Low Resource Integrated Platform for Production and Analysis of Capped mRNA.

The existing platform for large-scale mRNA production is fast, but consumable costs, process technicality, and complexity represent key bottlenecks limiting global mRNA biologics manufacturing. Another challenge is the lack of a consolidated platform for mRNA product characterization and assays that meet regulatory requirements. Bridging these innovation gaps to simplify processes and reduce cost would improve mRNA biologics manufacturability, especially in low-resource settings.

A novel sustainable platform for scaled manufacturing of double-stranded RNA biopesticides.

RNA interference (RNAi) represents one of the most conserved pathways evolved by eukaryotic cells for regulating gene expression. RNAi utilises non-translatable double-stranded RNA (dsRNA) molecules to sequester or degrade mRNA molecules gene. In RNAi, specifically designed exogenous dsRNA delivered to the cell can silence a target gene, a phenomenon that has been exploited in many functional studies and explored in biopesticide applications.

Correction: Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC.

Correction for 'Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC' by Alison O. Nwokeoji, et al., Analyst, 2019, 144, 4985-4994.

Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC.

Long double-stranded (ds) RNA is emerging as a novel alternative to chemical and genetically-modified insect and fungal management strategies. The ability to produce large quantities of dsRNA in either bacterial systems, by in vitro transcription, in cell-free systems or in planta for RNA interference applications has generated significant demand for the development and application of analytical tools for analysis of dsRNA. We have utilised atomic force microscopy (AFM) in conjunction with ion-pair reverse-phase high performance liquid chromatography (IP-RP-HPLC) to provide novel insight into dsRNA for RNAi applications.

High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography.

The emergence of new sustainable approaches for insect management using RNA interference (RNAi) based insecticides has created the demand for high throughput analytical techniques to fully characterise and accurately quantify double stranded RNA (dsRNA) prior to downstream RNAi applications. In this study we have developed a method for the rapid characterisation of single stranded and double stranded RNA using high resolution RNase mapping in conjunction with ion-pair reverse-phase chromatography utilising a column with superficially porous particles. The high resolution oligoribonucleotide map provides an important 'fingerprint' for identity testing and bioprocess monitoring.

Accurate Quantification of Nucleic Acids Using Hypochromicity Measurements in Conjunction with UV Spectrophotometry.

UV absorbance spectrophotometry is widely used for the quantification of nucleic acids. For accurate quantification, it is important to determine the hypochromicity of the oligonucleotide or complex nucleic acid structure. The use of thermal denaturation studies in conjunction with UV spectrophotometry to determine hypochromicity requires prolonged, elevated temperatures, which may cause partial hydrolysis of RNA.

Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry.

RNA interference has provided valuable insight into a wide range of biological systems and is a powerful tool for the analysis of gene function. The exploitation of this pathway to block the expression of specific gene targets holds considerable promise for the development of novel RNAi-based insect management strategies. In addition, there are a wide number of future potential applications of RNAi to control agricultural insect pests as well as its use for prevention of diseases in beneficial insects.

RNASwift: A rapid, versatile RNA extraction method free from phenol and chloroform.

RNASwift is an inexpensive, versatile method for the rapid extraction of RNA. Existing RNA extraction methods typically use hazardous chemicals including phenol, chloroform and formamide which are often difficult to completely remove from the extracted RNA. RNASwift uses sodium chloride and sodium dodecyl sulphate to lyse the cells and isolate the RNA from the abundant cellular components in conjunction with solid phase extraction or isopropanol precipitation to rapidly purify the RNA.

Nucleic acid separations using superficially porous silica particles.

Ion pair reverse-phase liquid chromatography has been widely employed for nucleic acid separations. A wide range of alternative stationary phases have been utilised in conjunction with ion pair reverse-phase chromatography, including totally porous particles, non-porous particles, macroporous particles and monolithic stationary phases. In this study we have utilised superficially porous silica particles in conjunction with ion pair reverse-phase liquid chromatography for the analysis of nucleic acids.

Analysis of crRNA Using Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC ESI MS).

Mass spectrometry is a powerful tool for characterizing RNA. Here we describe a method for the identification and characterisation of crRNA using liquid chromatography interfaced with electrospray ionization mass spectrometry (LC ESI MS). The direct purification of crRNA from the Cascade-crRNA complex was performed using denaturing ion pair reverse phase chromatography.

Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus.

Immunity against viruses and plasmids provided by CRISPR-Cas systems relies on a ribonucleoprotein effector complex that triggers the degradation of invasive nucleic acids (NA). Effector complexes of type I (Cascade) and II (Cas9-dual RNA) target foreign DNA. Intriguingly, the genetic evidence suggests that the type III-A Csm complex targets DNA, whereas biochemical data show that the type III-B Cmr complex cleaves RNA.