds true tested (see Figure S1 Supporting Information). Because has inherent activity, became interested increasing ability. For construction PCR conducted introduce random mutations into gene. Mutants expressed 96-well plates using synthetic codon-optimized gene non-codon-optimized approach failed. After heat denaturing host proteins, lysates directly primer-extension screening assay identify rNTP-active incorporate multiple recently described trials SYBRgreen I failed detect product formation solution, performed employing chip-based oligonucleotide addressed assay. Herein, 96 primerextension reactions parallel an immobilized (20 nt)/template (90 nt) complex rNTPs By partial substitution UTP biotinylated dUTP analogue, products case any activity (Figure 1). Subsequent incubation slide surface streptavidin-Alexa Fluor 546 conjugate results spatially resolved fluorescent signal binding reaction products. Thereby level fluorescence correlates activity. 512 be derived more 1700 screened mutants (examples S2). evaluation extension solution (data not shown), three (M1, M2, M3) enhanced obtained. Subsequently, purified thoroughly characterized. First 90-nt-long template. Reactions comparing polymerase, mutants. It turned out M3 longest 2). This extend nt)/DNA consecutive 50 ribonucleotides, thus generating >70 nt DNA–RNA product. contrast, [a] N. Staiger, Prof. Dr. A. Marx Department Chemistry Konstanz Research School Chemical Biology University Universit tsstrasse 10, 78457 (Germany) Fax: (+49)7531-88-5140 E-mail : andreas.marx@uni-konstanz.de information article WWW under http://dx.doi.org/10.1002/cbic.201000384.