Novel Mobile Phase to Control Charge States and Metal Adducts in the LC/MS for mRNA Characterization Assays.

Mass spectrometry is a widely used tool in the characterization of oligonucleotides. This analysis can be challenging due to the large number of possible charge states of oligonucleotides, which can limit the sensitivity of the assay, along with the propensity of oligonucleotides to readily form adducts with free alkali metals. To reduce the adduct formation, oligonucleotides are typically purified with desalting columns prior to analysis. We have developed a mobile phase that gives superior reduction in charge states and adduct formation compared to previously reported methods and, more importantly, obviates the requirement of desalting samples prior to mass spectrometric analysis, significantly decreasing the sample preparation time and amount of RNA required for analysis. We have applied this mobile phase to develop methods to quantify the 5'-capping efficiency and to characterize the polyadenosine (poly(A)) tail of mRNA synthesized in vitro: two critical quality attributes of mRNA therapeutics. Through this, we were able to demonstrate RNA that was co-transcriptionally capped to have capping efficiency equivalent (the percent total molecules that contain a cap) to other reports in the literature using materials that were generated using the same synthesis procedure. Furthermore, by using a mobile phase mixture comprised of hexafluoroisopropanol, triethylammonium acetate, triethylamine, and ethanol, we were able to determine the size distribution of the poly(A) tail in various mRNA samples from DNA templates that ranged from 50 to 150 nt poly(A) and verify that distribution with commercially available RNA standards, successfully demonstrating that this mobile phase composition could be used for characterization assays for both mRNA caps and tails.

Versatile separation of nucleotides from bacterial cell lysates using strong anion exchange chromatography.

Nucleotides exemplify some of the building blocks of life, comprising DNA & RNA, participating in processes such as cell signaling and metabolism, and serving as carriers of metabolic energy. The quantification of these compounds in biological samples is critical for researchers to understand complex systems. Herein, we demonstrate an anion exchange chromatography method utilizing a pH range of 8 to 10, which provides superior resolution and selectivity to previously reported methods and, more importantly, gives the flexibility to shift analyte selectivity if resolution between analytes is not optimal. We have applied the method to study the kinetics of the nucleotide pool in a bacterial cell-free lysate system that is producing RNA. Sample to sample runtimes are less than 18 min and recoveries greater than 96% were observed for all analytes through our methanol quench protocol with day-to-day variabilities less than 5%. This method reliably detects and quantifies all analytes that were expected to be observed in the process and helps lay the groundwork for future nucleotide research.

First Sprayable Double-Stranded RNA-Based Biopesticide Product Targets Proteasome Subunit Beta Type-5 in Colorado Potato Beetle (Leptinotarsa decemlineata).

Colorado potato beetle (CPB, Leptinotarsa decemlineata) is a major pest of potato and other solanaceous vegetables in the Northern Hemisphere. The insect feeds on leaves and can completely defoliate crops. Because of the repeated use of single insecticide classes without rotating active ingredients, many chemicals are no longer effective in controlling CPB. Ledprona is a sprayable double-stranded RNA biopesticide with a new mode of action that triggers the RNA interference pathway. Laboratory assays with second instar larvae fed Ledprona showed a dose-response where 25×10-6g/L of dsPSMB5 caused 90% mortality after 6days of initial exposure. We also showed that exposure to Ledprona for 6h caused larval mortality and decreased target messenger RNA (mRNA) expression. Decrease in PSMB5 protein levels was observed after 48h of larval exposure to Ledprona. Both PSMB5 mRNA and protein levels did not recover over time. Ledprona efficacy was demonstrated in a whole plant greenhouse trial and performed similarly to spinosad. Ledprona, currently pending registration at EPA, represents a new biopesticide class integrated pest management and insecticide resistance management programs directed against CPB.

Complete enzymatic digestion of double-stranded RNA to nucleosides enables accurate quantification of dsRNA.

The rapid growth of research focusing on RNA, especially for RNA interference applications, has created a need for a robust method that can accurately determine the concentration of long dsRNA. As it is difficult to find a source for pure dsRNA reference material, the most common method for quantitation is using a reversed-phase HPLC method to determine purity, which is linked to a calibration curve prepared by measurements obtained using UV absorbance at 260 nm. In this study we developed a nucleic acid digestion method that can digest both double- and single-stranded RNA and DNA to nucleosides. A reversed-phase HPLC/UV method was used to separate and quantitate the monomeric nucleosides. Using this method, we were able to calculate the absorptivity coefficient (proxy for the extinction coefficient) for dsRNA to be 45.9 ± 0.52 µg mL-1/A260. This value agrees with the one report we were able to find but uses an orthogonal method. Moreover, this study allowed us to understand that sequence design can dramatically change the extinction coefficient of the molecule. For molecules with ssRNA overhangs, we observed a 5% reduction in the calculated extinction coefficient.

Enhancing biological hydrogen production from cyanobacteria by removal of excreted products.

Hydrogen is produced by a [NiFe]-hydrogenase in the cyanobacterium Arthrospira (Spirulina) maxima during autofermentation of photosynthetically accumulated glycogen under dark anaerobic conditions. Herein we show that elimination of H2 backpressure by continuous H2 removal ("milking") can significantly increase the yield of H2 in this strain. We show that "milking" by continuous selective consumption of H2 using an electrochemical cell produces the maximum increase in H2 yield (11-fold) and H2 rate (3.4-fold), which is considerably larger than through "milking" by non-selective dilution of the biomass in media (increases H2 yield 3.7-fold and rate 3.1-fold). Exhaustive autofermentation under electrochemical milking conditions consumes >98% of glycogen and 27.6% of biomass over 7-8 days and extracts 39% of the energy content in glycogen as H2. Non-selective dilution stimulates H2 production by shifting intracellular equilibria competing for NADH from excreted products and terminal electron sinks into H2 production. Adding a mixture of the carbon fermentative products shifts the equilibria towards reactants, resulting in increased intracellular NADH and an increased H2 yield (1.4-fold). H2 production is sustained for a period of time up to 7days, after which the PSII activity of the cells decreases by 80-90%, but can be restored by regeneration under photoautotrophic growth.

Metabolic pathways for photobiological hydrogen production by nitrogenase- and hydrogenase-containing unicellular cyanobacteria Cyanothece.

Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H(2) effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H(2) production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H(2) is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O(2) by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO(2). Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H(2) production rate by 2-fold (at the expense of the dark-H(2) rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement.