Omicron, the current SARS-CoV-2 variant of concern, is much more contagious than other previous variants. Whether strict lockdown could effectively curb the transmission of Omicron is largely unknown. In this retrospective study, we compared the strictness of government lockdown policies in Shanghai and other countries. Based on the daily Omicron case number from 1 March 2022 to 30 April 2022, the effective reproductive numbers in this Shanghai Omicron wave were calculated to confirm the impact of strict lockdown on Omicron transmission. Pearson correlation was conducted to illustrate the determining factor of strict lockdown outcomes in the 16 different districts of Shanghai. After a very strict citywide lockdown since April 1st, the average daily effective reproductive number reduced significantly, indicating that strict lockdown could slow down the spreading of Omicron. Omicron control is more challenging in districts with higher population mobility and lockdown is more likely to decrease the number of asymptomatic carriers than the symptomatic cases. All these findings indicate that the strict lockdown could curb the transmission of Omicron effectively, especially for the asymptomatic spread, and suggest that differentiated COVID-19 prevention and control measures should be adopted according to the population density and demographic composition of each community. Since December 2019, the COVID-19 outbreak suddenly and rapidly spread all over the world .On 2 November 2021, the novel SARS-CoV-2 VOC Omicron was first collected in South Africa and then spread rapidly . It causIn this article, we compared the strictness of the Shanghai lockdown policy with that of other countries. We further evaluated the effect of strict lockdown on Omicron spread in Shanghai since 1 April 2022 and identified critical factors to make the lockdown strategy work optimally. The asymptomatic rate in Shanghai was also analyzed. We expect the experience of the Shanghai Omicron epidemic could provide valuable information to other countries encountering highly transmissible SARS-CoV-2 strains in the future. We would like to suggest authorities formulate differentiated lockdown strategies according to the population density and demographic composition of each community, in which way COVID-19 waves can be controlled effectively with minimal social, economic, and psychological costs.The daily case number in this Shanghai Omicron wave from 1 March 2022 to 30 April 2022 was retrieved from Shanghai Municipal Health Commission Database. Subway ridership dataset of Shanghai was shared by Chinese Software Developer Network (CSDN).Stage I: Normal COVID-19 Prevention and Control Measures Stage from March 1st to March 12th when minimum requirements were imposed on majority of the citizens; Stage II: Precise Epidemic Control Stage from March 13th to March 31st where only residents of certain communities at high risk were quarantined; Stage III: Citywide Lockdown Stage since April 1st when the whole city is shut down until gradually opened.To quantify the strictness of government policies, we followed the Oxford Coronavirus Government Response Tracker project  which orThe raw data of daily new infections during the three PHSM stages was smoothed following a 7-day averaged, 4th-order polynomial method. The asymptomatic rate was calculated in the following Equation (1) and smoothed:The trend of the Omicron wave in each district was plotted in the same way.Estimation of the effective reproductive number Rt is a reliable and common way to evaluate changes in disease transmission over time. It has been widely used in the COVID-19 pandemic to help policymakers and public health officials to assess the effectiveness of interventions . Based oIn order to better assess the effect of lockdown, we defined the leading time from the lockdown starting date to the daily Omicron case peaking date as the effective interval (EI) of lockdown. We further evaluated the impact of infected cases and population mobility on EI. In this correlation analysis, two algorithms, infection index, and active infection index were involved:The infection index only considers the number of daily Omicron cases on the day before lockdown.The active infection index considers both the number of daily Omicron cases on the day before lockdown and population mobility. In Equation (3) the daily subway ridership was used as an indicator of the population mobility .The correlation analysis between these two indices with the EI of 16 districts in Shanghai was examined by two-tailed Pearson correlation in GraphPad Prism 9.3.1.Shanghai is one of the largest cities in the world with a population of 25 million. On 1 March 2022, Omicron variant BA.2 hit Shanghai and the case number increased rapidly in the following weeks. The Shanghai government decided to implement a “static management” lockdown in the eastern half of the city on March 28th and then in the whole city on April 1st, to curb Omicron from rapid spreading. During the lockdown time, all schools and workplaces were closed except for necessary health care services including hospitals and COVID-19 testing providers. Public transport was closed, and a “stay-at-home” order was given to almost all residents in the city except those working on Omicron prevention and control. All public events were canceled, and gatherings were restricted. On June 1st, Shanghai started to open up gradually, tentatively, and cautiously.The calculated stringency index of the Shanghai lockdown is 97, which is only slightly lower than the 100 of the India lockdown, but higher than all other countries . ShanghaAs aforementioned in the Methods section, we classified three PHSM stages. The daily stringency index during Stage I was merely nine as only individuals with a travel history of the medium- or high-risk regions were monitored. As Shanghai started to control certain communities at high risk in a “2+12” manner in Stage II, the daily stringency index became 45. After the “static management” in the eastern half of Shanghai on March 28th, the daily stringency index further increased to 66. Implementation of lockdown policy in Stage III finally reached a daily stringency index of 97  of the active infection index, the daily subway ridership in each district of Shanghai was adopted as the indicator of population mobility [p = 0.0145), showing both the higher number of infected individuals and the extensive personnel movement before lockdown brings challenges to curbing the Omicron epidemic. This result demonstrates that timely lockdown before the surge of case number is important for rapid control of Omicron transmission, especially for those urban regions with high population mobility.We tried to interpret the significant difference in EI in the 16 districts under the same lockdown policy. As more infections will put a bigger population at risk of Omicron, we analyzed the impact of daily new infections before lockdown on EI. Infection index, an algorithm only considering daily new infections, was introduced and its calculation was given in Equation (2). The infection index of each district was then plotted against the corresponding EI as shown in mobility . The corAsymptomatic spread is a characteristic feature of Omicron . In thisIn order to understand the effect of lockdown on the asymptomatic transmission of Omicron, we analyzed the asymptomatic rate before and after lockdown B. The daIn the Shanghai Omicron wave, the municipal government implemented the strictest lockdown policy. These measures effectively stopped the spreading of Omicron, especially its asymptomatic transmission. Through correlation analysis, we found that timely lockdown before the surge of case numbers is critical for putting the Omicron epidemic under control in urban regions. Previous work also utilized the same Oxford Coronavirus Government Response Tracker project to evaluate the association between physical distancing interventions and the incidence of COVID-19 in 149 countries . Among tSome other previous studies investigated the impacts of New Zealand’s graduated, risk-informed national COVID-19 suppression measures in early 2020 on the epidemiology of COVID-19 . They diIn previous studies, Patricio et. al. conducted an empirical analysis of the impact of lockdown on SARS-CoV-2 transmission and death toll for a panel of 152 countries . This stDue to privacy concerns, COVID-19 data acquired and released by governments and healthcare authorities are mostly only available at a relatively coarse level, which is usually limited to the nationwide level instead of down to local areas ,28. Of nThe epidemiological data analyzed in this study were collected through numerous thoughtful PCR tests during the 2-month lockdown. Considering that majority of the countries have been adopting loose PHSM, this Omicron outbreak in Shanghai provides a precious chance to accurately evaluate the effectiveness of strict lockdown measures on curbing the spreading of the highly contagious Omicron variant. However, the estimation of population mobility in this study was limited to subway ridership, as other public transportation and personal vehicle information are unavailable.After its continuous evolution in the past two years, the new strains of SARS-CoV-2 keep presenting new features that bring challenges to COVID-19 prevention and control. This study confirmed that strict lockdown measures are capable of effectively curbing the transmission of Omicron, the current highly contagious VOC. The success of lockdown measures in Shanghai can be largely attributed to their potency in cutting the asymptomatic spread, which can trigger the COVID-19 explosion unexpectedly. Based on our studies, we suggest that differentiated lockdown strategies should be formulated for each community according to its population density and demographic composition in order to reduce the social, economic, and psychological costs of city-wide lockdown.


PPR), organelle zinc-finger (OZI), and multiple organellar RNA editing factors/RNA editing factor interacting proteins genes in control and treatment, results revealed upregulation of PPR and OZ1 genes in treated samples. This induction showed the role of these genes in RNA editing. The current findings report that RNA editing increased under alkaline stress which may contribute in adaptation for rice by changing amino acids in edited genes (88 genes). These findings will provide basis for identification of RES in other crops and also will be useful in alkaline tolerance development in rice.Ribonucleic acid editing (RE) is a post-transcriptional process that altered the genetics of RNA which provide the extra level of gene expression through insertion, deletions, and substitutions. In animals, it converts nucleotide residues C-U. Similarly in plants, the role of RNA editing sites (RES) in rice under alkaline stress is not fully studied. Rice is a staple food for most of the world population. Alkaline stress cause reduction in yield. Here, we explored the effect of alkaline stress on RES in the whole mRNA from rice chloroplast and mitochondria. Ribonucleic acid editing sites in both genomes (3336 RESs) including chloroplast (345 RESs) and mitochondria (2991 RESs) with average RES efficiency ∼55% were predicted. Our findings showed that majority of editing events found in non-synonymous codon changes and change trend in amino acids was hydrophobic. Four types of RNA editing A-G (A-I), C-T (C-U), G-A, and T-C were identified in treated and untreated samples. Overall, RNA editing efficiency was increased in the treated samples. Analysis of Gene Ontology revealed that mapped genes were engaged in many biological functions and molecular processes. We also checked the expression of pentatricopeptide repeat ( Arabidopsis , goatgrass (Aegilops tauschii), rice (Oryza sativa), maize (Zea mays), tobacco (Nicotiana tabacum), and grape (Vitis vinifera)  .The most common editing types in non-flowering land plants are adenosine (A) to inosine (I) in tRNA, cytidine (C) to uridine (U) in messenger tRNA and RNA, and uridine (U) to cytidine (C) in mRNA . Protein(ORRM) protein, plant polyphenol oxidase (PPO), PPR, OZ, and RIP/MORF  with the BioProject PRJNA414178.2 Reference genomes and annotation files of mitochondria (BA000029.3) and chloroplast (KT289404.1) were also accessed from NCBI in fasta format. Quality of sequence reads was assessed by using FastQC tool3 to identify low-quality reads and adapter sequences (Two rice genotypes Caidao (CD) which was alkaline-sensitive and WD20342 (WD) an alkaline-tolerant were assessed in control and alkaline environments . The traequences . Hisat2 equences . Number equences . The frap-values were adjusted  analysis was performed to identify over-represented biological processes (BP), molecular functions (MF), and cellular components (CC) using the identified genes. The gene list was submitted to the ClueGO is a Cytoscapenotation . Gene on5 (6) which utilize BAM files were used for SNP calling  was used to annotate the RNA editing site  was applied for 36 h to create alkaline stress. The leaf samples were collected in triplicates. The total RNA was extracted with the help of TRIzol method and complementary DNA was prepared by using reverse transcriptase-III, first strand cDNA Synthesis Kit . Real-time PCR for quantification (qRT-PCR) of selected genes of PPR, OZ1, and MORF/RIP gene family members was performed by using StepOne RT-PCR (Applied Biosystems® 7900 HT Fast RT-PCR). Three biological replicates from control and treated samples were used. OsActin was used as an internal control and for expression calculations 2ΔΔ CT method was used (Seeds of rice genotype (IRRI6) were sterilized with 2.5% sodium hypochlorite for 35 min followed by three washes and placed in 37°C Owen for 1 day. Uniformly germinated seeds were grown under normal conditions (16-h light/8-h dark at 26 ± 2°C) for 2 weeks. For this purpose, 96-wells plates supported by a container filled with nutrients media (Yoshida) were used. After this, one batch was kept as a control and in other batch 0.5% Nawas used . List ofPlants of two rice genotypes Caidao (CD) and WD20342 (WD) were raised under normal and stress conditions. The genome size of rice mitochondrial and chloroplast genomes is 490,520 and 134,556 bp with number of genes 81 and 129, respectively . The genPPR and OZ1) involved in RNA editing.To build the sequencing profundity and unwavering quality of editing sites, the subsequent bam documents of three replicates of each genotype under each condition were converged for resulting distinguishing proof of RESs. After the SNPs calling, we found total 3,336 RNA editing sites in both genomes including 345 in chloroplast and 2,991 in mitochondria, respectively . We notiWe employed VEP tool for the identification of characteristics summary of RNA editing sites. The results revealed that there was no RNA editing site detected in the genes of chloroplast while in mitochondrial genome RNA editing in third codon position was mainly occurred  whereas Based on the genetic properties, we divided RNA editing sites in the mitochondrial genome into six different types . These tp < 0.01) difference in CD untreated and treated samples while no difference was found in WD treated and untreated samples. In mitochondria, both genotypes CD and WD under treatment showed induction in editing efficiency. Overall, the tolerant genotype showed increased efficiency.We checked the statistics of RNA editing efficiency. In total, the average editing efficiency of RESs was about 0.54. In chloroplast, the genome efficiency was 0.31, 0.48, 0.49, and 0.52 . While iWe performed cluster profiling of the chloroplast  and mitoc oxidase gene family (cox1 and 2), and maturase R (matR) were detecting in this study and heatmap of randomly selected genes were generated  helps in aerobic energy development  is not fully clear but it converts the edited amino acid to its original form. In angiosperm, one matR was reported in the fourth intron of nad1 gene , Organelle Zinc finger 1 (OZ1), and multiple organellar RNA editing factors (MORF/RIP) genes. After the blast results, we found a total of 482 genes of PPR family, 12 genes of OZ1 family, and 7 MORF/RIP genes in rice. Previously, it was reported that rice have 477 PPR genes but one more study reported 491 PPR genes in rice  is a post-transcriptional mechanism in which transcripts modified by different mutations types such as insertion, deletion, and substitutions. The RE was firstly reported in  animals . It can  animals . For pro animals  and fold animals . Editing animals . Our stu animals . Previou animals . Some st animals . In grap animals . Similar animals . Further animals .With the innovation of new sequencing approaches such as RNA sequencing, RESs were distinguished mainly in living species, particularly in plants. Current study, reported many RESs, and the statics of altering types demonstrated that RE normally happens as G-A, T-C, C-U, and A-I changes in deciphered areas of cellular organs mRNAs. We found that changes in coding regions of the proteins were found at third and first positions which changed the physiochemical properties of the amino acids . Most ofPPR), organelle zinc finger 1 (OZ1), organelle RNA recognition motif-containing protein (ORRM), protoporphyrinogen IX oxidase (PPO), and multiple organellar RNA editing factors/RNA editing factor interacting protein (MORF/RIP) genes are reported in the RNA editing , C-T (C-U), G-A, and T-C, and most editing events were identified in non-synonymous codons which increased the hydrophobicity. Overall, RNA editing efficiency was increased due to stability of editing factors in the alkaline stressed samples. Furthermore, RNA editing changes the amino acids in the edited genes which help the plants to cope abiotic situations. These outcomes will contribute to improve understanding the molecular mechanism of dynamics of gene regulation at post-transcriptional level in rice. These findings will be useful in designing alkaline tolerance breeding programs in rice.https://www.ncbi.nlm.nih.gov//bioproject/PRJNA414178.The RNA-seq data was publically available at the followimg link OR, MU, and HC performed the analysis. OR and MU wrote the original draft. MK provided research facilities and guided during the whole study. MC supervised the whole study, reviewed, and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Artificial breeding makes traits move forward in one direction and reach the extreme, such as ultra-fine wool covering the whole body of fine wool sheep. Nevertheless, many other domestic sheep remain the coarse wool type, and some mendelian genome loci have been identified as having major genes for these traits; however, the epigenetic regulation is still unclear.EDAR, FGF5, TCHH, KRT2) were obtained by comprehensive analysis of the MeRIP-seq and RNA sequence, which are closely related to primary wool follicle morphogenesis and development. The PI3K/AKT pathway was enriched through different m6A-related genes. These results provided new insights to understand the role of epigenetics in wool sheep domestication and breeding.In our study, a set of lambs with coarse wool type all over their bodies were discovered within a full-sib family during an embryo transfer experiment of merino fine wool sheep. The difference between coarse and fine wool traits were studied from the perspective of RNA modification-N6-methyladenosine. A total of 31,153 peaks were collected, including 15,968 peaks in coarse skin samples and 15,185 peaks in fine skin samples. In addition, 7208 genes were differentially m6A methylated, including 4167 upregulated and 3041 downregulated in coarse skin samples. Four key genes ( N6-methyladenosine (m6A) is a type of RNA modification which affects RNA stability, nuclear transport, splicing, and translation . m6A wasSheep can produce wool fibers for clothing and textiles. Different sheep breeds have different wool characteristics, and there are great differences in fiber diameter, crimp, and elasticity . ChineseIn the merino, therefore, as a result of long-time selective breeding, the woolly coat has been almost exclusively developed, and today it forms homogeneous fleeces covering the whole body which are produced by primary and secondary wool follicles . However2 of middle-side dorsal skin of sheep was quickly collected, and immediately frozen in liquid nitrogen for total RNA extraction. After RNA extraction, the total RNA of each sample was divided into two pools, including coarse and fine for m6A immunoprecipitation (IP). Each pool contained three mixed total RNA. Six original extractions from each pool were collected for quantitative Real-Time PCR (qRT-PCR).The one-month-old merino lambs with coarse wool were found in a merino fine wool lamb population during an embryo transfer experiment of Chinese Merino in Chifeng city, Inner Mongolia, China. Four coarse wool and three fine wool full-sib sheep with same gender   were sam2 added to the RNA tube, mixed well, and incubated at 95 °C for 5–10 min until the RNA fragments were mainly distributed in 100~200 nt. Then, 10% RNA fragments were saved as “Input” and the rest was used for m6A immunoprecipitation (IP). The specific anti-m6A antibody (Synaptic Systems) was applied for m6A IP. After that, the enriched RNA was extracted by using TRIzol reagent (Invitrogen). The stranded RNA sequencing library was constructed by KC-Digital Stranded mRNA Library Prep Kit for Illumina , as per the manufacturer’s instruction. The library products of 200–500 nt were enriched and quantified, and finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model.Total RNA was extracted from skin tissue (samples same as transcriptome sequencing) using TRIzol Reagent (Invitrogen). After RNA extraction, DNaseI was used to remove residual gDNA. RNA quality was examined by Nanodrop onec spectrophotometer , and RNA integrity was visualized by 1.0% agarose gel electrophoresis. Qualified RNA was finally quantified by Qubit3.0 with Qubit RNA Broad Range Assay kit . Subsequently, 50 μg total RNA of each sample was calculated for polyadenylated RNA enrichment by using VAHTS mRNA Capture Beads (VAHTS). This was followed by 20 mM ZnClClean reads were harvested by filtering low-quality reads and removing adaptor sequence. Clean reads were further treated with in-house scripts to eliminate duplication bias introduced in library preparation and sequencing. In brief, clean reads were first clustered according to the UMI sequences, in which reads with the same UMI sequence were grouped into the same cluster. Reads in the same cluster were compared to each other by pairwise alignment, and then reads with sequence identity over 95% were extracted to a new sub-cluster. After all sub-clusters were generated, a multiple sequence alignment was performed to get one consensus sequence for each sub-cluster. After these steps, any errors and biases introduced by PCR amplification or sequencing were eliminated. The deduplicated consensus sequences were used for m6A site analysis. They were mapped to the reference genome of Oar_rambouillet_v1.0 from NCBI database using STAR software (version 2.5.3a)  with default parameters . The exo−∆∆Ct method [A total of four differently m6A methylated genes  were selected for qRT-PCR, which was performed with SYBR Premix ExTaq  on ABI 7500 Real-Time PCR System . The cDNA synthesis of coding genes was performed using Quantscript RT Kit Quant cDNA  with approximately 500 ng of total RNA as the template; SYBR Premix ExTaq  was used for real-time PCR, which was available for an ABI7500 Real-Time PCR System (Applied Biosystems). The Tm values of each reaction were list in t method .In order to remove adaptor and low-quality data, raw data of IP and input were trimmed by perl and cutadapt. Then, the clean data were available. A total of 65,836,752 and 65,814,440 raw data were obtained from coarse and fine skin samples, respectively, including 60,538,940 and 61,402,346 clean reads, respectively. The effective clean reads accounted for 66.83 and 65.06, respectively. In the input library (RNA-seq library), 59,483,600 and 66,350,948 raw data were obtained from coarse and fine skin samples, respectively, including 52,447,544 and 59,261,076 clean reads, respectively, which accounted for 75.24% and 74.39%, respectively. The relative results are presented in The clean reads were mapped to sheep genome (Oar_rambouillet_v1.0). The results were as follows: the mapping ratios of m6A-seq library between IP samples of coarse and fine were 96.52% and 96.29%, respectively, while the mapping ratios of RNA-seq library between coarse and fine input samples were 96.03% and 96.04%, respectively. More details are shown in p < 0.05), it meant that the region was bound by protein. Then, the adjacent region was merged together as the final protein binding sites. That would be the final peaks. A total of 15,968 peaks were obtained in coarse skin samples, while 15,185 peaks were obtained in fine skin samples  was used to calculate the gene expression level in coarse and fine wool skin samples . A totalhttp://www.geneontology.org, accessed on 5 January 2021) and KEGG  databases were used to explore the related pathways of m6A modified genes [To analyze the biological function of m6A modified genes, GO (Gene Ontology, ed genes . The topIn this study, we identified some important candidate genes that were related to coarse wool development, such as EDAR, FGF5, and TCHH. Q-PCR was used to verify gene expression differences between coarse and fine wool sheep skin samples. The result of Q-PCR analysis for four differentially expressed genes were consistent with those of RNA-seq, according to which EDAR and KRT2 were upexpressed in coarse skin samples, while FGF5 and TCHH were downexpressed in coarse skin samples .In the phenotypic analysis, we found that, although the coarse and fine wool lambs are siblings from the same family, they had different wool characteristics, one group presenting heterogeneous wool characteristics and the other presenting homogenous wool characteristics. The difference between the above two groups is the presence or absence of medullated wool developed from primary wool follicles . This inFGF5 gene is a well-known gene that affects hair length, and is closely related to hair follicle development and hair shaft length in mammals, such as humans , cats 3, and sheOverall, in this study, fine and coarse wool sheep were used to study the molecular mechanism of wool follicle development. From the perspective of mRNA modification, EDAR, TCHH, and FGF5 genes, as well as PI3K/AKT signaling pathway were firstly found to be related to the generation of coarse hair phenotype. We provide a new perspective for studying the role of epigenetics in the domestication and breeding of fine wool sheep.


