Comprehensive chromatographic assessment of forced degraded in vitro transcribed mRNA.

Heightened interest in messenger RNA (mRNA) therapeutics has accelerated the need for analytical methodologies that facilitate the production of supplies for clinical trials. Forced degradation studies are routinely conducted to provide an understanding of potential weak spots in the molecule that are exploited by stresses encountered during bulk purification, production, shipment, and storage. Consequently, temperature fluctuations and excursions are often experienced during these unit operations and may accelerate mRNA degradation. Here, we present a concise panel of chromatography-based stability-indicating assays for evaluating thermally stressed in vitro transcribed (IVT) mRNA as part of a forced degradation study. We found that addition of EDTA to the mRNAs prior to heat exposure reduced the extent of degradation, suggesting that transcripts may be fragmenting via a divalent metal-ion mediated pathway. Trace divalent metal contamination that can accelerate RNA instability is likely carried over from upstream steps. We demonstrate the application of these methods to evaluate the critical quality attributes (CQAs) of mRNAs as well as to detect intrinsic process- and product-related impurities.

Deadenylase-dependent mRNA decay of GDF15 and FGF21 orchestrates food intake and energy expenditure.

Hepatokines, secretory proteins from the liver, mediate inter-organ communication to maintain a metabolic balance between food intake and energy expenditure. However, molecular mechanisms by which hepatokine levels are rapidly adjusted following stimuli are largely unknown. Here, we unravel how CNOT6L deadenylase switches off hepatokine expression after responding to stimuli (e.g., exercise and food) to orchestrate energy intake and expenditure. Mechanistically, CNOT6L inhibition stabilizes hepatic Gdf15 and Fgf21 mRNAs, increasing corresponding serum protein levels. The resulting upregulation of GDF15 stimulates the hindbrain to suppress appetite, while increased FGF21 affects the liver and adipose tissues to induce energy expenditure and lipid consumption. Despite the potential of hepatokines to treat metabolic disorders, their administration therapies have been challenging. Using small-molecule screening, we identified a CNOT6L inhibitor enhancing GDF15 and FGF21 hepatokine levels, which dramatically improves diet-induced metabolic syndrome. Our discovery, therefore, lays the foundation for an unprecedented strategy to treat metabolic syndrome.

Quantitative and Qualitative Assessment of Small RNA Preparations.

Recent advances in high-throughput sequencing have shed new light on the diversity of small noncoding RNA (sncRNA) classes and their crucial roles in gene regulation and disease. One key step in sncRNA profiling consists in their quantification and assessment of their degradation extent. In this chapter, we will describe different gold standard methods used to achieve both purposes before using the sncRNAs in downstream applications.

Stability of SARS-CoV-2 RNA in Nonsupplemented Saliva.

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.

Computational assessment of thermostability in miRNA:CNT system using molecular dynamics simulations.

BACKGROUND: Carbon nanotubes (CNTs) show great promise as theranostic agents due to their drug delivery properties, intrinsic near-infrared radiation-responsiveness, and magnetic functionalization. However, temperature elevation caused by these external stimuli during drug delivery should be considered for the evaluation of CNT-based systems loaded with temperature-sensitive biomolecules. METHODS: We examine the thermal stability of a 33 nucleotides long hairpin miRNA encapsulated in (20,20) CNT using all-atom molecular dynamics simulations in explicit water. We systematically increase the temperature as 298, 310, 327, and 343 K, reaching the melting temperature of miRNA. To emphasize the effect of the aromatic confined space, we compare the dynamics of miRNA inside the CNT to its dynamics free in the solution at the same temperatures, reaching a total simulation time of 7.9 µs. RESULTS: miRNA hairpin mostly maintains its double-stranded structure in the confined CNT, even at elevated temperatures. Binding free energies and potential of mean force calculations also underline the strong π-π interactions between the biomolecule and the CNT for 298-343 K. CONCLUSION: The let-7 miRNA mimic, which represents a wide family of RNAi-based therapeutics, can be transported in the CNT under medically applied hyperthermic conditions. GENERAL SIGNIFICANCE: This study shows how the structure and dynamics of miRNA hairpin are affected when encapsulated in an aromatic tube, during a systematic increase of temperature. It also indicates the high potential of CNT-based systems for the delivery of oligonucleotide therapeutics while simultaneous imaging/magnetic field guiding to the target tissue is achieved.

