Simultaneous Dual-Site Identification of 5mC/8oG in DNA Triplex Using a Nanopore Sensor.

DNA triplex participates in delivering site-specific epigenetic modifications critical for the regulation of gene expression. Among these marks, 5mC with 8oG functions comprehensively on gene expression. Recently, few research studies have emphasized the necessity of incorporation detection of 5mC with 8oG using one DNA triplex at the same time. Herein, DNA triplex structure was designed and tailored for the site-specific identification of 5mC with 8oG by means of nanopore electroanalysis. The identification was associated with the distinguishable current modulation types caused by DNA unzipping through the nanopore in an electrical field. Results demonstrated that the epigenetic modification proximity to the latch zone or constriction area of the nanopore enables differentiation of modification series at single nucleotide resolution in one DNA triplex, at both physiological and mildly acidic environment. In addition, our nanopore method enables the kinetic and thermodynamic studies to calculate the free energy of modified DNA triplex with applied potentials. Gibbs' energy provided the direct evidence that the DNA triplex with these epigenetic modifications is more stable in acidic environment. Considering modified DNA functions significantly in gene expression, the presented method may provide future opportunities to understand incorporating epigenetic mechanisms of many dysregulated biological processes on the basis of accurate detection.

De novo profiling of insect-resistant proteins of rice via nanopore peptide differentiation.

Nanopore is used as a single-molecule detector for proteins, peptides and amino acids identification of biomedical importance. We, on the first try, extended its profiling application for peptidome encoded by the insect-resistance gene in rice. Taking brown planthopper (Bph) for example, we previously cloned and verified Bph32 gene, which encodes SCR ("Bph32") protein as direct executor against this injurious insect. However, the homology protein expressed in susceptible line doesn't have this antibiosis resistance. Hence, profiling the structural modulars (peptide domains) of Bph32 proteins may provide essential basis to understand rice in response to insects. Herein, we combined approaches of bioinformatics, biochemistry and nanopore analysis to profile the rice peptides with diverse properties. Bph32 proteins were theoretically modeled into 24 functional peptide domains using Swiss-Model workspace. Next, 22 water-soluble peptides were identified by biuret-chemistry amplified nanopore current modulations. Among those, 16 ones were distinguished at one amino acid resolution via reading the current modulation spectrum, consequently providing the peptidome fingerprints. In addition, the current modulations were evidenced as quadratic function of peptide's molecular masses. These findings suggest that nanopore may work as a new generation of mass detector for more omics analysis, especially in agricultural field where demands strongly.

Nanopore detects γ-radiation inhibited HIV-1 protease activity.

Inhibition of HIV-1 protease (PR) activity is realized by exposure to 60Co γ-radiation. The radiation effects on enzyme kinetics of HIV-1 PR are subsequently monitored using nanopore sensor. Substantial loss of proteolytic efficiency towards GagPol polypeptide is observed due to the radiation treatment. Results shows ~50% of GagPol polypeptide was not involved in HIV-1 PR proteolysis by exposure to ultra-low intensity of γ-radiation (0.1K Gy), and the values reach to over 90% with high γ-ray treatment. Besides, the inactivation effect is also verified in blood samples which contain the virus protease. Our finding provides the potential benefits of γ-radiation to inactivate viral proteinic function, and might be a complementary to the designation of HIV-1 PR inhibitors.