The discovery of ribozymes has inspired exploration of RNA’s potential to serve as primordial catalysts in a hypothesized RNA world. Modern oxidoreductase enzymes employ differential binding between reduced and oxidized forms of redox cofactors to alter cofactor reduction potential and enhance the enzyme’s catalytic capabilities. The utility of differential affinity has been underexplored as a chemical strategy for RNA. Here we show an RNA aptamer that preferentially binds oxidized forms of flavin over reduced forms and markedly shifts flavin reduction potential by −40 mV, similar to shifts for oxidoreductases. Nuclear magnetic resonance structural analysis revealed π–π and donor atom–π interactions between the aptamer and flavin that cause unfavorable contacts with the electron-rich reduced form, suggesting a mechanism by which the local environment of the RNA-binding pocket drives the observed shift in cofactor reduction potential. It seems likely that primordial RNAs could have used similar strategies in RNA world metabolisms. 2 shifted the reduction potential of the bound cofactor, similar to flavoproteins, and was shown through structural characterization to use π–π and donor–π interactions to drive the shift.An RNA aptamer that selectively binds FAD over FADH A hypothesized RNA world4 could have made extensive use of diverse RNAs to support replication and metabolism. Natural ribozymes carry out RNA cleavage, ligation and transesterification reactions1, and the ribosome catalyzes peptide bond formation5. In vitro selection experiments7 have expanded the chemical repertoire of RNA to include nucleotide synthesis8, RNA polymerization9, carbon–carbon bond formation10 and several other reactions. For modern protein enzymes, the chemical environments of substrate-binding pockets are powerful determinants of enzyme reactivity. RNA has a remarkable ability to form binding pockets for potential substrates or cofactors, as seen in the aptamer portions of riboswitches and in artificial aptamers selected in vitro12. In a few cases, structural and mechanistic studies have provided examples of how local RNA environments can influence the intrinsic activity of bound molecules. For example, fluorogenic aptamers demonstrate how global RNA folding and local environments can enhance intrinsic fluorescence by desolvating the ligand and constraining rotation of the excited state13, and several ribozymes provide examples of RNA environments that shift pKa values of bound ligands16. It seems likely that RNA binding pockets can employ additional mechanisms to influence ligand properties for catalytic use, such as by leveraging thermodynamic properties in ways proteins do through ground state perturbation and differential affinities for substrate and product.The discovery of natural RNA enzymes (ribozymes)+) and flavin adenine dinucleotide (FAD), are often used by key enzymes in metabolic pathways. They are often viewed as strong candidates for having been part of an early RNA world17, and several studies have suggested that flavin-type molecules could have formed under plausible prebiotic conditions18. Nevertheless, RNA’s ability to exploit these cofactors for redox catalysis remains underexplored. A cardinal rule of redox metabolism is that electrons flow down the energy gradient, which is defined by the midpoint reduction potentials (Em) of electron donor and acceptor species. Free flavins have Em values around −210 mV and, hence, can only transfer electrons to substrates with Em values that are more positive than this value and can only receive electrons from substrates with Em values that are more negative. Protein enzymes markedly shift the Em values of bound flavins by exploiting the energy of differential recognition of oxidized and reduced cofactors . This wide range of observed Em among flavoenzymes illustrates the tremendous power of local chemical environments within protein-binding pockets to expand the reactive capabilities of flavins so that they can participate in diverse reactions, such as the oxidation of succinate to fumarate20 or the dehalogenation of chlorophenols21. If RNA can similarly leverage differential recognition of oxidized and reduced cofactors to shift Em values, this would provide a mechanism by which primordial RNA could have catalyzed diverse metabolic reactions in an RNA world, in addition to providing a starting point for engineering diverse oxidoreductase ribozymes for synthetic biology applications.Redox reactions are foundational for modern metabolism, both for biosynthesis and catabolism. Adenosine monophosphate-containing cofactors, such as nicotinamide adenine dinucleotide  were found to leave the Em of bound PQQ unaltered in cyclic voltammetry experiments, whereas DNA aptamers binding to the adenosine of NAD+ also had no impact on Em (ref. 24). In vitro selection experiments identified an NAD+-dependent alcohol dehydrogenase ribozyme that uses NAD+ to oxidize a benzylic alcohol to an aldehyde and performs the reverse reaction with the reduced form, NADH27. This ribozyme possesses the ability to alter the rate of the oxidation and reduction reactions, a kinetic property, but changes to the Em of the cofactor, a thermodynamic property, were not reported. Ribozymes that use flavin redox cofactors, such as FAD, have not been reported. Our previous work provided an early example of an RNA that recognizes the FAD isoalloxazine ring, but the aptamer from that study, Ftest1, did not differentiate between oxidized and reduced flavins28. These examples demonstrate that differential binding to cofactors is not a generic property for nucleic acids and that appropriate binding pockets capable of impacting cofactor properties do not automatically emerge during selection experiments. In contrast, we report in this study an RNA aptamer that preferentially binds oxidized flavins and markedly shifts their Em values. Nuclear magnetic resonance (NMR) structural studies provide key insights that reveal the mechanisms by which the local chemical environments within the RNA binding pocket achieve these shifts.Previous in vitro selection experiments have identified aptamers that recognize nicotinamides and flavins, but preferential binding to the oxidized over the reduced forms was not enforced during the selections. From those previous efforts, a preference for oxidation state was reported only for an NAD2  at 450 nm with a smaller second peak at 377 nm. In the presence of aptamer X2B2, λmax of the bound FAD was red-shifted to 456 nm (Δλmax = +8 nm), with shoulders at 482 nm and 430 nm and a second peak at 384 nm  Ca2+  hexammine, which mimics hydrated Mg2+ and for which the NH3 ligands are exchange inert, did not support FAD binding, indicating at least one required inner-sphere contact with a partially dehydrated metal ion  spectroscopy. Free FAD absorbs maximally  than for X2B2 itself  hexammine, indicating the presence of at least one inner-sphere contact interaction in the mutant  observed for X2B2-C14U with FMN  as the reference dye, Em values for free FAD, FMN and Rb were −209 ± 1 mV, −211 ± 2 mV and −210 ± 1 mV, respectively, which agreed with literature values33. A non-FAD-binding RNA (Neg) was tested with FAD, and an Em of −209 ± 1 mV was measured, showing that non-FAD-binding RNA does not affect the Em of the flavin. Additionally, Ftest1, an RNA aptamer that does not distinguish between the oxidized and reduced forms of FAD28, was tested with FAD, and an Em of −212 ± 3 mV was measured, showing that non-preferential binding to one form of FAD toward the other does not substantially alter Em  was used as the reference dye, shifted Em to −234 ± 1 mV, which was a ΔEm of −25 mV versus free FAD ; the deoxy substitution at U18 had Em = −225 mV (ΔEm = −14 mV); and the deoxy substitution at both G13 and U18 had Em = −223 mV (ΔEm = −12 mV)  ensemble placed the 2′-OH of either G13 or U18 within 3.3 Å of the N5 of FMN, indicating a possible source of hydrogen bonding between the RNA and flavin. Mutant X2B2-C14U with deoxy substitutions at one or both of these positions were tested for their ability to bind to FMN. Each variant still produced a red-shift in the UV-Vis spectra, with a modest loss of the shoulder at 482 nm when the deoxy substitution was included at U18 Fig. . The impmV) Fig. . These s2 , which allows the FMN to react with other molecules, such as the methyl viologen mediator used in the xanthine oxidase assay, and potentially with substrates in engineered oxidoreductase ribozymes.The resulting model Fig.  indicatem, we introduced a purine (X2B2-C14A and X2B2-C14G) or deletion (X2B2-ΔC14) at that position. From the UV-Vis spectra, X2B2-ΔC14 lost all flavin-binding ability, and the purine mutants retained only a minor amount of binding  on affinity and ΔE2 and successfully minimized the aptamer to a length of 38 nucleotides, resulting in