Quality assessment of RNA in long-term storage: The All Our Families biorepository.

BACKGROUND: The All Our Families (AOF) cohort study is a longitudinal population-based study which collected biological samples from 1948 pregnant women between May 2008 and December 2010. As the quality of samples can decline over time, the objective of the current study was to assess the association between storage time and RNA (ribonucleic acid) yield and purity, and confirm the quality of these samples after 7-10 years in long-term storage. METHODS: Maternal whole blood samples were previously collected by trained phlebotomists and stored in four separate PAXgene Blood RNA Tubes (PreAnalytiX) between 2008 and 2011. RNA was isolated in 2011 and 2018 using PAXgene Blood RNA Kits (PreAnalytiX) as per the manufacturer's instruction. RNA purity (260/280), as well as RNA yield, were measured using a Nanodrop. The RNA integrity number (RIN) was also assessed from 5-25 and 111-130 months of storage using RNA 6000 Nano Kit and Agilent 2100 BioAnalyzer. Descriptive statistics, paired t-test, and response feature analysis using linear regression were used to assess the association between various predictor variables and quality of the RNA isolated. RESULTS: Overall, RNA purity and yield of the samples did not decline over time. RNA purity of samples isolated in 2011 (2.08, 95% CI: 2.08-2.09) were statistically lower (p<0.000) than samples isolated in 2018 (2.101, 95% CI: 2.097, 2.104), and there was no statistical difference between the 2011 (13.08 µg /tube, 95% CI: 12.27-13.89) and 2018 (12.64 µg /tube, 95% CI: 11.83-13.46) RNA yield (p = 0.2964). For every month of storage, the change in RNA purity is -0.01(260/280), and the change in RNA yield between 2011 and 2018 is -0.90 µ g / tube. The mean RIN was 8.49 (95% CI:8.44-8.54), and it ranged from 7.2 to 9.5. The rate of change in expected RIN per month of storage is 0.003 (95% CI 0.002-0.004), so while statistically significant, these results are not relevant. CONCLUSIONS: RNA quality does not decrease over time, and the methods used to collect and store samples, within a population-based study are robust to inherent operational factors which may degrade sample quality over time.

Longitudinal assessment and stability of long non-coding RNA gene expression profiles measured in human peripheral whole blood collected into PAXgene blood RNA tubes.

OBJECTIVE: Long non-coding RNAs (lncRNAs) are emerging as novel biomarkers for a variety of chronic conditions including autoimmune disease. PAXgene Blood RNA tubes are routinely used in clinical research and molecular diagnostic development to capture RNA profiles in peripheral whole blood. While the stability of mRNA expression profiles captured using PAXgene tubes has been documented previously, no previous work has determined the stability of lncRNA expression profiles observed in PAXgene tubes stored at - 80 °C. Here we sought to determine the effects on lncRNA expression profiles following - 80 °C storage of total RNA templates, cDNA synthesized using fresh or frozen total RNA template, and the impact of freeze-thaw cycles on both total RNA and cDNA obtained from PAXgene tubes. RESULTS: We find that storage of whole blood in PAXgene tubes, total RNA and cDNA for up to 1 year at - 80 °C or up to ten total RNA or cDNA freeze-thaw cycles do not significantly alter lncRNA expression profiles compared to baseline. As monthly expression profiles were determined, some month to month lncRNA expression variability was observed. However, all monthly observations fell within the 95% confidence interval calculated at baseline.

Rapid and Cost-Effective RNA Extraction of Rat Pancreatic Tissue.

