Discrimination of Ribonucleoside Mono-, Di-, and Triphosphates Using an Engineered Nanopore

Abstract

Ribonucleotides, which widely exist in all living organisms and are essential to both physiological and pathological processes, can naturally appear as ribonucleoside mono-, di-, and triphosphates. Natural ribonucleotides can also dynamically switch between different phosphorylated forms, posing a great challenge for sensing. A specially engineered nanopore sensor is promising for full discrimination of all canonical ribonucleoside mono-, di-, and triphosphates. However, such a demonstration has never been reported, due to the lack of a suitable nanopore sensor that has a sufficient resolution. In this work, we utilized a phenylboronic acid (PBA) modified Mycobacterium smegmatis porin A (MspA) hetero-octamer for ribonucleotide sensing. Twelve types of ribonucleotides, including mono-, di-, and triphosphates of cytidine (CMP, CDP, CTP), uridine (UMP, UDP, UTP), adenosine (AMP, ADP, ATP), and guanosine (GMP, GDP, GTP) were simultaneously discriminated. A machine-learning algorithm was also developed, which achieved a general accuracy of 99.9% for ribonucleotide sensing. This strategy was also further applied to identify ribonucleotide components in ATP tablets and injections. This sensing strategy provides a direct, accurate, easy, and rapid solution to characterize ribonucleotide components in different phosphorylated forms.

Single-Molecule Interconversion between Chiral Configurations of Boronate Esters Observed in a Nanoreactor

“…These measurements further confirmed that our observation of telegraphic switching from cis -diol binding to PBA is a consequence of dynamic interconversion between chiral boronate esters when observed in a single structure, and this conclusion is general when a large variety of analytes were tested. Though the same phenomenon may as well be carried out with a previously reported MspA nanoreactor however with the PBA placed at site 90, − a significant reduction of the event amplitude was observed with halogenated catechols, suggesting that MspA-91PBA is optimum for the demonstration described in this paper (Figure S25). Though the observed two current levels are a result of interconversion between two chiral configurations, it is still a challenge to directly assign their absolute chiral states solely from the nanopore readout.…”

“…For simplicity, this modified MspA hetero-octamer is referred to in this paper as MspA-91PBA (Figure a). The modification site of MspA-91PBA differs from the previously reported MspA hetero-octamer, which is named MspA-90PBA. − Since this MspA hetero-octamer has never been previously reported, a pore preparation protocol (Methods) along with plasmid design (Figure S1), pore purification by nickel affinity chromatography (Figure S2), and hetero-octamer purification by gel electrophoresis and gel extraction (Figure S3) are described here in detail.…”

“…Boronic acids have been previously applied as adapters of an α-HL or an MspA − nanoreactor and used for the detection of saccharides, alditols, and nucleotides. However, none of these projects focused on the chiral configurational interconversion of boronate esters.…”

Nanopore Discrimination of Nucleotide Sugars

“…A nucleotide sugar molecule, which simultaneously contains a nucleotide and a saccharide moiety, can in principle demonstrate two binding modes when monitored by MspA-90PBA. Binding to different moieties of the same molecule can also be regulated by the pH of the environment, a phenomenon that has also never been seen in any previous demonstrations using MspA-90PBA. − ,, …”

“…Binding to different moieties of the same molecule can also be regulated by the pH of the environment, a phenomenon that has also never been seen in any previous demonstrations using MspA-90PBA. [33][34][35]40,41 A variety of nucleotide and monosaccharide moieties and the linkage between them all contribute to the diversity of nucleotide sugars. Inspired by the success of UDP-Glc sensing, we expect that the same strategy may also be applied to the sensing of other nucleotide sugars.…”