aptamer X2B2. Using UV-Vis spectroscopy, we observed aptamer-induced changes to the flavin spectra, specifically red-shifted peaks and new shoulders. These changes facilitated analysis of aptamer mutants and allowed us to identify a key mutant, X2B2-C14U, with greater binding affinity for the flavin. Red-shifts of similar magnitude have been observed in some flavoproteins, where they are attributed to protein–flavin interactions34, and the appearance of shoulders at ±26 nm relative to λmax indicates a lack of hydrogen bonding with the flavin relative to the free flavin in water37. Both of these aptamers readily bound to FAD, FMN and Rb, which suggested that the interactions between the aptamers and the cofactors were concentrated on the isoalloxazine ring, and this binding was maintained under a wide range of divalent metal ion conditions.Using in vitro selection, we isolated an RNA aptamer with preferential binding to FAD over FADHm for aptamer-bound flavin relative to free flavin, demonstrating, to our knowledge for the first time, that an RNA is capable of shifting the Em of a redox cofactor. Our observed ΔEm values corresponded to a greater than twofold increase in Kdred relative to Kdox for X2B2 and a nearly sevenfold increase for X2B2-C14U, indicating that X2B2-C14U was better able than the parent X2B2 aptamer to distinguish between FAD and FADH2  and the ribitol plus ADP (FAD) attached to the isoalloxazine ring, these two flavins bound with similar affinity to each aptamer and produced similar, more modest ΔEm values relative to FMN. Nevertheless, all six complexes (two aptamers × three flavins) substantially shifted Em for the bound flavin cofactors, and X2B2-C14U was especially considerable in decreasing Em values by as much as ΔEm = −40 mV , whereas the U14 in X2B2-C14U interacts via the O4 atom (electron donating) . Thus, subtle changes within the binding pocket impact ΔEm without disrupting the overall structure of the aptamer–FMN complex.The identity of the pyrimidine at position 14 clearly influenced the Eng) Fig. . In compm) of a bound redox cofactor, flavin. Based on UV-Vis spectral changes and NMR structural analysis, the X2B2 aptamer and its X2B2-C14U mutant use interactions similar to those observed in flavoproteins, including π−π stacking and donor−π interactions with the isoalloxazine ring, to achieve the differential binding required for substantially shifting Em. Functional groups within the X2B2-binding pocket further modulate differential binding and the resulting ΔEm values. Collectively, these results suggest catalytic strategies that early RNAs could have harnessed, providing them with a broadened range of cofactor reduction potentials, such as those now seen in flavoproteins and other oxidoreductase enzymes, and allowing them to develop metabolism in an RNA world to enable assembly of new functional RNAs with expanded catalytic abilities.Our results demonstrate that the local chemical environment of an RNA-binding pocket is not merely a passive interaction surface but, instead, is capable of substantially shifting the midpoint reduction potential (E−1), glucose oxidase (24.8 U mg−1), catalase , phenosafranin, methyl viologen and riboflavin- were purchased from Sigma-Aldrich, and hexaamminecobalt (III) chloride was purchased from Tokyo Chemical Industry (TCI). The FAD resin (Sigma-Aldrich) was agarose derivatized with cyanogen bromide–activated adipic dihydrazide. Radiolabeled α-32P GTP used for in vitro transcriptions during selections was purchased from ICN. For NMR spectroscopy studies, the nucleotide-specific 2H-labeled samples, including AC-, AG-, GU- and A2RURCG-X2B2-C14U, were prepared by incorporating the corresponding deuterated and protonated NTPs in T7 transcriptions, as previously described46. Fully deuterated NTPs and H5, H6-deuterated CTP and UTP were purchased from Silantes and Cambridge Isotope Laboratories (CIL). H8-deuterated ATP (A2R) and GTP (GR) were prepared in-house47, and 13C/15N NTPs were purchased from CIL. For all buffers used, the pH is defined at room temperature unless otherwise stated. Spectral data analysis was done using Microsoft Excel 365 and GraphPad Prism 9.DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT) and were purified by denaturing PAGE. 9,10-anthraquinone-2-sulfonic acid sodium salt, hydrate (AQS) was purchased from Alfa Aesar. FAD, FMN and Rb were purchased from Chem-Impex. Xanthine, xanthine oxidase  and ethanol precipitated before denaturing PAGE purification. Desired products were excised from the gel and extracted via the crush-and-soak method in TEN elution buffer, followed by ethanol precipitation. Recovered RNA oligonucleotides were quantified using a NanoDrop 2000c (Thermo Fisher Scientific). For RNAs used in ITC and NMR spectroscopy, in vitro transcriptions were carried out in 40 mM Tris-HCl pH 8.0, 5 mM DTT, 10 mM spermidine, 0.01% (v/v) Triton X-100, 20 mM MgCl2, 12 mM of each NTP  and Ribolock RNase Inhibitor . Transcription reactions were quenched with 1 M urea and 25 mM EDTA. RNAs were purified by denaturing PAGE, visualized by UV shadowing, electroeluted from the gel using elutrap (Whatman) and washed in Amicon ultra-centrifugal filters . 2H- and 13C/15N-labeled RNAs were synthesized by incorporating corresponding 2H- or 13C/15N-labeled NTPs into in vitro transcriptions.RNAs were generated via in vitro transcription49. The initial RNA pool for selection  at room temperature and then passed through 200 µl of an ADP-modified agarose column to remove adenosine-binding RNAs and to enforce flavin binding (counter selection). The flow-through was passed through an FAD-modified agarose resin. Non-binding RNAs were washed off with 2 ml of 1× TKNCM buffer, and weak binders were ‘fast’ eluted with 1 ml of 5 mM FAD in 1× TKNCM buffer. ‘Slow elutions’ were done for high-affinity RNAs by incubating the column four times for extended periods  with 300 µl of 5 mM FAD in 1× TKNCM buffer before collecting each flow-through. A final wash of 5 mM FAD in 1× TKNCM buffer was done after the fourth elution step. The ‘fast’ and ‘slow’ elutions and final wash were combined until the end of round 6, and only ‘slow’ elutions were recovered and carried forward from round 7 onward. The recovered RNA at the end of each round was pooled and passed through a YM-30 spin column to remove excess FAD before RT–PCR. PCR amplification was carried out using 15 cycles of 30 seconds at 94 °C, 60 seconds at 37 °C and 45 seconds at 72 °C with a final 5 minutes at 72 °C after the 15th cycle. RNA for the next selection round was transcribed in vitro from these DNA templates and gel purified as described above. Cherenkov counting of all fractions, including the selection columns, was measured in a scintillation counter, and the percent of eluted RNA was calculated  for 15 minutes. Pre-folded aptamers were then introduced to appropriate binding buffers as described below.2+, which we found to be dispensable at the concentrations used) unless otherwise stated. A typical assay contained 50 µM of pre-folded RNA aptamer and 50 µM of flavin . Absorbance was measured in a 10-mm quartz cuvette between 290 nm and 650 nm in 1-nm increments using a NanoDrop 2000c. For aptamer mutants, 40 µM of RNA aptamer and 40 µM of FAD were used. All measurements were normalized so that the maximum absorbance was equal to 1.UV-Vis-based assays were carried out in 1× TKNM buffer 6]Cl3 at concentrations between 1 µM and 100 mM. Another sample containing FMN but no aptamer was measured in parallel to subtract fluorescence quenching by the metal ion. Fluorescence measurements were conducted on a plate reader in black 96-well plates using an excitation wavelength of 450 nm and an emission wavelength of 530 nm, and samples were tested in triplicate. Data were analyzed by calculating the fraction bound using (F − F0)/(Fc − F0), where F was the measured fluorescence of the aptamer–FMN complex at a particular divalent metal ion concentration; F0 was the measured fluorescence of only the FMN at a particular divalent metal ion concentration; and Fc was the fluorescence of the fully complexed aptamer with FMN and was set to equal the fluorescence of the highest divalent metal ion concentration53. Binding curves were generated using specific binding with a variable Hill slope (Y = (Bmax)(Xh)/(Kdh + Xh)) and constraining Bmax to be equal to 1 , 3 µM of pre-folded aptamer (450 pmol), 50 mM Tris pH 7.0, 140 mM KCl, 10 mM NaCl and MgClmax  and glycerol  were then added to each sample. The gel was prepared with 12% acrylamide, 90 mM Tris pH 8.0, 70 mM KCl, 5 mM NaCl and 15 mM MgCl2 and was pre-run at 100 V for 2 hours at room temperature. Samples were loaded, and the gel was run at 100 V for 4.5 hours at room temperature, with frequent monitoring to maintain a temperature of ~24 °C for the entirety of the run. RNAs were imaged on the gel using UV shadow