High Sensitivity and Specificity Platform to Validate MicroRNA Biomarkers in Cancer and Human Diseases.

We developed a technology for detecting and quantifying trace nucleic acids using a bracketing protocol designed to yield a copy number with approximately ± 20% accuracy across all concentrations. The microRNAs (miRNAs) let-7b, miR-15b, miR-21, miR-375 and miR-141 were measured in serum and urine samples from healthy subjects and patients with breast, prostate or pancreatic cancer. Detection and quantification were amplification-free and enabled using osmium-tagged probes and MinION, a nanopore array detection device.

Ready-to-use nanopore platform for the detection of any DNA/RNA oligo at attomole range using an Osmium tagged complementary probe.

Nanopores can serve as single molecule sensors. We exploited the MinION, a portable nanopore device from Oxford Nanopore Technologies, and repurposed it to detect any DNA/RNA oligo (target) in a complex mixture by conducting voltage-driven ion-channel measurements. The detection and quantitation of the target is enabled by the use of a unique complementary probe.

Nanopore device-based fingerprinting of RNA oligos and microRNAs enhanced with an Osmium tag.

Protein and solid-state nanopores are used for DNA/RNA sequencing as well as for single molecule analysis. We proposed that selective labeling/tagging may improve base-to-base resolution of nucleic acids via nanopores. We have explored one specific tag, the Osmium tetroxide 2,2'-bipyridine (OsBp), which conjugates to pyrimidines and leaves purines intact.

HPLC methods for purity evaluation of man-made single-stranded RNAs.

Synthetic RNA oligos exhibit purity decreasing as a function of length, because the efficiency of the total synthesis is the numerical product of the individual step efficiencies, typically below 98%. Analytical methods for RNAs up to the 60 nucleotides (nt) have been reported, but they fall short for purity evaluation of 100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted as pharmaceuticals. In an attempt to exploit a single HPLC method and obtain both identity as well as purity, ion-pair reversed-phase chromatography (IP-RP) at high temperature in the presence of an organic cosolvent is the current analytical strategy.

False positives and false negatives measure less than 0.001% in labeling ssDNA with osmium tetroxide 2,2'-bipyridine.

Osmium tetroxide 2,2'-bipyridine (OsBp) is known to react with pyrimidines in ssDNA and preferentially label deoxythymine (T) over deoxycytosine (C). The product, osmylated DNA, was proposed as a surrogate for nanopore-based DNA sequencing due to OsBp's "perfect" label attributes. Osmylated deoxyoligos translocate unassisted and measurably slow via sub-2 nm SiN solid-state nanopores, as well as via the alpha-hemolysin (α-HL) pore.

Single pyrimidine discrimination during voltage-driven translocation of osmylated oligodeoxynucleotides via the α-hemolysin nanopore.

The influence of an electric field on an isolated channel or nanopore separating two compartments filled with electrolytes produces a constant ion flux through the pore. Nucleic acids added to one compartment traverse the pore, and modulate the current in a sequence-dependent manner. While translocation is faster than detection, the α-hemolysin nanopore (α-HL) successfully senses base modifications in ssDNA immobilized within the pore.

Osmium-Based Pyrimidine Contrast Tags for Enhanced Nanopore-Based DNA Base Discrimination.

Nanopores are a promising platform in next generation DNA sequencing. In this platform, an individual DNA strand is threaded into nanopore using an electric field, and enzyme-based ratcheting is used to move the strand through the detector. During this process the residual ion current through the pore is measured, which exhibits unique levels for different base combinations inside the pore.

Osmylated DNA, a novel concept for sequencing DNA using nanopores.

Saenger sequencing has led the advances in molecular biology, while faster and cheaper next generation technologies are urgently needed. A newer approach exploits nanopores, natural or solid-state, set in an electrical field, and obtains base sequence information from current variations due to the passage of a ssDNA molecule through the pore. A hurdle in this approach is the fact that the four bases are chemically comparable to each other which leads to small differences in current obstruction.

Capillary electrophoretic separation-based approach to determine the labeling kinetics of oligodeoxynucleotides.

With the recent advances in electron microscopy (EM), computation, and nanofabrication, the original idea of reading DNA sequence directly from an image can now be tested. One approach is to develop heavy atom labels that can provide the contrast required for EM imaging. While evaluating tentative labels for the respective nucleobases in synthetic oligodeoxynucleotides (oligos), we developed a streamlined CE protocol to assess the label stability, reactivity, and selectivity.

Eutectic phase polymerization of activated ribonucleotide mixtures yields quasi-equimolar incorporation of purine and pyrimidine nucleobases.

The RNA world hypothesis requires a plausible mechanism by which RNA itself (or precursor RNA-like polymers) can be synthesized nonenzymatically from the corresponding building blocks. Simulation experiments have exploited chemically reactive mononucleotides as monomers. Solutions of such monomers in the prebiotic environment were likely to be very dilute, but in experimental simulations of polymerization reactions dilute solutions of activated mononucleotides in the millimolar range hydrolyze extensively, and only trace amounts of dimers and trimers are formed.

Influence of ionic inorganic solutes on self-assembly and polymerization processes related to early forms of life: implications for a prebiotic aqueous medium.

A commonly accepted view is that life began in a marine environment, which would imply the presence of inorganic ions such as Na+, Cl-, Mg2+, Ca2+, and Fe2+. We have investigated two processes relevant to the origin of life--membrane self-assembly and RNA polymerization--and established that both are adversely affected by ionic solute concentrations much lower than those of contemporary oceans. In particular, monocarboxylic acid vesicles, which are plausible models of primitive membrane systems, are completely disrupted by low concentrations of divalent cations, such as magnesium and calcium, and by high sodium chloride concentrations as well.

Unique Catalysis and Regioselectivity Observed in the Poly(C)-Directed RNA Dimer Formation from 2-MeImpG: Kinetic Analysis as a Function of Monomer and Polymer Concentration.

Polycytidylate, poly(C), serves as a scaffold or template to direct and catalyze the synthesis of long oligoguanylates from guanosine 5'-phosphate 2-methylimidazolide, 2-MeImpG. In the absence of poly(C), small amounts of three isomeric dimers, i.e.