Preprint Short Note Version 4 Preserved in Portico This version is not peer-reviewed

Single Molecule Protein Sequencing Based on the Superspecificity of tRNA Synthetases

Version 1 : Received: 22 November 2019 / Approved: 26 November 2019 / Online: 26 November 2019 (11:52:57 CET)
Version 2 : Received: 4 December 2019 / Approved: 4 December 2019 / Online: 4 December 2019 (12:32:26 CET)
Version 3 : Received: 13 January 2020 / Approved: 13 January 2020 / Online: 13 January 2020 (09:43:28 CET)
Version 4 : Received: 17 February 2020 / Approved: 18 February 2020 / Online: 18 February 2020 (11:50:29 CET)

How to cite: Sampath, G. Single Molecule Protein Sequencing Based on the Superspecificity of tRNA Synthetases. Preprints 2019, 2019110318 (doi: 10.20944/preprints201911.0318.v4). Sampath, G. Single Molecule Protein Sequencing Based on the Superspecificity of tRNA Synthetases. Preprints 2019, 2019110318 (doi: 10.20944/preprints201911.0318.v4).

Abstract

Single molecule de novo protein sequencing based on the 'superspecificity' of amino-acyl tRNA synthetases (aaRS) is proposed. An unfolded protein molecule is threaded through a nanopore in an electrolytic cell (e-cell) to expose the terminal residue in the e-cell's trans chamber. After the residue is cleaved with an exopeptidase, a set of tRNAs, their aaRSs, and ATP are added to trans. An aaRS charges a cognate tRNA molecule with the residue. The charged tRNA (along with the other reactants) is transferred to an extended e-cell with N (20 ≤ N ≤ 61) pores in N individual cis chambers and a single trans chamber. Each pore holds an RNA molecule ending in a unique codon that is exposed in trans. The charged tRNA's anticodon base-pairs with the terminal codon of an RNA. If tRNAs and residues are fluorescently tagged with two different colors, the residue can be identified from the observed position of the resulting color pair. As charging is 'superspecific' identification is unambiguous. The protein molecule in the first e-cell is advanced by one residue and the process repeated. In this approach there is no need for precise pore current or optical intensity measurements. Potential implementation issues are discussed. Other possibilities, including one in which the terminal residue is cleaved after charging, are also examined.

Subject Areas

protein sequencing; single molecule; nanopore; tRNA; amino acyl tRNA synthetase; codon; optical tag

Comments (1)

Comment 1
Received: 18 February 2020
Commenter: G Sampath
Commenter's Conflict of Interests: Author
Comment: - Added paragraph on post-translational modifications - Added paragraph on shotgun sequencing from peptides - Item 5 in discussion section of version 3 not applicable, has been removed - References added - Other minor modifications, typos fixed
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