imaging with a UV lamp at 254 nm and a TLC plate with a fluorescent dye. FMN was imaged using FMN fluorescence with a UV lamp at 365 nm. Images were overlayed for analysis  were pre-folded as described above. Then, 300 pmol of FMN, buffer . RNA and flavin samples were each prepared separately in 10 mM Tris-HCl pH 7.5 and 20 mM MgCl32. Assays were conducted in a sealable quartz cuvette with a septum and a low flow rate of argon flowing through the headspace via a needle punctured through the septum. Reactions contained 100 mM HEPES pH 7.5, 200 mM KCl, 15 mM MgCl2, 700 µM xanthine, 30 µM methyl viologen, 20 µM reference dye, 20 µM flavin, 20 µM pre-folded RNA aptamer, 5 mM glucose, 75 µg ml−1 (1.86 U ml−1) of glucose oxidase and 20 µg ml−1 (44.4 U ml−1) of catalase. The glucose, glucose oxidase and catalase created an anaerobic environment by removing molecular oxygen dissolved in solution54. The reaction was initiated by anaerobic addition of xanthine oxidase  using a syringe, and the sample was mixed. The amount of xanthine oxidase was kept low to ensure that the reduction of both the flavin–aptamer complex and the reference dye were at equilibrium throughout the assay. UV-Vis absorbance between 290 nm and 750 nm was collected every 1 minute for 120–180 minutes using an Agilent Cary 60 UV-Vis spectrophotometer.The method for determining the flavin-binding RNA aptamer reduction potentials was adapted from the xanthine–xanthine oxidase reducing system developed by Masseyy-intercept (b) being equal to the difference in reduction potential between the flavin and the reference dye  in millivolts55. All samples were run in triplicate, and the reported reduction potentials were the averages of the triplicates. All Em values are with respect to the standard hydrogen electrode. The absorbance spectrum did not indicate any formation of the semiquinone flavin (λmax ~600 nm) in these assays, indicating that, in all examples, the measured reduction potential was for the 2-electron reduction of the oxidized flavin to the hydroquinone form.Reduction potentials for free flavins and for aptamer–flavin complexes were calculated by measuring the oxidized-to-reduced ratios for each assay component and assuming that both the flavin and the reference dye were 100% oxidized for the first measurement and 100% reduced when the spectra no longer changed. The percent oxidized for each species was determined by the change in absorbance for the opposing species’ isosbestic point. For assays with AQS, the AQS was measured at 335 nm (flavin isosbestic point), and the flavins and aptamer–flavin complexes were measured at 355 nm (AQS isobestic point). For assays with phenosafranin, the phenosafranin was measured at 540 nm, and the aptamer–flavin complex was measured at 456 nm. Contributions from phenosafranin were subtracted from this measurement. The Nernst concentration terms were then plotted against each other, and a linear best-fit line was determined based on the plotted data with the 2 at 37 °C for 30 minutes. 2D 1H-1H NOESY data for fully protonated and site-specifically deuterated samples were collected in D2O  at 308 K and 293 K. 1D and 2D imino proton spectra were collected for X2B2-C14U–FMN in buffer containing 10% D2O + 90% H2O. Nucleotide-specific 2H-labeled samples, including AC-, AG-, GU- and A2RURCG-X2B2-C14U, facilitated peak assignments. HNNCOSY data were collected for 13C/15N-labeled X2B2-C14U–FMN and 13C/15N-labeled X2B2-C14U–Rb-. All NMR data were collected on a Bruker Avance III 800-MHz spectrometer equipped with TCI cryoprobe . The NMR data were processed by NMRPipe56 and analyzed by NMRViewJ57.X2B2-C14U–FMN and X2B2–FMN complexes were prepared (200–600 µM) and pre-incubated in 10 mM Tris-d11 pD = 7.5 and 20 mM MgCl59). NOE-derived distance restraints were applied for the remaining nucleotides and FMN. Eight structures with the lowest target function were generated and served as initial structures for the MD simulations using Amber 18 (ref. 60). Simulated annealing (SA) MD simulations were performed 20 times with the last structure extracted from each trajectory. Then, the total 160 structures were clustered into ten groups using the k-means clustering method, and the ten centroid structures were selected as the refined structures sorted in descending order of cluster size.The X2B2-C14U structure was initially calculated by CYANA using NMR-derived restraints. Standard torsion angle restraints, hydrogen bonding restraints for Watson–Crick base pairs and phosphate distance restraints in RNA helices were employed to maintain the A-form helical geometry in base pairs 1–8, 10–12, 28–30 and 31–38 (refs. For SA MD simulations, the RNA molecule was simulated using the RNA.OL3 force field, and the ligand FMN was treated using the GAFF force field generated by the Antechamber package built in Amber. RNA and FMN molecules were solvated in a truncated octahedron periodic box of TIP3P water, and the distance between the edge of the water box and the solute was no less than 12 Å. Moreover, the RNA and FMN system was simulated in roughly 1 M NaCl. After energy minimization, the system was heated from 0 K to 300 K in 10 ps with constant volume and then equilibrated for 10 ps under 300 K and 1 atm pressure. Finally, the SA MD simulation was performed by heating the system to 400 K in 10 ps and then cooling gradually from 400 K to 0 K in about 2.5 ns with constant volume, and the last structure was collected. NOE-derived distance restraints and standard hydrogen bond distance restraints were used during energy minimization, heating, equilibration and SA MD simulation. Eight torsional restraints were employed to keep the nucleotides G15, U18 and A26 coplanar, and positional restraints were used to fix the RNA helical part including the nucleotides 1–12 and 28–38. Statistics of NMR restraints and the calculated structures are summarized in Supplementary Table Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41589-022-01121-4.Supplementary InformationSupplementary Tables 1–5, Supplementary Figs. 1 and 2 and Supplementary Information References.Reporting Summary


Determining the structuraldynamics of RNA and DNA is essentialto understanding their cellular function, but direct measurement ofstrand association or folding remains experimentally challenging.Here we illustrate a temperature-jump/drop method able to reveal refoldingdynamics. Time-resolved temperature-jump/drop infrared spectroscopyis used to measure the melting and refolding dynamics of a 12-nucleotideRNA sequence comprising a UACG tetraloop and a four-base-pair double-strandedGC stem, comparing them to an equivalent DNA (TACG) sequence. Stem-loopmelting occurred an order of magnitude more slowly in RNA than DNA(6.0 ± 0.1 μs versus 0.8 ± 0.1 μs at 70 °C).In contrast, the refolding dynamics of both sequences occurred onsimilar time scales (200 μs). While the melting and refoldingdynamics of RNA and DNA hairpins both followed Arrhenius temperaturedependences, refolding was characterized by an apparent negative activationenergy, consistent with a mechanism involving multiple misfolded intermediatesprior to zipping of the stem base pairs. Ribonucleicacid (RNA) sequencesand their deoxy counterparts (DNA) are fundamental to the storage,regulation, and expression of genetic information in organisms. Revealingthe underlying molecular mechanisms is central to our understandingof biological processes as well as supporting development of new chemicaland biomedical technologies, such as nucleic acid aptamers and antisensetherapeutics.10 T-jump methods have been used to investigate the impact of a numberfactors upon nucleic acid dynamics, including base sequence, chainlength, and the presence of small molecule ligands.6 Typically, T-jump studiesemploy fast (nanosecond) initiation, but the samples cool on multi-millisecondtime scales. The latter are too slow to observe refolding directly,meaning that refolding (association) rates have to be inferred fromtwo state models linking the dissociation rate to the equilibriumconstant.7 Here, we demonstrate an approach that exploitsa much faster sample cooling time to enable observation of both themelting and refolding dynamics of nucleic acid tetraloop hairpinsfollowing T-jump initiation.While the structures of nucleic acid sequencesare well understood,the dynamic fluctuations and interactions within and between nucleicacid strands in solution remain a source of uncertainty. Insightshave been gained by nonequilibrium temperature jump (T-jump) spectroscopyexperiments, which induce a rapid rise in temperature as a means toinitiate and follow conformational changes in real time.10 but RNA demonstrates greatercomplexity in the biological setting due to its propensity to existas single strands that fold into functional three-dimensional structures.Hairpins, consisting of a Watson–Crick base paired