Real-Time Sensing and Discrimination of Single Chemicals Using the Channel of Phi29 DNA Packaging Nanomotor

Abstract

A highly sensitive and reliable method to sense and identify a single chemical at extremely low concentrations and high contamination is important for environmental surveillance, homeland security, athlete drug monitoring, toxin/drug screening, and earlier disease diagnosis. This article reports a method for precise detection of single chemicals. The hub of the bacteriophage phi29 DNA packaging motor is a connector consisting of 12 protein subunits encircled into a 3.6 nm channel as a path for dsDNA to enter during packaging and to exit during infection. The connector has previously been inserted into a lipid bilayer to serve as a membrane-embedded channel. Herein we report the modification of the phi29 channel to develop a class of sensors to detect single chemicals. The lysine-234 of each protein subunit was mutated to cysteine, generating 12-SH ring lining the channel wall. Chemicals passing through this robust channel and interactions with the SH group generated extremely reliable, precise, and sensitive current signatures as revealed by single channel conductance assays. Ethane (57 Da), thymine (167 Da), and benzene (105 Da) with reactive thioester moieties were clearly discriminated upon interaction with the available set of cysteine residues. The covalent attachment of each analyte induced discrete stepwise blockage in current signature with a corresponding decrease in conductance due to the physical blocking of the channel. Transient binding of the chemicals also produced characteristic fingerprints that were deduced from the unique blockage amplitude and pattern of the signals. This study shows that the phi29 connector can be used to sense chemicals with reactive thioesters or maleimide using single channel conduction assays based on their distinct fingerprints. The results demonstrated that this channel system could be further developed into very sensitive sensing devices.

Single pore translocation of folded, double-stranded, and tetra-stranded DNA through channel of bacteriophage phi29 DNA packaging motor

“…Recently, a reengineered connector with reduced channel size has shown the capability to discriminate ss-DNA and RNA [24]. By selectively introducing probes either at the terminal ends or within the channel lumen, single antibodies or single chemicals can be identified with high confidence at ultra-low concentrations based on their characteristic current fingerprints [27,28]. Procedures for large scale production and purification of the connector have been well developed [18,29-33].…”

Binomial distribution for quantification of protein subunits in biological Nanoassemblies and functional nanomachines

“…Medical applications of these or their derivatives could be used for pathogen detection, disease diagnosis, and drug and therapeutic RNA delivery. 19,20,24,25 Also common to living system is the transportation of dsDNA. In the ASCE (additional strand catalytic E) family including the AAA+ (ATPases Associated with diverse cellular Activities) and the FtsK-HerA superfamily, there are nanomotors that facilitate a wide range of functions involved in dsDNA riding, tracking, packaging, and translocation.…”

Biological Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism

“…The revolution mechanism offers a hint for the design of nanomolecular motors that can manipulate biomolecules in a controlled fashion. These nanomachines can also be applied for the construction of sophisticated nanodevices, including molecular sensors (221)(222)(223), bioreactors (224), chips (225), DNA-sequencing apparatuses (169,221,(226)(227)(228)(229), or other electronic and optical devices (230,231). In addition, these multisubunit biocomplexes could serve as drug targets for therapeutics and have potential for diagnostic applications (222,223,232,233), especially as the biomotors have an advantage of a high stoichiometry that could be applied to solve the drug resistance issue (234,235).…”