Strategies for the production of dsRNA biocontrols as alternatives to chemical pesticides.

Current crop pest control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes and agricultural practices. However, many insects, plant viruses, and fungi have no current means of control or have developed resistance against traditional pesticides. dsRNA is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. The successful commercialisation of dsRNA based biocontrols for effective pest management strategies requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. A number of methods exist for the production and delivery of dsRNA based biocontrols and here we review alternative methods currently employed and emerging new approaches for their production. Additionally, we highlight potential challenges that will need to be addressed prior to widespread adoption of dsRNA biocontrols as novel sustainable alternatives to traditional chemical pesticides.

Chemically modified dsRNA induces RNAi effects in insects in vitro and in vivo: A potential new tool for improving RNA-based plant protection.

Global agriculture loses over $100 billion of produce annually to crop pests such as insects. Many of these crop pests either are not currently controlled by artificial means or have developed resistance against chemical pesticides. Long dsRNAs are capable of inducing RNAi in insects and are emerging as novel, highly selective alternatives for sustainable insect management strategies. However, there are significant challenges associated with RNAi efficacy in insects. In this study, we synthesized a range of chemically modified long dsRNAs in an approach to improve nuclease resistance and RNAi efficacy in insects. Our results showed that dsRNAs containing phosphorothioate modifications demonstrated increased resistance to southern green stink bug saliva nucleases. Phosphorothioate-modified and 2'-fluoro-modified dsRNA also demonstrated increased resistance to degradation by soil nucleases and increased RNAi efficacy in Drosophila melanogaster cell cultures. In live insects, we found chemically modified long dsRNAs successfully resulted in mortality in both stink bug and corn rootworm. These results provide further mechanistic insight into the dependence of RNAi efficacy on nucleotide modifications in the sense or antisense strand of the dsRNA in insects and demonstrate for the first time that RNAi can successfully be triggered by chemically modified long dsRNAs in insect cells or live insects.

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. 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.

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.

Quantification of dsRNA using stable isotope labeling dilution liquid chromatography/mass spectrometry.

RATIONALE: Recent developments in RNA interference (RNAi) have created a need for cost-effective and large-scale synthesis of double-stranded RNA (dsRNA), in conjunction with high-throughput analytical techniques to fully characterise and accurately quantify dsRNA prior to downstream RNAi applications. METHODS: Stable isotope labeled dsRNA was synthesised both in vivo (15 N) and in vitro (13 C,15 N-guanosine-containing dsRNA) prior to purification and quantification. The stable isotope labeled dsRNA standards were subsequently spiked into total RNA extracted from E. coli engineered to express dsRNA. RNase mass mapping approaches were subsequently performed using liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) for both the identification and absolute quantification of the dsRNA using the ratios of the light and heavy oligonucleotide pairs. RESULTS: Absolute quantification was performed based on the resulting light and heavy oligoribonucleotides identified using MS. Using this approach we determined that 624.6 ng/µL and 466.5 ng/µL of dsRNA was present in 80 µL total RNA extracted from 108 E. coli cells expressing 765 bp and 401 bp dsRNAs, respectively. CONCLUSIONS: Stable isotope labeling of dsRNA in conjunction with MS enabled the characterisation and quantification of dsRNA in complex total RNA mixtures.

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.

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.

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.

MassCascade: Visual Programming for LC-MS Data Processing in Metabolomics.

Liquid chromatography coupled to mass spectrometry (LC-MS) is commonly applied to investigate the small molecule complement of organisms. Several software tools are typically joined in custom pipelines to semi-automatically process and analyse the resulting data. General workflow environments like the Konstanz Information Miner (KNIME) offer the potential of an all-in-one solution to process LC-MS data by allowing easy integration of different tools and scripts. We describe MassCascade and its workflow plug-in for processing LC-MS data. The Java library integrates frequently used algorithms in a modular fashion, thus enabling it to serve as back-end for graphical front-ends. The functions available in MassCascade have been encapsulated in a plug-in for the workflow environment KNIME, allowing combined use with e.g. statistical workflow nodes from other providers and making the tool intuitive to use without knowledge of programming. The design of the software guarantees a high level of modularity where processing functions can be quickly replaced or concatenated. MassCascade is an open-source library for LC-MS data processing in metabolomics. It embraces the concept of visual programming through its KNIME plug-in, simplifying the process of building complex workflows. The library was validated using open data.

Metabolic differences in ripening of Solanum lycopersicum 'Ailsa Craig' and three monogenic mutants.

Application of mass spectrometry enables the detection of metabolic differences between groups of related organisms. Differences in the metabolic fingerprints of wild-type Solanum lycopersicum and three monogenic mutants, ripening inhibitor (rin), non-ripening (nor) and Colourless non-ripening (Cnr), of tomato are captured with regard to ripening behaviour. A high-resolution tandem mass spectrometry system coupled to liquid chromatography produced a time series of the ripening behaviour at discrete intervals with a focus on changes post-anthesis. Internal standards and quality controls were used to ensure system stability. The raw data of the samples and reference compounds including study protocols have been deposited in the open metabolomics database MetaboLights via the metadata annotation tool Isatab to enable efficient re-use of the datasets, such as in metabolomics cross-study comparisons or data fusion exercises.

Target list building for volatile metabolite profiling of fruit.

Fruit flavour is the combination of numerous biochemicals: sugars for sweetness, acids for sourness and volatile metabolites for aroma. The objective of this study was to establish a method to develop a target list of statistically relevant compounds for the characterization of melon from non-targeted data, while preserving the profile information. Five different varieties were sampled (sampling 12 biological replicates from 12 plants) using dynamic headspace extraction, then analysed by gas chromatography-mass spectrometry in full scan mode. Using Metalign and SIMCA-P software the raw data was spectrally aligned and then subjected to principal component analysis (PCA). The principal component analysis plot showed good separation of the five varieties based on their full scan GC-MS profile. Mass spectral data points responsible for the differences between varieties were highlighted by further statistical analysis. The mass spectra were then reconstructed and the corresponding chemicals identified using library search or reference standards were available to create a new target component list. To validate the new target list, the initial data set was re-processed using the targeted approach and the results subjected again to principal component analysis. The two representations showed excellent agreement on the separation of the five varieties. The new target list obtained from this study can be applied to differentiate and characterize the volatile profile of melon varieties using a list of statistically significant compounds.