stem anda single-stranded loop, are among the most common RNA structural motifs,occurring in a range of prokaryotic and eukaryotic RNAs.11 Tetraloops in particular provide nucleationsites for folding and recognition sites for RNA binding proteins.13 The two most common loop motifs are GNRA and UNCG, where R = purineand N = any nucleotide.12 The latter aremore thermodynamically stable, displaying melting points some 20 °Chigher than other sequences, whereas the former are more prevalent,participating in tertiary interactions and protein recognition.12To date, much emphasis has beenplaced upon elucidating the dynamicsof double-stranded DNA,15 but the folding dynamics have yet to be resolved. To address thisimportant issue, we combine a nanosecond T-jump, to initiate melting,with a subsequent sample cooling rate that is fast enough to allowobservation of strand refolding. Faster cooling is achieved usinga thin cell to enhance heat transfer. Using IR spectroscopy as a probeof vibrational modes of the nucleic acid bases provides real-timeinsight into stem base pairing and stacking. Although DNA tetraloopsare not known biologically, comparing RNA tetraloop dynamics withthose of an analogous DNA sequence reveals inherent differences betweenmolecules with overtly similar molecular structures.The melting characteristics of UNCG loopshave been determinedpreviously by static spectroscopy experiments,R) observed at 1575 cm–1 uponbase stacking provided a spectral marker for stem melting and reassociation.20The salt-free,lyophilized DNA and RNA oligomer sequences 5′-GCGC(XACG)GCGC-3′” indicates the loopposition) were purchased from Eurogentec. Other chemicals were purchasedfrom Sigma-Aldrich and used without modification. The GCGC stem sequenceof the tetraloops was chosen to minimize base pair slippage. The reductionin the molar extinction coefficient of the guanine ring vibrationalmode equipped with CaF2 windows. A 50 μm PTFE spacer definedthe path length for IR absorption measurements while a 12 μmpath length was used for T-jump experiments.All DNA and RNA samples were prepared to astrand concentrationof 10 mM, which is below the threshold for duplex formation, in 1M deuterated phosphate buffer (pD 6.8).22 Briefly, a 4 ns duration T-jumppump pulse (125 Hz), generated by a Nd:YAG-pumped OPO, centered at2750 cm–1, resonant with the high-frequency wingof the OD-stretching vibration of the solvent initiated a T-jump of10 °C in the sample (averaged across the sample), as determinedby trifluoroacetic acid (TFA) calibration.22 A series of probe pulses centered at 1650cm–1 (bandwidth: 300 cm–1) generatedby a Ti:sapphire-pumped OPA (10 kHz) with difference frequency mixingof signal and idler were used to capture the nanosecond to millisecondnucleic acid dynamics using the time-resolved multiple probe (TRMPS)method.22 Data werecollected at T-jump-probe time delays from 1 ns to 8 ms, though thecooling of the sample was complete by 4 ms (vide infra) such thatdata obtained at time delays from 4 to 8 ms were used for backgroundcorrection.1IR absorption spectrawere measured using a Bruker Vertex 70 Fouriertransform (FT)-IR spectrometer. The T-jump measurements were performedusing the STFC Central Laser Facility’s ULTRA spectrometer.–1 region that arebroadly similar to those observed for DNA samples (S1). The temperature-induced spectral changes are representedas difference spectra relative to the 20 °C measurement. By comparisonwith previous work the changes in this spectral region can be assignedto the melting of double-stranded GC-rich nucleic acids.16 We will focus on the guanine ring vibrationalmode (GR) at 1575 cm–1 (purple panelsin S1). This band increases markedly in intensitywith temperature, and we assign this to the loss of base stackingand pairing in the stem of the loop.16 Byextension, we assume that loss of the stem structure leads to meltingof the hairpin loop.Infrared absorptionspectroscopy showed that increasing the temperatureof the RNA hairpin sample from 20 to 80 °C under equilibrium(non T-jump) conditions induced changes in band position and intensityacross the 1550–1700 cmR mode as a function of temperature. A Van’t Hoff analysiswas used to obtain thermodynamic parameters associated with the meltingtransition (15Melting curves for the RNA andDNA hairpins were obtained by plottingthe absorbance of the Gansition 1. BecausT0), 5 deg belowthe melting temperature of the sample (T0 = Tm – 5 °C). The spectraare represented as T-jump on–T-jump off difference spectraand show changes in band positions and intensities that emerge overa period of up to 20 μs following the T-jump (blue–red).For both RNA and DNA samples, the spectral changes following the T-jumpwere observed to be identical with those in the difference IR absorptionspectra The resultsof T-jump spectroscopy measurements on RNA and DNAhairpins are shown in spectra 1a,band T0).In each case, the GR band intensity was observed to rise,peaking near 20 and 6 μs for RNA and DNA hairpins, respectively,before decaying on millisecond time scales (S4). In eachcase, the temporal dependence of the GR band was fittedusing a triple-exponential function (Table S1). The majority of the temporal profile was accounted for by twoprocesses with lifetimes quantifying the rise (τ1) and decay (τ2) of the signal. The third exponentialterm was generally of low amplitude (<25%) but was essential toachieve a good quality fit. The triple-exponential method was foundto provide more robust results than fitting the data with a stretchedexponential function. The results from the fittingprocess are shown in 2O gives rise to a small, broadbandsolvent-dependent contribution to the data. In the analysis that followsthis has not been subtracted, but comparisons showed that applyingthis correction led to changes in lifetime parameters that fell withinthe errors stated.To examine the dynamics of thehairpins following the T-jump, experimentswere conducted on the RNA and DNA sequences starting from a rangeof different initial temperatures  was foundto be in good agreement with an Arrhenius representation (2O), yielding positive activation energies , as would be expected forstrand melting. These values are in good agreement with prior work.23In accordance with previous work, we associate the time scaleofthe rising G2 was also well-representedby Arrhenius behavior. In contrast to τ1, however,the time scales of τ2 were found to increase withincreasing T0, yielding values from 150to 400 μs, from 60 to 80 °C for both RNA and DNA, consistentwith a process with an apparent negative activation energy (2O).Examining the response of the hairpins on microsecond to millisecondtime scales, as the sample cools following the T-jump, revealed thatthe temperature dependence of τ2O solvent were characterized using T-jump spectroscopyof a D2O/TFA solution. Fitting the temporal profile ofa vibrational mode of the TFA calibrant molecule at 1670 cm–1 to a biexponential function (S5) has been shownto provide a reliable measure of sample temperature.1 A biexponential function was used to mirror the approachused for nucleic acid dynamics while accounting for the fact thatthe rise time of the T-jump in the solvent is essentially instantaneous,following the profile of the nanosecond laser pulse. For the TFA data,the second time scale recovered from the exponential function wasequivalent to the small τ3 contribution observedfor RNA and DNA, and so we use the major TFA relaxation time scaleas an indicator of sample cooling time. The results showed that thecooling time scale of the solvent was invariant with T0 (2O (1470 cm–1) present in both the TFA calibrationsample and the hairpin samples were compared and found to displaysimilar dynamics .For comparison with the RNA and DNA data, the cooling dynamicsof the Dfunction 3 and S5  with T0 3, blue. 2O coolingtakes place on <150 μs time scales, the hairpin dynamicsof both RNA and DNA show dynamics on several hundreds of microsecondtime scales, increasing exponentially as T0 increases .In addition to showing no Arrheniusbehavior, it is clear from ncreases 3b, which–1 for theactivation energies of refolding  of RNA and DNA, respectively. These values are consistent withnegative activation energies that have been calculated for the refoldingof oligonucleotides in other studies, confirming that our approachis directly measuring hairpin refolding as well as the validity ofthe models used to obtain refolding time scales.27 It is noteworthy that although RNA and DNA show very different meltingbehaviors, the refolding process is consistent between the two—anoutcome which has been reported by previous studies on different sequencesand suggests the key rate-limiting steps are conserved in both.28 It is important to note that the refolding processas measured is a convolution of the solvent cooling and the nucleicacid dynamics. When these time