Label-free amino acid identification forde novoprotein sequencing via tRNA charging and current blockade in a nanopore

Abstract

Label-free identification of amino acids (AAs) based on superspecific transfer RNAs (tRNAs) and amino-acyl tRNA synthetase (AARS) is explored. Molecules of a specific AA, a tRNA and its cognate AARS, and adenosine triphosphate (ATP) from a reservoir are confined to a micro-sized cavity by hydraulic pressure to counter the effects of diffusion. In this confined space a cognate tRNA in the cavity gets charged with AA and adenosine monophosphate (AMP) is released. The products are transferred to an electrolytic cell with a nanopore where AA, AMP, and ATP cause current blockades of different sizes. If an AMP-sized blockade occurs it means that a tRNA molecule has been charged with AA, this identifies AA; otherwise the procedure is repeated with a different tRNA and AARS until AA is identified. Unlike in most other nanopore-based methods, there is no need for precise measurement of current blockade levels. The efficacy of the procedure is assessed by simulating the movement of particles in a reservoir-cavity structure and analyzing the blockades that occur in the nanopore. In the former the probability of reactants escaping the cavity is found to be near 0. In the latter the probability of an error in identifying an AMP blockade is found to be less than 10% in the worst case (which occurs with the largest volume AA, namely Tryptophan). Detailed information and data are provided in a Supplementary File.

Slowing down an analyte in a nanopore

“…With AAs since different pH values are used to achieve slowdown, they need to be probed in different e-cells. A method to separate AAs from a sample by routing them to 20 different e-cells (one for each of the 20 different AAs) is described in [18]. The following is a brief summary.…”

“…Binding is accompanied by the release of adenosine monophosphate (AMP). In the method introduced in [18] an AA is fed into a micro-sized reservoir-cavity structure. An AA is bound by a cognate aminoacyl tRNA synthetase to its cognate tRNA in the cavity; the resulting charged (amino-acyl) tRNA is filtered out and deacylated.…”

“…The slowdown method described above can be used in each of the 20 cells to enable detection of a blockade with a bandwidth of a few Khz. As discussed at length in [18] each of the cells is isolated from the others so that a cell is like a silo in which the behavior of the analyte can be controlled independently based on the specific properties of the analyte. Thus, for example, the pore voltage can be set based on the volume of the analyte to maximize blockade size.…”

“…Here a different approach that is based on the use of hydraulic pressure [25] is used. As described in [18] a piston at the top of cis with a pressure of ΔP results in flow of the electrolyte to and through the pore. Assuming hydraulic flow to be Poiseuille flow, the velocities of the analyte in cis are given by: vhyd = ΔP Rcis 2 / 8ηLcis (cis) (13a) = ΔP Rpore 2 / 8ηLpore (pore)…”

“…However other than the solution in [17], which is limited to the sequencing by synthesis (SBS) method, the problem remains truculent and continues to resist general solution. The second problem, namely separation of the 20 AAs in measurement, is addressed later when introducing a method for AA identification that is designed to achieve such separation and make physical and chemical differences less of a problem [18]. A fourth, less severe, problem is that of delivering the analyte from the top of the cis chamber of the e-cell to the pore entrance.…”