Resolved Single-Molecule Detection of Individual Species within a Mixture of anti-Biotin Antibodies Using an Engineered Monomeric Nanopore

Abstract

Oligomeric protein nanopores with rigid structures have been engineered for the purpose of sensing a wide range of analytes including small molecules and biological species such as proteins and DNA. We chose a monomeric β-barrel porin, OmpG, as the platform from which to derive the nanopore sensor. OmpG is decorated with seven flexible loops that move dynamically to create a distinct gating pattern when ionic current passes through the pore. Biotin was chemically tethered to the most flexible one of these loops. The gating characteristic of the loop’s movement in and out of the porin was substantially altered by analyte protein binding. The gating characteristics of the pore with bound targets were remarkably sensitive to molecular identity – even providing the ability to distinguish between homologues within an antibody mixture. A total of five gating parameters were analyzed for each analyte to create a unique fingerprint for each biotin binding protein. Our exploitation of gating noise as a molecular identifier may allow more sophisticated sensor design while OmpG’s monomeric structure greatly simplifies nanopore production.

Electrostatic Interactions between OmpG Nanopore and Analyte Protein Surface Can Distinguish between Glycosylated Isoforms

“…The effect of bound avidin on the loops was remarkably different from the effect of a mouse monoclonal anti-biotin antibody (mAb) used in a previous study. 30 Under identical conditions, mAb binding increased both the gating frequency and event duration, which led to a decrease in open probability. Furthermore, one polyclonal anti-biotin antibody eliminated all gating events and resulted in a constantly open pore.…”

“…Recently, we have explored an alternate nanopore sensing scheme based on outer membrane protein G (OmpG). 30 OmpG from Escherichia coli (E. coli) is a β-barrel protein with 14 strands connected by seven short turns on the periplasmic side and seven long loops on the extracellular side (Figure 1a). 31–33 Loop 6 moves dynamically, switching the protein between open and closed conformations resulting in gating signal in the current recording (Figure 1b).…”

“…34–36 Previously we showed that the flexible architecture of the OmpG nanopore can be used to resolve between structurally homologous protein analytes in mixtures. 30 Specifically, upon binding to a biotin group tethered to loop 6, a mixture of anti-biotin antibodies raised from different host species was clearly distinguished. Each antibody species was identified by its unique gating fingerprint and no interference from the other antibodies was observed.…”

Mechanism of OmpG pH-Dependent Gating from Loop Ensemble and Single Channel Studies

“…19 All primers (Eurofins MWG Operon) for each mutagenesis PCR are listed in Supplemental Information Table S1. Paired forward and reverse primers were used in a 1:1 molar ratio.…”

“…14,18–20 Our recent works have demonstrated the use of OmpG gating behavior for highly specific protein homologue sensing. 19,21,22 Thus, understanding of OmpG gating may also guide the design of OmpG-based nanopore sensors for new applications.…”