Multiplex single-molecule kinetics of nanopore-coupled polymerases

Abstract

DNA polymerases have revolutionized the biotechnology field due to their ability to precisely replicate stored genetic information. Screening variants of these enzymes for unique properties gives the opportunity to identify polymerases with novel features. We have previously developed a single-molecule DNA sequencing platform by coupling a DNA polymerase to a α-hemolysin pore on a nanopore array. Here, we use this approach to demonstrate a single-molecule method that enables rapid screening of polymerase variants in a multiplex manner. In this approach, barcoded DNA strands are complexed with polymerase variants and serve as templates for nanopore sequencing. Nanopore sequencing of the barcoded DNA reveals both the barcode identity and kinetic properties of the polymerase variant associated with the cognate barcode, allowing for multiplexed investigation of many polymerase variants in parallel on a single nanopore array. Further, we develop a robust classification algorithm that discriminates kinetic characteristics of the different polymerase mutants. As a proof of concept, we demonstrate the utility of our approach by screening a library of ~100 polymerases to identify variants for potential applications of biotechnological interest. We anticipate our screening method to be broadly useful for applications that require polymerases with unique or altered physical properties.

Site‐Specific Introduction of Bioorthogonal Handles to Nanopores by Genetic Code Expansion

“…[8] The noncovalent pore-analyte interactions, governed by the side chains of 20 proteinogenic amino acids in the pore lumen, determine the molecular recognition events, and thus the resistive-pulse readout. To this end, chemical and biological strategies such as site-directed mutagenesis, [9] native chemical ligation, [10] internal adaptor installation, [6b, 11] SpyTag-SpyCatcher chemistry, [12] sulfhydryl chemistry, [13] and 2-cyanobenzothiazole (CBT) chemistry, [14] have been developed and used to facilitate nanopore engineering and functionalization. [15] The ideal scenario to manipulate the properties of nanopore building blocks without the limitation of amino acid site, type, number or reactivity however has not been achieved.…”