Thermostable Polymerase Variants for Nanopore Sequencing
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Solution Overview
Problem
Current DNA polymerases used in recombinant DNA technologies face challenges such as rapid DNA strand movement through nanopores, making sequencing difficult and prone to background noise, and lack improved properties like thermostability and modified nucleotide incorporation.
Innovation Solution
Modified DNA polymerases with specific amino acid substitutions identified through directed evolution experiments, such as at sites G12, K114, N194, A451, and D681, which enhance thermostability and sequencing performance by improving stability and kinetic characteristics.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DNA strand moves rapidly through nanopore at 1-5 bases per second, then sequencing throughput is high, but recording becomes difficult and prone to background noise
Solution Approach 1:
The patent modifies the polymerase enzyme's kinetic parameters through directed evolution to reduce its processivity and slow down DNA translocation speed through the nanopore. This parameter change allows the DNA strand to move slower (within detectable speed ranges), enabling accurate recording of each base while maintaining sequencing throughput through optimized enzyme characteristics.
2Reliability
If wild-type polymerase is used, then basic polymerization function is maintained, but thermostability and modified nucleotide incorporation are insufficient
Solution Approach 1:
The patent employs directed evolution to systematically change the polymerase enzyme's parameters, including thermostability, processivity, and substrate specificity. Through multiple rounds of mutation and selection, the enzyme acquires improved thermostability and enhanced ability to incorporate modified nucleotides while preserving its fundamental polymerization function.
Solution Approach 2:
The directed evolution process allows the polymerase enzyme to self-optimize its properties for specific applications. The enzyme undergoes spontaneous mutations and is selected under controlled conditions to enhance desired traits such as thermostability and modified nucleotide incorporation capability, without requiring external engineering intervention for each specific improvement.
3Productivity
If polymerase processivity is high, then DNA synthesis efficiency is improved, but control over DNA sequencing rate is reduced
Solution Approach 1:
The patent modifies the polymerase's processivity parameter through directed evolution to achieve an optimal balance. The engineered polymerase has reduced processivity compared to wild-type, which provides better control over the DNA sequencing rate through the nanopore, while maintaining sufficient synthesis efficiency for practical applications.
Data Source
AI summary
Described herein is a variant pol6 polymerase having improved thermostability. The disclosed polymerases each contain one or more substitutions relative to SEQ ID NO: 1 selected from the group consisting of G12W/Y/F, K114W/F/I, L117L/F/P, N194F/W/V, M232W/G/R, G313E/Q/L, A451F/W/Y, K490W/F/Y, Q565Y/I/V, Q590P/V/Y, and D681G/N/H. Additionally, the present specification discloses substitutions that can reduce stuttering in such polymerases, the substitutions selected from the group consisting of N298L, L538R, P542A, I570H/T/W/R/N/G, N574L, E633W/F, S636F, E639K, and K655G.