Regardless of the extraction method, optimized RNA extraction of tissues and cell lines are carried out in four stages: 1) homogenization, 2) effective denaturation of proteins from RNA, 3) ribonuclease inactivation, and 4) removal of contamination from DNA, proteins, and carbohydrates. However, it is very laborious to maintain the integrity of RNA when there are high levels of RNase in the tissue. Spontaneous autolysis makes it very difficult to extract RNA from pancreatic tissue without damaging it. Thus, a practical RNA extraction method is needed to maintain the integrity of pancreatic tissues during the extraction process. An experimental and comparative study of existing protocols was carried out by obtaining 20-30 mg of rat pancreatic tissues in less than 2 minutes and extracting the RNA. The results were assessed by electrophoresis. The experiments were carried out three times for generalization of the results. Immersing pancreatic tissue in RNA stabilization reagent at -80 °C for 24 h yielded high integrity RNA, when the RNA extraction reagent was used as the reagent. The results obtained were comparable to the results obtained from commercial kits with spin column bindings.

SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification.

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has had an enormous impact on society worldwide, threatening the lives and livelihoods of many. The effects will continue to grow and worsen if economies begin to open without the proper precautions, including expanded diagnostic capabilities. To address this need for increased testing, we have developed a sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay compatible with current reagents, which utilizes a colorimetric readout in as little as 30 min. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was optimized to increase sensitivity and sample stability. This protocol, combined with the RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with the RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. This simple inactivation and purification pipeline is inexpensive and compatible with other downstream RNA detection platforms and uses readily available reagents. It should increase the availability of SARS-CoV-2 testing as well as expand the settings in which this testing can be performed.

Invention and Early History of Gapmers.

Gapmers are antisense oligonucleotides composed of a central DNA segment flanked by nucleotides of modified chemistry. Hybridizing with transcripts by sequence complementarity, gapmers recruit ribonuclease H and induce target RNA degradation. Since its concept first emerged in the 1980s, much work has gone into developing gapmers for use in basic research and therapy. These include improvements in gapmer chemistry, delivery, and therapeutic safety. Gapmers have also successfully entered clinical trials for various genetic disorders, with two already approved by the U.S. Food and Drug Administration for the treatment of familial hypercholesterolemia and transthyretin amyloidosis-associated polyneuropathy. Here, we review the events surrounding the early development of gapmers, from conception to their maturity, and briefly conclude with perspectives on their use in therapy.

Contributions and competition of Mg2+ and K+ in folding and stabilization of the Twister ribozyme.

Native folded and compact intermediate states of RNA typically involve tertiary structures in the presence of divalent ions such as Mg2+ in a background of monovalent ions. In a recent study, we have shown how the presence of Mg2+ impacts the transition from partially unfolded to folded states through a "push-pull" mechanism where the ion both favors and disfavors the sampling of specific phosphate-phosphate interactions. To further understand the ion atmosphere of RNA in folded and partially folded states results from atomistic umbrella sampling and oscillating chemical potential grand canonical Monte Carlo/molecular dynamics (GCMC/MD) simulations are used to obtain atomic-level details of the distributions of Mg2+ and K+ ions around Twister RNA. Results show the presence of 100 mM Mg2+ to lead to increased charge neutralization over that predicted by counterion condensation theory. Upon going from partially unfolded to folded states, overall charge neutralization increases at all studied ion concentrations that, while associated with an increase in the number of direct ion-phosphate interactions, is fully accounted for by the monovalent K+ ions. Furthermore, K+ preferentially interacts with purine N7 atoms of helical regions in partially unfolded states, thereby potentially stabilizing the helical regions. Thus, both secondary helical structures and formation of tertiary structures leads to increased counterion condensation, thereby stabilizing those structural features of Twister. Notably, it is shown that K+ can act as a surrogate for Mg2+ by participating in specific interactions with nonsequential phosphate pairs that occur in the folded state, explaining the ability of Twister to self-cleave at submillimolar Mg2+ concentrations.

Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay.

BACKGROUND: The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation. METHODS: IAV was irradiated with 253.7 nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays. RESULTS: A long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3' termini of each genomic segment and subsequent qPCR of the 5' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R2 = 0.931, P = 0.000066). CONCLUSIONS: This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.

Low-cost and user-friendly biosensor to test the integrity of mRNA molecules suitable for field applications.

The use of mRNA in biotechnology has expanded with novel applications such as vaccines and therapeutic mRNA delivery recently demonstrated. For mRNA to be used in patients, quality control assays will need to be routinely established. Currently, there is a gap between the highly sophisticated RNA integrity tests available and broader application of mRNA-based products by non-specialist users, e.g. in mass vaccination campaigns. Therefore, the aim of this work was to develop a low-cost biosensor able to test the integrity of a mRNA molecule with low technological requirements and easy end-user application. The biosensor is based on a bi-functional fusion protein, composed by the λN peptide that recognizes its cognate aptamer encoded on the 5' end of the RNA under study and ß-lactamase, which is able to produce a colorimetric response through a simple test. We propose two different mechanisms for signal processing adapted to two levels of technological sophistication, one based on spectrophotometric measurements and other on visual inspection. We show that the proposed λN-ßLac chimeric protein specifically targets its cognate RNA aptamer, boxB, using both gel shift and biolayer interferometry assays. More importantly, the results presented confirm the biosensor performs reliably, with a wide dynamic range and a proportional response at different percentages of full-length RNA, even when gene-sized mRNAs were used. Thus, the features of the proposed biosensor would allow to end-users of products such as mRNA vaccines to test the integrity of the product before its application in a low-cost fashion, enabling a more reliable application of these products.

Central dogma rates and the trade-off between precision and economy in gene expression.

Steady-state protein abundance is set by four rates: transcription, translation, mRNA decay and protein decay. A given protein abundance can be obtained from infinitely many combinations of these rates. This raises the question of whether the natural rates for each gene result from historical accidents, or are there rules that give certain combinations a selective advantage? We address this question using high-throughput measurements in rapidly growing cells from diverse organisms to find that about half of the rate combinations do not exist: genes that combine high transcription with low translation are strongly depleted. This depletion is due to a trade-off between precision and economy: high transcription decreases stochastic fluctuations but increases transcription costs. Our theory quantitatively explains which rate combinations are missing, and predicts the curvature of the fitness function for each gene. It may guide the design of gene circuits with desired expression levels and noise.

Assessment of RNA Stability in Postmortem Tissue from New-Born Lambs.

The recovery of high quality RNA from postmortem tissue is crucial to gene expression analyses. The acquisition of postmortem tissue has inherent time delays and, hence, understanding the temporal variation in the stability of total RNA is imperative. This experiment aimed: ( 1 ) to qualitatively and quantitatively assess the integrity of total RNA derived from a range of new-born ovine tissues (liver, spleen, thyroid, skeletal muscle, ileum, and perirenal adipose tissue) which were stored at ambient temperature until extraction at 0, 3, 6, and 9 h postmortem; and ( 2 ) to analyze the stability of the reference gene(s) and expression of specific target genes in these tissues. Postmortem sampling time resulted in variable reductions in the relative integrity number (RIN) values across the tissues, ranging from 0.9 to 1.8% in liver, spleen, skeletal muscle, and ileum to 5.7-11.1% in the thyroid and perirenal adipose tissues, respectively (P < 0.05). In conclusion, tissues with small reductions in RIN value can exhibit disproportionately large differences in the normalization factor used to calculate the target gene expression. Hence, changes in transcript abundance due to RNA degradation are not always sufficiently buffered through normalization with reference genes. The normalization factor should be presented alongside the RIN value in postmortem tissue studies.

Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation.

