Label-free amino acid identification for de novo protein sequencing via tRNA charging and current blockade in a nanopore

Author:

Sampath G.

Abstract

AbstractA label-free procedure to identify single amino acids (AAs) for protein sequencing is developed in theory and simulated in part. A terminal AA cleaved from a protein/peptide, a tRNA, its cognate amino-acyl tRNA synthetase (AARS), and adenosine triphosphate (ATP) are brought together in a container where tRNA, if cognate, gets charged with AA and adenosine monophosphate (AMP) is released. The released AMP (and any free AA and ATP molecules) filters into the cis chamber of an adjoining electrolytic cell (e-cell) from where they pass through a nanopore into the trans chamber. Addition of NaOH to the container deacylates the tRNA if it is charged. The resulting free AA passes into the cis chamber of the e-cell, translocates into trans, and causes a current blockade; AA is immediately known from the identity of the tRNA (the two are cognate). If the tRNA is not charged there is no AA bound to it so AA remains unidentified. In this approach there is no need to distinguish among the 20 AAs by blockade size; it suffices to distinguish blockades from noise: thus a high-precision analog measurement has been transformed into a low-precision binary one. Identification is accurate because of tRNA superspecificity (the tRNA charging error rate is < 1/350); parallel execution with 20 different tRNAs can identify AA in one cycle. This is a de novo method in which no prior information about the protein is used or needed.

Publisher

Cold Spring Harbor Laboratory

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