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.

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.

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/A 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.

Highlightsrapid purification of dsRNA in a single step protocol.high throughput purification and analysis of a wide range of dsRNAs.developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA.developed a novel method utilising RNase T1 for RNase mass mapping of dsRNA.

HighlightsSuperficially porous silica particles enable high resolution separation of nucleic acids.The pore size of the C18 superficially porous silica particles significantly effects resolution.Optimum separations of small oligonucleotides obtained with 80 Å pore sizes.Optimum resolution of oligonucleotides (>19 mers) was observed with pore sizes of 150 Å.Improved resolution of larger dsDNA/RNA molecules was achieved with pore sizes of 400 Å