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. Here we aimed to investigate NA specificity and mechanism of CRISPR interference for the Streptococcus thermophilus Csm (III-A) complex (StCsm). When expressed in Escherichia coli, two complexes of different stoichiometry copurified with 40 and 72 nt crRNA species, respectively. Both complexes targeted RNA and generated multiple cuts at 6 nt intervals. The Csm3 protein, present in multiple copies in both Csm complexes, acts as endoribonuclease. In the heterologous E. coli host, StCsm restricts MS2 RNA phage in a Csm3 nuclease-dependent manner. Thus, our results demonstrate that the type III-A StCsm complex guided by crRNA targets RNA and not DNA.

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. The AFM analysis enabled direct structural characterisation of the A-form duplex dsRNA and accurate determination of the dsRNA duplex length. Moreover, further analysis under non-denaturing conditions revealed the presence of heterogeneous dsRNA species. IP-RP-HPLC fractionation and AFM analysis revealed that these alternative RNA species do not arise from different lengths of individual dsRNA molecules in the product, but represent misannealed RNA species that present as larger assemblies or multimeric forms of the RNA. These results for the first time provide direct structural insight into dsRNA produced both in vivo in bacterial systems and in vitro, highlighting the structural heterogeneity of RNA produced. These results are the first example of detailed characterisation of the different forms of dsRNA from two production systems and establish atomic force microscopy as an important tool for the characterisation of long dsRNA.

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.

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. In addition, dsRNA is difficult to denature even at elevated temperature, and the extinction coefficients of nucleic acids are also affected by temperature, which makes it difficult to accurately determine the nucleic acid concentration. To overcome these caveats, we have utilized the chemical denaturant dimethyl sulfoxide which, in conjunction with a short thermal denaturation, prevents renaturation of the duplex nucleic acids (dsDNA/RNA). Using this approach, we have measured the absorbance of both the unstructured and structured nucleic acids to accurately measure their hypochromicity and determine their extinction coefficients. For a range of different dsRNA, we have for the first time determined values of 46.18-47.29 µg/mL/A260 for the quantification of dsRNA using UV spectrophotometry. Moreover, this approach enables the accurate determination of the relative proportion of duplex nucleic acids in mixed ds/ss nucleic acid solutions, demonstrating significant advantages over current methods.

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. Moreover, the purified RNA is directly compatible with downstream analysis. Using spectrophotometry in conjunction with ion pair reverse phase chromatography to analyse the extracted RNA, we show that RNASwift extracts and purifies RNA of higher quality and purity in comparison to alternative RNA extraction methods. The RNASwift method yields approximately 25 µg of RNA from only 10(8)Escherichia coli cells. Furthermore, RNASwift is versatile; the same simple reagents can be used to rapidly extract RNA from a variety of different cells including bacterial, yeast and mammalian cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods.

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. Reproducible RNA mapping chromatograms were generated from replicate analyses. Moreover, this approach was used to provide a method to rapidly distinguish different RNA sequences of the same size, based on differences in the resulting chromatograms. Principal components analysis of the high resolution RNA mapping data enabled us to rapidly compare multiple HPLC chromatograms and distinguish two dsRNA sequences of different size which share 72% sequence homology. We used the high resolution RNase mapping method to rapidly fingerprint biomanufactured dsRNA across a number of different batches. The resulting chromatograms in conjunction with principal components analysis demonstrated high similarity in the dsRNA produced across the different batches highlighting the potential ability of this method to provide information for batch release in a high throughput manner.

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. The potential to synthesise large quantities of dsRNA by in-vitro transcription or in bacterial systems for RNA interference applications has generated significant demand for the development and application of high throughput analytical tools for the rapid extraction, purification and analysis of dsRNA. Here we have developed analytical methods that enable the rapid purification of dsRNA from associated impurities from bacterial cells in conjunction with downstream analyses. We have optimised TRIzol extractions in conjunction with a single step protocol to remove contaminating DNA and ssRNA, using RNase T1/DNase I digestion under high-salt conditions in combination with solid phase extraction to purify the dsRNA. In addition, we have utilised and developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA. Furthermore, we have optimised base-specific cleavage of dsRNA by RNase A and developed a novel method utilising RNase T1 for RNase mass mapping approaches to further characterise the dsRNA using liquid chromatography interfaced with mass spectrometry.

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. We have investigated a range of different pore-sizes and phases for the analysis of a diverse range of nucleic acids including oligonucleotides, oligoribonucleotides, phosphorothioate oligonucleotides and high molecular weight dsDNA and RNA. The pore size of the superficially porous silica particles was shown to significantly affect the resolution of the nucleic acids. Optimum separations of small oligonucleotides such as those generated in RNase mapping experiments were obtained with 80Å pore sizes and can readily be interfaced with mass spectrometry analysis. Improved resolution of larger oligonucleotides (>19mers) was observed with pore sizes of 150Å. The optimum resolution for larger dsDNA/RNA molecules was achieved using superficially porous silica particles with pore sizes of 400Å. Furthermore, we have utilised 150Å pore size solid-core particles to separate typical impurities of a fully phosphorothioated oligonucleotide, which are often generated in the synthesis of this important class of therapeutic oligonucleotide.

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. Following purification of the crRNA, the intact mass was determined by LC ESI MS. Using this approach, a significant reduction in metal ion adduct formation of the crRNA was observed. In addition, RNase mapping of the crRNA was performed using RNase digestion in conjunction with liquid chromatography tandem MS analysis. Using the intact mass of the crRNA, in conjunction with RNase mapping experiments enabled the identification and characterisation of the crRNA, providing further insight into crRNA processing in a number of type I CRISPR-Cas systems.