Multiplex Single-Molecule Kinetics of Nanopore-Coupled Polymerases

DNA
polymerases have revolutionized the biotechnology field due
to their ability to precisely replicate stored genetic information.
Screening variants of these enzymes for specific properties gives
the opportunity to identify polymerases with different features. We
have previously developed a single-molecule DNA sequencing platform
by coupling a DNA polymerase to an 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 altered physical properties.