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We developed a technology to measure microRNA (miRNA) copies in serum and tested it on a commercially available combined human serum (H6914 from Sigma-Aldrich). Copies of miR-15b and miR-16, believed to be constant among healthy and diseased individuals, were measured and agreed with the ones reported by Mitchell PS et al. (2008). Cancer biomarkers let7-b, miR-21, miR-141 and miR-375 varied 3,000 to 6,000 copies per 1 microLiter H6914 (5-10 femtomolar (fM)). Detection and quantification of oligos and miRNAs at such low concentration was shown earlier. It is accomplished by repurposing the commercial MinION nanopore platform to conduct single-molecule voltage-driven ion-channel measurements, employing osmium-tagged oligo probes, and using a publicly available algorithm. These miRNAs were quantified in the serum of healthy individuals or cancer patients using novel optimized probes and a detailed protocol that delivers miRNA copies with better than 85% confidence across all concentrations. A linear correlation, the same with healthy and cancer serum samples, is observed between miR-15b copies and RNA concentration in serum. The assay’s simplicity, readiness, sensitivity, and precision advocate for its use as a Laboratory Developed Test (LDT) for disease-screening based on miRNA dysregulation.

Nanopores can serve as single molecule sensors. We exploited the MinION, a portable nanopore device from Oxford Nanopore Technologies (ONT), 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. Using a validated labeling technology, probes are tagged with a bulky Osmium tag (Osmium tetroxide 2,2’-bipyridine), in a way that preserves strong hybridization between probe and target. Intact oligos traverse the MinION’s nanopore relatively quickly compared to the device’s acquisition rate, and exhibit count of events comparable to the baseline. Counts are reported by a publicly available software,OsBp_detect. Due to the presence of the bulky Osmium tag, probes traverse more slowly, produce multiple counts over the baseline, and are even detected at single digit attomole (amole) range. In the presence of the target the probe is “silenced”. Silencing is attributed to a 1:1 double stranded (ds) complex that doesn’t fit and can’t traverse this nanopore. This ready-to-use platform can be tailored as a diagnostic test to meet the requirements for point-of-care cell-free tumor DNA (ctDNA) and microRNA (miRNA) detection and quantitation in body fluids.

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. We report our protocol using osmium tetroxide 2,2'-bipyridine (Osbipy) as a thymidine (T) specific label. The observed rates show that the labeling process is kinetically independent of both the oligo length, and the base composition. The conditions, i.e. temperature, optimal Osbipy concentration, and molar ratio of reagents, to promote 100% conversion of the starting oligo to labeled product were established. Hence, the optimized conditions developed with the oligos could be leveraged to allow osmylation of effectively all Ts in ssDNA, while achieving minimal mislabeling. In addition, the approach and methods employed here may be adapted to the evaluation of other prospective contrasting agents/labels to facilitate next-generation DNA sequencing by EM.

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. 'Base calling' becomes even more challenging because most nanopores sense a short sequence and not individual bases. Perhaps sequencing DNA via nanopores would be more manageable, if only the bases were two, and chemically very different from each other; a sequence of 1s and 0s comes to mind. Osmylated DNA comes close to such a sequence of 1s and 0s. Osmylation is the addition of osmium tetroxide bipyridine across the C5-C6 double bond of the pyrimidines. Osmylation adds almost 400% mass to the reactive base, creates a sterically and electronically notably different molecule, labeled 1, compared to the unreactive purines, labeled 0. If osmylated DNA were successfully sequenced, the result would be a sequence of osmylated pyrimidines (1), and purines (0), and not of the actual nucleobases. To solve this problem we studied the osmylation reaction with short oligos and with M13mp18, a long ssDNA, developed a UV-vis assay to measure extent of osmylation, and designed two protocols. Protocol A uses mild conditions and yields osmylated thymidines (1), while leaving the other three bases (0) practically intact. Protocol B uses harsher conditions and effectively osmylates both pyrimidines, but not the purines. Applying these two protocols also to the complementary of the target polynucleotide yields a total of four osmylated strands that collectively could define the actual base sequence of the target DNA.

CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is overexpressed and exposed on the cell surface in a large fraction of solid malignancies. We constructed a single-chain fragment (scFv) based on CC49 and fused it to beta-lactamase (BLA). Following optimization of the scFv domain by combinatorial consensus mutagenesis (CCM) for increased expression and stability, we characterized the protein variant for binding, in vivo pharmacokinetics (PK), and antitumor efficacy. The fusion protein TAB2.5 possessed a similar binding specificity relative to the parent antibody CC49. TAB2.5 also showed prolonged retention (T(1/2) = 36.9 h) in tumor-bearing mice with tumor/plasma ratios of up to 1000. Preliminary evaluation of TAB2.5, in combination with a novel prodrug, GC-Mel, resulted in significant efficacy in a colorectal xenograft tumor model and supports the utility of the protein as an agent for tumor-selective prodrug activation.

