Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection

Abstract

Any system, natural or human-made, is better understood if we analyze both its history and its structure. Here we combine structural analyses with a "Reconstructed Evolutionary Adaptive Path" (REAP) analysis that used the evolutionary and functional history of DNA polymerases to replace amino acids to enable polymerases to accept a new class of triphosphate substrates, those having their 3′-OH ends blocked as a 3 0 -ONH 2 group (dNTP-ONH 2 ). Analogous to widely used 2′,3′-dideoxynucleoside triphosphates (ddNTPs), dNTP-ONH 2 s terminate primer extension. Unlike ddNTPs, however, primer extension can be resumed by cleaving an O-N bond to restore an -OH group to the 3′-end of the primer. REAP combined with crystallographic analyses identified 35 sites where replacements might improve the ability of Taq to accept dNTP-ONH 2 s. A library of 93 Taq variants, each having replacements at three or four of these sites, held eight variants having improved ability to accept dNTP-ONH 2 substrates. Two of these (A597T, L616A, F667Y, E745H, and E520G, K540I, L616A) performed notably well. The second variant incorporated both dNTP-ONH 2 sand ddNTPs faithfully and efficiently, supporting extension-cleavage-extension cycles applicable in parallel sequencing and in SNP detection through competition between reversible and irreversible terminators. Dissecting these results showed that one replacement (L616A), not previously identified, allows Taq to incorporate both reversible and irreversible terminators. Modeling showed how L616A might open space behind Phe-667, allowing it to move to accommodate the larger 3′-substituent. This work provides polymerases for DNA analyses and shows how evolutionary analyses help explore relationships between structure and function in proteins.DNA polymerases | evolutionary analysis | sequencing technology | single nucleotide polymorphisms | synthetic biology

Directed Evolution of Polymerases To Accept Nucleotides with Nonstandard Hydrogen Bond Patterns

“…Such sequence/ phylogenetic patterns can arise because of shifts in the selective constraints acting at individual sites throughout the evolutionary history of a gene family, and by extension, the precise biomolecular behaviors of the homologous proteins are not identical across the phylogeny. 25,27 A qualitative analysis is given in Table 1.…”

Engineered DNA Polymerases in Biotechnology

“…[64] A very recent approach to increase acceptance of 3'-OH modifications useful for sequencing-by-synthesis techniques is described by Benner and co-workers. [65] They have used "reconstructing evolutionary adaptive paths" (REAP) [66] and analysed the history of DNA polymerase evolution particularly between viral and nonviral DNA polymerases, because viral polymerases are empirically known to have a higher acceptance for modified nucleotides than their nonviral counterparts. [67] After construction and screening of a small combinatorial library of interesting sites (93 variants), they identified Taq DNA polymerase variants that accept the reversible terminators more efficiently than the wild-type.…”

Site-Directed Mutagenesis as Applied to Biocatalysts

“…REAP identified sites that may have changed during the separation of viral and non-viral polymerases. In one example, mutations of the residues identified by REAP analysis for the DNA polymerase of Thermus aquaticus yielded 8 mutants that showed a change in the substrate specificity for unnatural dNTP´s (Chen et al, 2010).…”

“…The findings are used to generate focused and functionally enriched enzyme libraries (Chen et al, 2010).…”