SBIR-STTR Award

The main objective of this SBIR Phase I application is to develop a generic approach for construction by methods of gene engineering hybrid DNA metabolizing enzymes with enhanced processivity. Particularly, a set of processive thermostable DNA polymerases, capable of extensive rapid replication, efficient recycling between substrate molecules and suitable for both LA-PCR and most challenging DNA sequencing applications will be developed. A number of plasmid constructs will be made and the encoding chimeric proteins will be expressed in E. coli, purified and assayed for enhanced processivity, i.e for capability to extend a primer for 1 kb or more prior to dissociating from a template, and for significantly higher affinity to a primer-template comparing to current commercial thermostable polymerases. Such improvement in basic enzyme properties will enhance both molecular biology research at universities and boost industrial genome research as well. The availability of a specialized enzyme for long PCR and advanced genomic sequencing will enhance diagnostic procedures for cancer, genetic defects and pathogens.

Thesaurus Terms:
DNA directed DNA polymerase, genetic manipulation, hybrid enzyme, method development, protein engineering, technology /technique development chemical model, molecular dynamics, plasmid polymerase chain reaction

Thermostable DNA modifying enzymes play a crucial role in current methods for DNA amplification and sequencing. Major improvements in these methods have been made in recent years, largely as a result of a marked increase in our understanding of how these enzymes and accessory proteins function. There is, however, an untapped potential in the use of genetically modified DNA polymerases, reverse transcriptases, ligases, restriction enzymes and DNA binding proteins, which may provide significant advantages over their natural counterparts used today. The goal of the proposed project is to develop such modified enzymes and proteins that would have an immediate impact on improving efficiency and accuracy of DNA amplification applications. The work will focus primarily on the optimization of hybrid DNA polymerases, which are by far the leading enzymes in the research reagent and DNA diagnostics markets. A distinguishing feature of the strategy we are pursuing is the incorporation into target enzymes of protein domains that are not naturally associated with their targets, instead of simple domain swapping of homologous proteins or random mutagenesis. Fidelity Systems has successfully pursued this strategy by recognizing the role of helixhairpin- helix (HhH) protein domains in sequence non-specific interactions with DNA. Under Phase I, a number of expression vectors were constructed that permitted the fusing of multiple HhH domains of the unique DNA topoisomerase V (isolated from Methanopyrus kandleri) with different DNA polymerases. Critical functional features (processivity, inhibitor resistance, specificity, thermostability) of the expressed chimeras have already been validated in DNA sequencing and PCR. Under Phase II funding, we propose to implement our hybrid strategy more broadly, characterize and prioritize not only the thermophilic DNA processing enzymes but important mesophilic targets as well, and prove the universal applicability of our strategy. The crystal structure of Topo V with HhH domains that has been solved in Phase I provides a rock solid platform for further optimization of chimeras' feature-rich phenotypes for specific DNA applications. The proposed Phase II work will complement and enhance the value of internally funded discovery and characterization of new proteins at Fidelity Systems, and will help promote the transfer of our technology to non-DNA processing enzymes