Polymeric compounds similar to oligonucleotides are relevant to the origin of life and particularly to the concept of an RNA world. Although short oligomers of RNA can be synthesized nonenzymatically under laboratory conditions by second-order reactions in concentrated solutions, there is no consensus on how these polymers could have been synthesized de novo on the early Earth from dilute solutions of monomers. To address this question in the context of an RNA world, we have explored ice eutectic phases as a reaction medium. When an aqueous solution freezes, the solutes become concentrated in the spaces between the ice crystals. The increased concentration offsets the effect of the lower temperature and accelerates the reaction. Here we show that in the presence of metal ions in dilute solutions, frozen samples of phosphoimidazolide-activated uridine react within days at -18 degrees C to form oligouridylates up to 11 bases long. Product yields typically exceed 90%, and approximately 30% of the oligomers include one or more 3'-5' linkages. These conditions facilitate not only the notoriously difficult oligouridylate synthesis, but also the oligomerization of activated cytidylate, adenylate, and guanylate. To our knowledge, this represents the first report to indicate that ice matrices on the early Earth may have accelerated certain prebiotic polymerization reactions.

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., the 2'-5'-, the 3'-5'-, and the pyrophosphate-linked, are formed slowly. In the presence of poly(C) oligomers that are primarily 3'-5'-linked are formed quickly and in high yield. Product analysis suggests that the oligomers are elongation products of the 3'-5'-linked dimer, abbreviated D. Assuming that D is formed slowly from two molecules of 2-MeImpG (Scheme 1) and elongates relatively fast, the initial rate of dimerization, d[D]/dt in M h(-1), was determined using two independent methods. The first method is based on the approximation that at the onset of the reaction the substrate is consumed only via hydrolysis and dimerization, and thus elongation can be neglected. The second, more accurate, method exploits the assertion that every oligomer was once a 3'-5'-linked dimer. Hence the concentration of D was obtained indirectly from the concentration of the oligomer products. These two methods gave comparable results. Experiments were run in aqueous solution in the presence of 1.0 M NaCl, 0.2 M MgCl(2) at pH 7.9 +/- 0.1 and 23 degrees C. Controls were run in the absence of poly(C) and in the presence of other polynucleotides. The kinetics were determined as a function of both monomer and polymer concentration the latter expressed in C equivalents. The kinetic data obtained in the presence of poly(C) confirmed an earlier conclusion regarding the remarkable effect of poly(C) on the formation of the 3'-5'-linked diguanylate. Initial dimerization rates were quantitatively correlated using a simple template-directed (TD) model that presumes cooperative binding (two association constants) of 2-MeImpG on poly(C) and reaction between adjacent template-bound molecules. The model allows for the estimation of the association constants and the intrinsic rate constant of dimerization, k(2). Insights into the detailed mechanism are also gained from this analysis. The fact that the proposed model can successfully correlate kinetic data that vary by more than 5000-fold between the slowest and the fastest reaction adds confidence and suggests the suitability of this model for describing TD reactions in general. It is anticipated that similar analysis of other known TD reactions may lead to clues that will facilitate the design of more efficient polynucleotide-synthesizing systems.

To identify key parameters which influence the efficiency of nonenzymatic template-directed oligonucleotide reactions, a kinetic study of oligoguanylate synthesis on a polycytidylate (poly(C)) template has been performed. This oligomerization is satisfactorily described by three kinetic processes: (i) dimerization to form the first primer (k2), (ii) elongation of a preformed primer (ki, i ≥ 3), and (iii) hydrolysis of the monomer to form deactivated material (kh), with kh < k2 < ki. This is the first reported study that includes rate determinations of ki as a function of the concentration of both poly(C) template and the activated monomer, guanosine 5‘-monophosphate-2-methylimidazolide (2-MeImpG), in the range 2 mM ≤ [poly(C)] ≤ 50 mM and 5 mM ≤ [2-MeImpG] ≤ 50 mM. ki values determined under conditions where the template is fully saturated with monomer are practically independent of both monomer and polymer concentration and thus strongly support a template-directed elongation model. Values of ki determi...