Fluorometric determination of microRNA by using an entropy-driven three-dimensional DNA walking machine based on a catalytic hairpin assembly reaction on polystyrene microspheres.

An entropy-driven 3-D DNA walking machine is presented which involves catalytic hairpin assembly (CHA) for detection of microRNA. A 3-D DNA walking machine was designed that uses streptavidin-coated polystyrene microspheres as track carriers to obtain reproducibility. The method was applied to microRNA 21 as a model analyte. Continuous walking on the DNA tracks is achieved via entropy increase. This results in a disassembly of ternary DNA substrates on polystyrene microspheres and leads to cycling of microRNA 21. The release of massive auxiliary strands from ternary DNA substrates induces the CHA. This is accompanied by in increase in fluorescence, best measured at excitation/emission wavelengths of 480/520 nm. On account of entropy-driven reaction, the assay is remarkably selective. It can differentiate microRNA 21 from homologous microRNAs in giving a signal that is less than 5% of the signal for microRNA 21 except for microRNA-200b. The assay works in the 50 pM to 20 nM concentration range and has a 41 pM detection limit. The method displays good reproducibility (between 1.1 and 4.2%) and recovery (from 99.8 to 104.0%). Graphical abstract An entropy-driven 3-D DNA walking machine is described. It is based on the use of polystyrene microspheres and of a catalytic hairpin assembly reaction for sensitive microRNA detection. Figure Notes: AS represents auxiliary strand; S represents substrate strand; LS represents link strand; F represents fuel nucleic acid; RepF represents nucleic acid labeled with FAM; RepQ represents nucleic acid labeled with BHQ1.

Electrochemical competitive immunodetection of messenger RNA modified with N6-methyladenosine by using DNA-modified mesoporous PtCo nanospheres.

N6-Methyladenosine (m6A) is the most abundant RNA modification in eukaryotic messenger RNA (mRNA). A highly sensitive electrochemical immunosensor is described for the determination of m6A-RNA. The method is based on the use of antibody (anti-m6A) and PtCo mesoporous nanospheres (MPNs). The analogously modified probe of type m6A-DNA-PtCo competes with m6A-RNA for antibodies on the gold electrode as an electrical signal probe. The electrical signal, best acquired at a working potential of -0.37 V (vs. Ag/AgCl) reflects the concentration of m6A. The PtCo MPNs catalyze the reduction of H2O2, and this amplifies the current and enhances sensitivity. The detection time of the assay is <1.5 h. Under optimal conditions, response is linear in the 0.005 to 100 nM m6A RNA concentration range, and the detection limit is 2.1 pM. The results obtained by this immunoassay with human cell lines are comparable to those obtained with a commercial kit. Graphical abstractSchematic representation of a method for electrochemical determination of m6A-modified mRNA. Anti-m6A Ab: antibody against m6A; BSA: bovine serum albumin; PtCo: PtCo mesoporous nanospheres; SH-m6A-DNA: DNA modified with both m6A and thiol groups; DPV: differential pulse voltammetry.