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.

Massive Chondrolysis and Joint Destruction after Artificial Anterior Cruciate Ligament Repair.

The Ligament Augmentation Reconstruction System (LARS) is an artificial ligament made of polyethylene terephthalate (PET) used for anterior cruciate ligament (ACL) reconstruction in Australia. Poor results with previous generations of synthetic grafts causing synovitis, graft failure, and premature osteoarthritis have encouraged the production of the newer LARS ligament with good results. We present a case of massive chondrolysis and joint destruction after LARS implantation requiring total knee replacement in a 23-year-old male. This case documents a rare and severe complication to the LARS ligament as caution for the implementation of this device in young athlete.

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.

Amino acid misincorporation in recombinant biopharmaceutical products.

Microbial and mammalian host systems have been used extensively for the production of protein biotherapeutics. Generally these systems rely on the production of a specific gene sequence encoding one therapeutic product. Analysis of these protein products over many years has proven that this was not always the case, with multiple species of the intended product being produced due to amino acid misincorporation or mistranslation during biosynthesis of the protein. This review is the first to give a comprehensive overview of the occurrence and analysis of these misincorporations. Furthermore, using the latest data on misincorporation in native human proteins we explore potential considerations for producing a specification for misincorporation for the development of a human biotherapeutic protein product in a production environment.

Characterising microbial protein test substances and establishing their equivalence with plant-produced proteins for use in risk assessments of transgenic crops.

Most commercial transgenic crops are genetically engineered to produce new proteins. Studies to assess the risks to human and animal health, and to the environment, from the use of these crops require grams of the transgenic proteins. It is often extremely difficult to produce sufficient purified transgenic protein from the crop. Nevertheless, ample protein of acceptable purity may be produced by over-expressing the protein in microbes such as Escherichia coli. When using microbial proteins in a study for risk assessment, it is essential that their suitability as surrogates for the plant-produced transgenic proteins is established; that is, the proteins are equivalent for the purposes of the study. Equivalence does not imply that the plant and microbial proteins are identical, but that the microbial protein is sufficiently similar biochemically and functionally to the plant protein such that studies using the microbial protein provide reliable information for risk assessment of the transgenic crop. Equivalence is a judgement based on a weight of evidence from comparisons of relevant properties of the microbial and plant proteins, including activity, molecular weight, amino acid sequence, glycosylation and immuno-reactivity. We describe a typical set of methods used to compare proteins in regulatory risk assessments for transgenic crops, and discuss how risk assessors may use comparisons of proteins to judge equivalence.

Determination and control of low-level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system.

Escherichia coli is used extensively in the production of proteins within biotechnology for a number of therapeutic applications. Here, we discuss the production and overexpression of the potential biopharmaceutical human thioredoxin protein (rhTRX) within E. coli. Overexpression of foreign molecules within the cell can put an enormous amount of stress on the translation machinery. This can lead to a misfiring in the construction of a protein resulting in populations differing slightly in amino acid composition. Whilst this may still result in a population of active molecules being expressed, it does present significant problems with molecules that are destined for clinical applications. Amino acid misincorporation of this subset could potentially result in antibodies being raised to these unnatural proteins. Cross-reaction with a patient's endogenous thioredoxin could then lead to an autoimmune phenomena and serious health implications. Generally, the issue of misincorporation appears not to be a routine regulatory concern (see ICH Q6B guidelines). Therefore, amino acid misincorporation may not have been detected, much less explored in the clinic as the occurrence or absence of these random errors is not routinely reported. Using current technologies based on proteomics, the ability to find misincorporation critically depends upon the criteria for matching theoretical and experimental mass spectrometry data. Additionally, isolation and extraction of these mistranslated proteins from the production process is both difficult and expensive. Therefore, it is advantageous to find routes for removing their production during the upstream phase. In this study, we show how modern proteomic technology can be used to identify and quantify amino acid misincorporation. Using these techniques we have shown how manipulation of gene sequence and scoping of fermentation media composition can lead to the reduction and elimination of these misincorporations in rhTRX.

Capillary liquid chromatography/atmospheric-pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry: a comparison with liquid chromatography/matrix-assisted laser desorption/ionisation time-of-flight and liquid chromatography/electrospray ionisation quadrupole time-of-flight for the identification of tryptic peptides.

The atmospheric-pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI-QIT) analysis of tryptic peptides is reported following capillary liquid chromatographic (LC) separation and direct analysis of a protein digest. Peptide fragments were identified by peptide mass fingerprinting from mass spectrometric data and sequence analysis obtained by tandem mass spectrometry of the principal mass spectral peaks using a data-dependent scanning protocol. These data were compared with those from mass spectrometric analysis using capillary LC/MALDI-time-of-flight (TOF) and capillary LC/electrospray ionisation (ESI)-quadrupole TOF. For all three configurations the resulting data were searched against the MSDB database, using MASCOT and the sequence coverage compared for each technique. Complementary data were obtained using the three techniques.