The effectiveness and economics of polyvinyl sulfonic acid (PVSA) as a ribonuclease inhibitor for in vitro systems is reported. PVSA was shown to inhibit RNA cleavage in the presence of RNase A as well as in the presence of Escherichia coli lysate, suggesting that PVSA can act as a broader ribonuclease inhibitor. In addition, PVSA was shown to improve the integrity of mRNA transcripts by up to 5-fold in vitro as measured by their translational viability. Improved preservation of mRNA transcripts in the presence of PVSA under common RNA storage conditions is also reported. A cost comparison with commercially available RNAse inhibitors indicates the economic practicality of PVSA which is approximately 1,700 times less expensive than commonly used ribonuclease inhibitors. PVSA can also be separated from RNA by alcohol precipitation for applications that may be sensitive to the presence of PVSA.

Assessment and comparison of thermal stability of phosphorothioate-DNA, DNA, RNA, 2'-F RNA, and LNA in the context of Phi29 pRNA 3WJ.

The question of whether RNA is more stable or unstable compared to DNA or other nucleic acids has long been a subject of extensive scrutiny and public attention. Recently, thermodynamically stable and degradation-resistant RNA motifs have been utilized in RNA nanotechnology to build desired architectures and integrate multiple functional groups. Here we report the effects of phosphorothioate deoxyribonucleotides (PS-DNA), deoxyribonucleotides (DNA), ribonucleotides (RNA), 2'-F nucleotides (2'-F), and locked nucleic acids (LNA) on the thermal and in vivo stability of the three-way junction (3WJ) of bacteriophage phi29 motor packaging RNA. It was found that the thermal stability gradually increased following the order of PS-DNA/PS-DNA < DNA/DNA < DNA/RNA < RNA/RNA < RNA/2'-F RNA < 2'-F RNA/2'-F RNA < 2'-F RNA/LNA < LNA/LNA. This proposition is supported by studies on strand displacement and the melting of homogeneous and heterogeneous 3WJs. By simply mixing different chemically modified oligonucleotides, the thermal stability of phi29 pRNA 3WJ can be tuned to cover a wide range of melting temperatures from 21.2°C to over 95°C. The 3WJLNA was resistant to boiling temperature denaturation, urea denaturation, and 50% serum degradation. Intravenous injection of fluorescent LNA/2'-F hybrid 3WJs into mice revealed its exceptional in vivo stability and presence in urine. It is thus concluded that incorporation of LNA nucleotides, alone or in combination with 2'-F, into RNA nanoparticles derived from phi29 pRNA 3WJ can extend the half-life of the RNA nanoparticles in vivo and improve their pharmacokinetics profile.

Nuclear Magnetic Resonance Study of RNA Structures at the 3'-End of the Hepatitis C Virus Genome.

The 3'-end of the genomic RNA of the hepatitis C virus (HCV) embeds conserved elements that regulate viral RNA synthesis and protein translation by mechanisms that have yet to be elucidated. Previous studies with oligo-RNA fragments have led to multiple, mutually exclusive secondary structure predictions, indicating that HCV RNA structure may be context-dependent. Here we employed a nuclear magnetic resonance (NMR) approach that involves long-range adenosine interaction detection, coupled with site-specific 2H labeling, to probe the structure of the intact 3'-end of the HCV genome (385 nucleotides). Our data reveal that the 3'-end exists as an equilibrium mixture of two conformations: an open conformation in which the 98 nucleotides of the 3'-tail (3'X) form a two-stem-loop structure with the kissing-loop residues sequestered and a closed conformation in which the 3'X rearranges its structure and forms a long-range kissing-loop interaction with an upstream cis-acting element 5BSL3.2. The long-range kissing species is favored under high-Mg2+ conditions, and the intervening sequences do not affect the equilibrium as their secondary structures remain unchanged. The open and closed conformations are consistent with the reported function regulation of viral RNA synthesis and protein translation, respectively. Our NMR detection of these RNA conformations and the structural equilibrium in the 3'-end of the HCV genome support its roles in coordinating various steps of HCV replication.