Real-Time and Accurate Identification of Single Oligonucleotide Photoisomers via an Aerolysin Nanopore

Abstract

Identification of the configuration for the photoresponsive oligonucleotide plays an important role in the ingenious design of DNA nanomolecules and nanodevices. Due to the limited resolution and sensitivity of present methods, it remains a challenge to determine the accurate configuration of photoresponsive oligonucleotides, much less a precise description of their photoconversion process. Here, we used an aerolysin (AeL) nanopore-based confined space for real-time determination and quantification of the absolute cis/ trans configuration of each azobenzene-modified oligonucleotide (Azo-ODN) with a single molecule resolution. The two completely separated current distributions with narrow peak widths at half height (<0.62 pA) are assigned to cis/ trans-Azo-ODN isomers, respectively. Due to the high current sensitivity, each isomer of Azo-ODN could be undoubtedly identified, which gives the accurate photostationary conversion values of 82.7% for trans-to- cis under UV irradiation and 82.5% for cis-to- trans under vis irradiation. Further real-time kinetic evaluation reveals that the photoresponsive rate constants of Azo-ODN from trans-to- cis and cis-to -trans are 0.43 and 0.20 min, respectively. This study will promote the sophisticated design of photoresponsive ODN to achieve an efficient and applicable photocontrollable process.

Resolving the Dynamic Non-Covalent Interaction inside Membrane Protein Channel by Single-Molecule Interaction Spectrum

“…To support this suggestion, we carried out voltage-dependent experiments, changed counterions and varied ionic strength. based on our previous work 56 . All data were acquired in the presence of 2.0 μM at pH 8.0, 20 ± 2 ℃.…”

Single-entity electrochemistry at confined sensing interfaces

“…The K238Q Aerolysin successfully discriminate the cysteine and homocysteine due to the strong interaction provided by the substitution by Glutamine (Q). [61] Azobenzene is a UV-sensitive molecule, by modifying it to the oligonucleotide (Azo-ODN), the cisto-trans or trans-to-cis modification under the UV-Vis control could be real-time monitored by Aerolysin composed confined space [62] ( (Figure 5(e)).…”

“…Examples of nanopore based single entity measurements. (a) ssDNA [63] (b) dsDNA [64] (c) RNA [34] (d) Peptide [65] (e) Light driven molecular structure modification [62] (f) Chemical reaction [66] (g) Enzyme cleavage monitoring [67] Peptides are usually chosen as the biomarkers to diseases. Unlike the DNA segments which are usually negatively charged, the charge density of peptides becomes an important descriptor as well as the 20 amino acid components.…”

The aerolysin nanopore: from peptidomic to genomic applications

“…Aerolysin is now emerging as a new nanopore system, with distinct differences from the widely used alpha-hemolysin nanopore system. ARP was first used for α-helical peptide translocation studies in 2006 3 and has since been used intensively for peptide and protein sensing, including small protein analysis, 4 to study protein unfolding dynamics, 5 to distinguish unfolded protein states, 6 for polysaccharide detection, 7 to analyze enzymatic degradation of long polysaccharide chains, 8 to detect botulinum toxin, 9 to analyze protein unfolding and translocations, 10 to detect low molecular weight (140 Da) poly(ethylene glycol) (PEG) at the single-molecule level, 11 for high-resolution size discrimination of PEG molecules, 12 to evaluate the physical parameters governing an unfolded protein translocation, 13 to identify single oligo-nucleotide photoisomers, 14 and for ultrasensitive detection of cancer cells by enzymatic amplification. 15 Most recently, Drs Pelta and Oukhaled’s group has reported that ARP can be used to identify amino acids in short peptides, detecting and characterizing a few dozen impurities in a high-purity (>98%) commercial peptide sample.…”