InDevR (United States)

ABSTRACTThe rapid development of mRNA vaccines since the COVID-19 pandemic has highlighted the potential of mRNA technology for vaccine and therapeutic applications. However, evaluating mRNA quality, including its integrity, 5′ capping efficiency, and 3′ poly(A) tailing, remains complex and challenging. Current analytical methods provide detailed insights, but are often limited by long analysis times, complex sample preparation, and the need for specialized expertise. The 5’ CapQassay addresses these challenges and provides a novel, rapid, and straightforward method that offers a single, unique measurement of intact mRNA with both a 5′cap and 3′poly(A) tail. This 2-hour benchtop-based assay provides quantitative or relative analysis, with high accuracy and precision without extensive sample processing. The 5’ CapQassay offers a significant advancement in mRNA analytics to alleviate bottlenecks in applications includingin vitrotranscription (IVT) reaction optimization, post-transcriptional capping optimization, and assessment of batch-to-batch IVT bioprocess consistency.

ABSTRACTThe COVID-19 pandemic highlighted mRNA as a promising platform for vaccines and therapeutics. Many of the analytical tools used to characterize the critical quality attributes of mRNA are inherently singleplex and are not necessarily optimal from a labor and cost perspective. Here we demonstrate feasibility of a multiplexed platform (VaxArray) for efficient identity verification and concentration determination for both monovalent and multivalent mRNA formulations. A model system comprised of mRNA constructs for influenza hemagglutinin and neuraminidase was used to characterize the analytical performance metrics for a VaxArray mRNA assay. The assay presented herein had a time to result of less than 2 hours, required no PCR-based amplification nor extraction of mRNA from lipid nanoparticles, and exhibited high construct specificity that enabled application to the bivalent mixture. The sensitivity for influenza hemagglutinin and neuraminidase mRNA was sub-µg/mL, which is vaccine-relevant, and the average accuracy (%recovery) and precision were 104%±2%and 9%±2%, respectively.

A systematic study was conducted characterizing the effect of furfural, 5-hydroxymethylfurfural (5-HMF), and acetic acid concentration on the production of xylitol and ethanol by a novel endophytic yeast, Rhodotorula mucilaginosa strain PTD3. The influence of different inhibitor concentrations on the growth and fermentation abilities of PTD3 cultivated in synthetic nutrient media containing 30 g/l xylose or glucose were measured during liquid batch cultures. Concentrations of up to 5 g/l of furfural stimulated production of xylitol to 77 % of theoretical yield (10 % higher compared to the control) by PTD3. Xylitol yields produced by this yeast were not affected in the presence of 5-HMF at concentrations of up to 3 g/l. At higher concentrations of furfural and 5-HMF, xylitol and ethanol yields were negatively affected. The higher the concentration of acetic acid present in a media, the higher the ethanol yield approaching 99 % of theoretical yield (15 % higher compared to the control) was produced by the yeast. At all concentrations of acetic acid tested, xylitol yield was lowered. PTD3 was capable of metabolizing concentrations of 5, 15, and 5 g/l of furfural, 5-HMF, and acetic acid, respectively. This yeast would be a potent candidate for the bioconversion of lignocellulosic sugars to biochemicals given that in the presence of low concentrations of inhibitors, its xylitol and ethanol yields are stimulated, and it is capable of metabolizing pretreatment degradation products.

Shiga toxin-producing Escherichia coli O157 is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype E. coli O157 strains, the present study evaluated the use of ampliPHOX, a novel colorimetric detection method based on photopolymerization, for pathogen identification with DNA microarrays. A low-density DNA oligonucleotide microarray was designed to target stx1 and stx2 genes encoding Shiga toxin production, the eae gene coding for adherence membrane protein, and the per gene encoding the O157-antigen perosamine synthetase. Results from the validation experiments demonstrated that the use of ampliPHOX allowed the accurate genotyping of the tested E. coli strains, and positive hybridization signals were observed for only probes targeting virulence genes present in the reference strains. Quantification showed that the average signal-to-noise ratio values ranged from 47.73 ± 7.12 to 76.71 ± 8.33, whereas average signal-to-noise ratio values below 2.5 were determined for probes where no polymer was formed due to lack of specific hybridization. Sensitivity tests demonstrated that the sensitivity threshold for E. coli O157 detection was 100-1000 CFU/mL. Thus, the use of DNA microarrays in combination with photopolymerization allowed the rapid and accurate genotyping of E. coli O157 strains.

The design and optimization of a vapor-phase analyte deposition method for limit of detection (LOD) studies on engineered surface-enhanced Raman scattering (SERS)-active substrates is presented. The vapor deposition method was designed to overcome current challenges in quantitative analysis of lithographically produced SERS substrates that are relatively small (hundreds of square micrometers). A custom-built flow cell was used to deposit benzenethiol from the vapor phase onto SERS-active Ag thin films, as the control substrates, and nanoaperture arrays that were generated by electron-beam lithography. The surface coverage of benzenethiol as a function of time was monitored using the ring stretching mode 1070-cm(-1) band and the trend was fit to Langmuir adsorption kinetics. The method was deemed reliable based on agreement between the LOD determined on the control substrates and previously reported values for those substrates. Application of the new method to a 20 x 20 microm(2) nanoaperture array yielded a LOD of 4.2 +/- 0.3 amol.