Hi-fidelity discrimination of isomiRs using G-quadruplex gatekeepers.

Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short sequences of miRNA make single-base isomiR mismatches very difficult to be discriminated. Non-specific hybridizations often arise when DNA probe-miRNA target hybridization is the primary, or initial, mode of detection. These errors then become exacerbated through subsequent amplification steps. Here, we present the design of DNA probes modified with poly-guanine (PG) tracts that were induced to form G-quadruplexes (G4) for hi-fidelity discrimination of miRNA core target sequence from single-base mismatched isomiRs. We demonstrate that, when compared to unmodified probes, this G4 'gate-keeping' function within the G4-modified probes enables more stringent hybridization of complementary core miRNA target transcripts while limiting non-specific hybridizations. This increased discriminatory power of the G4-modified probes over unmodified probes is maintained even after further reverse transcriptase extension of probe-target hybrids. Enzymatic extension also enhanced the clarity and sensitivity of readouts and allows different isomiRs to be distinguished from one another via the relative positions of the mismatches.

Hollow cross-linked enzyme aggregates (h-CLEA) of laccase with high uniformity and activity.

Hollow cross-linked enzyme aggregates of laccase (h-CLEA laccase) can be prepared by employing a millifluidic reactor carrying two coaxial laminar flows. In a confluence zone where acetonitrile and an aqueous solution of laccase meet, diffusion of acetonitrile into the aqueous solution gives rise to rapid precipitation of laccase aggregates at the water/acetonitrile interface, as is evidenced by fluorescence images. By controlling the flow rates carefully in the laminar flow regions, h-CLEA laccase around 220±10nm can be obtained, and the size of the h-CLEA laccase increases with increasing flow rates of both solutions. The h-CLEA laccase particles are distinctly different from CLEA laccase prepared in batch processes. The former only consist a crust of cross-linked enzymes (with a hollow core) whereas the latter has a highly porous structure. When the h-CLEA laccase is used as biocatalysts, their activity (0.26U/mg) is comparable to that of free enzymes at neutral pH due to the hollow structure. Moreover, the activity of h-CLEA laccase is higher than that of free laccase at high pH. For example, trypan blue (a dye molecule) can be decolorized completely in the presence of h-CLEA laccase within 270min even at pH 10.0, at which the free enzyme completely loses its activity. Because of their uniform sizes, h-CLEA laccase can be trapped in a membrane for continuous degradation of trypan blue up to 96h without losing any activity. This study shows the superiority of h-CLEA laccase compared to other types of immobilized enzymes.

Detection of G-Quadruplex Formation via Light Scattering of Defined Gold Nanoassemblies Modulated by Molecular Hairpins.

G-quadruplexes are of great scientific interest, as these unique DNA structures play key regulatory roles in cell replication, such as safeguarding against uncontrolled cellular divisions. The quadruplexes have also been applied for detecting DNA and protein biomarkers via methods like fluorescence resonance energy transfer (FRET) and gold nanoparticle (AuNP) aggregation. As an alternative and complementary platform to the established molecular techniques for the study of quadruplexes, we have developed a strategy coupling poly-G (PG)-mediated quadruplex formation with AuNP assembly detectable via dynamic light scattering (DLS). The presence of quadruplex-forming sequences also uniquely modifies the AuNP nanoassembly readout on DLS. In addition, molecular hairpins co-attached onto the AuNP together with PG successfully modulated the quadruplex-induced nanoassembly. Through molecular beacon-based fluorescence restoration and light scattering signal changes, the open/closed conformations of the hairpins are leveraged to tune the size of the quadruplex-mediated nanoassembly.

DNA-Directed Assembly of Nanogold Dimers: A Unique Dynamic Light Scattering Sensing Probe for Transcription Factor Detection.

We have developed a unique DNA-assembled gold nanoparticles (AuNPs) dimer for dynamic light scattering (DLS) sensing of transcription factors, exemplified by estrogen receptor (ER) that binds specifically to a double-stranded (ds) DNA sequence containing estrogen response element (ERE). Here, ERE sequence is incorporated into the DNA linkers to bridge the AuNPs dimer for ER binding. Coupled with DLS, this AuNP dimer-based DLS detection system gave distinct readout of a single 'complex peak' in the presence of the target molecule (i.e., ER). This unique signature marked the first time that such nanostructures can be used to study transcription factor-DNA interactions, which DLS alone cannot do. This was also unlike previously reported AuNP-DLS assays that gave random and broad distribution of particles size upon target binding. In addition, the ERE-containing AuNP dimers could also suppress the light-scattering signal from the unbound proteins and other interfering factors (e.g., buffer background), and has potential for sensitive detection of target proteins in complex biological samples such as cell lysates. In short, the as-developed AuNP dimer probe coupled with DLS is a simple (mix and test), rapid (readout in ~5 min) and sensitive (low nM levels of ER) platform to detect sequence-specific protein-DNA binding event.

Gold nanostructures for the multiplex detection of glucose-6-phosphate dehydrogenase gene mutations.

We describe a gold nanoparticle-based technique for the detection of single-base mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene, a condition that can lead to neonatal jaundice and hemolytic anemia. The aim of this technique is to clearly distinguish different mutations frequently described within the Asian population from their wild-type counterparts and across different mutant variants. Gold nanoparticles of different sizes were synthesized, and each was conjugated with a single-strand DNA (ssDNA) sequence specific for a particular mutation in the G6PD gene. It was found that only mutant targets presented a characteristic band on the agarose gel, indicating the successful formation of dimeric nanostructures. No such dimer bands were observed for the wild-type targets. The difference in the relative dimer band levels allowed different mutant variants to be distinguished from one another. The technique was further validated using G6PD-deficient patient samples. This simple mutation detection method with direct result readout is amenable for rapid and mass screening of samples.