scales are well separated, as at hightemperatures here, the results will be dominated by the nucleic aciddynamics. At lower temperatures a degree of convolution may affectthe absolute quantitative accuracy, but even under these conditions,a direct experimental measurement obtained for different moleculesunder identical experimental conditions will yield valuable comparisondata.Usingan Arrhenius analysis produced values of −48.2 ±5.0 and −45.2 ± 5.6 kJ mol3) wasfoundto be on the order of 800 μs. On the basis of its magnitude,in nucleic acid samples, which was greater than the solvent signal,and its nonconstant temperature dependence, this contribution couldnot be attributed simply to solvent response or a cooling propertyof the cell. As such it may indicate a slower dynamic process thatfollows the initial refolding, but the small nature of the contributionprecludes clear conclusions.The small time scale component . This is accompanied by a negativeactivation entropy (−324 ± 37 and −315 ± 43J K–1 mol–1), such that the Gibbsfree energy of activation is positive at elevated T, consistent with the observations. The fact that this negative activationenergy and indeed the time scale of refolding are shared between boththe RNA and DNA hairpin suggests that the rate-limiting step for bothis the same—the re-forming of the base stacks. Furthermore,this slow rate of formation of the base stacks has a more significantimpact on the refolding process than the differing features of RNAand DNA.In contrast to melting,the refolding processes for the RNA andDNA hairpins occur on the same time scales. This observation of anapparent negative activation energy, in accordance with various studies,shows that this follows a more complex mechanism than melting.40 We have shown previously that the cooling timescale for a sample varies sensitively with the sample path length,meaning that this strategy can be used to tune the cooling time toa certain degree to match the dynamics of the analyte molecule.21In conclusion, we have demonstrated that it is possibleto applyT-jump time-resolved spectroscopy strategies to observe the refoldingdynamics of nucleic acid hairpins. Using a system in which the solventcooling time is shorter than the refolding time of the sample effectivelyproduces a combined T-jump followed by a T-drop experiment, makingit possible to measure the native dynamics of both RNA and DNA hairpinmelting and refolding. This provides a direct measurement of parametersthat previously had to be calculated from the melting rates and equilibriumconstants. The use of IR spectroscopy for label-free detection ofthe conformational changes will also enable future comparisons with,for example, refolding rates obtained via microfluidic temperaturedrop techniques employing incorporation of fluorescent labels.Comparing our results obtained on similarRNA and DNA hairpinsreveals distinct melting dynamics, with RNA melting an order of magnitudemore slowly than DNA, which we attribute to the influence of the helicalconformation on the stabilization of the base stacking. However, therefolding dynamics both occur on similar time scales and show behaviorwhich is consistent with a negative activation energy. These observationsare in good agreement with previous studies and give insights intothe energy landscape that result in the similar refolding dynamicsof the DNA and RNA hairpins.


Thr). We introduced random mutations within this element and found that almost every change from the normal sequence leads to reduced binding by ThrRS. Furthermore, point mutations at six key positions that abolish the predicted ASL-like structure showed a significant decrease in ThrRS binding with a decrease in RPC10 protein levels. Concomitantly, tRNAThr levels were reduced in the mutated strain. These data suggest a novel regulatory mechanism in which cellular tRNA levels are regulated through a mimicking element within an RNA polymerase III subunit in a manner that involves the tRNA cognate aaRS. Aminoacyl tRNA synthetases (aaRSs) are a well-studied family of enzymes with a canonical role in charging tRNAs with a specific amino acid. These proteins appear to also have non-canonical roles, including post-transcriptional regulation of mRNA expression. Many aaRSs were found to bind mRNAs and regulate their translation into proteins. However, the mRNA targets, mechanism of interaction, and regulatory consequences of this binding are not fully resolved. Here, we focused on yeast cytosolic threonine tRNA synthetase (ThrRS) to decipher its impact on mRNA binding. Affinity purification of ThrRS with its associated mRNAs followed by transcriptome analysis revealed a preference for mRNAs encoding RNA polymerase subunits. An mRNA that was significantly bound compared to all others was the mRNA encoding RPC10, a small subunit of RNA polymerase III. Structural modeling suggested that this mRNA includes a stem-loop element that is similar to the anti-codon stem loop (ASL) structure of ThrRS cognate tRNA (tRNA Aminoacyl tRNA synthetases (aaRS) are a family of enzymes that recognize multiple cognate tRNAs and a single amino acid and charge the amino acid at the 3’ end of the tRNA with the expanse of an ATP . Most ceThr to ThrRS in E.coli leads to displacement of the target mRNA and relief of the inhibitory effect [His leads to reduced binding of HisRS to its target mRNA and increased translation [Interestingly, many aaRSs were found to have non-canonical functions in addition to their primary role in tRNA charging. These include cytokine activity, cellular signaling, or binding to DNA to regulate transcription ,5. It apy effect . In yeasnslation . S. cerevisiae cytosol. Interestingly, ThrRS shows an exceptional binding to an mRNA encoding a component of RNA polymerase III (RPC10). Point mutations revealed that binding occurs through a tRNAThr ASL-like element and has a role in the expression regulation of tRNAThr by RNA polymerase III. Here, we tested the repertoire of mRNAs bound by ThrRS in THS1) strain to RNA-binding protein immuno-purification (RIP) by anti-GFP magnetic beads. Isolated RNA (‘Bound’) from two independent biological repeats was subjected to high-throughput sequencing (RNA-seq) (pr) ~1) was observed between the two biological repeats of the Input and between the two of the Bound samples (pr < 0.22), suggesting that the reads are not merely reflections of mRNA expression. Furthermore, the correlation with the Bound signals from the untagged samples was similarly low (data not shown), which is consistent with low non-specific RNA binding. Overall, 364 mRNAs appeared enriched by more than two-fold in the Bound compared to the Input of the tagged strain, and 229 mRNAs were enriched in the Bound sample of ThrRS-GFP compared to the Bound of the untagged strain  A (SuppleRNA-seq) . RNA samRNA-seq) . A high  samples B. Howeved strain C,D. Seved strain E. Functid strain F (Suppled strain . RPC10 mRNA is significantly enriched in the bound sample (~16 fold) compared to control mRNA (ACT1)  B. RPC10 mRNA. LocARNA [RPC10 mRNA and all tRNAThr isoacceptors. The analysis suggests a prominent anticodon stem loop (ASL)-like structure in RPC10 mRNA at positions 135–151 that resembles the ASL of tRNAThr . The use of CRIPSR/Cas9 mutagenesis protocol does not introduce any other change within this genomic locus, such as a selection marker [To examine the importance of this element for binding, in an unbiased manner, we introduced random mutations to this region D. This wn marker . Only a 3 recombinant yeast colonies  were grown to logarithmic phase in a rich medium and collected in a batch ). The sample was subjected to RIP with anti-GFP beads, and RNA was reverse transcribed and subjected to Sanger sequencing for RPC10. The fluorescent signal of each base is a proxy for its abundance within the analyzed population. A clear preference towards binding the WT transcript was apparent  is the second most bound. Overall, any change within the predicted ASL-like region leads to a reduced association. The enrichment towards the normal sequence is further appreciated considering the fact that the CRISPR/Cas9 scheme imposes a negative selection towards insertion of normal sequences. Thus, the predicted ASL-like sequence in RPC10 is important for ThrRS binding.About 10apparent E: in almRPC10 with six specific mutations in this region: A135G, C138G, U141G, U144C, G147A, and C150U (RPC10-6M). This combination was selected based on the RIP followed by Sanger sequencing results . Interestingly, two of them are proteins involved in metabolic pathways (ACB1 and CYS3). Recently, we revealed that aaRS interacts with mRNAs that encode proteins involved in amino acid metabolism, suggesting that aaRS can post-transcriptionally regulate amino acid biosynthesis [We hypothesized that the changes in protein levels are due to translation regulation specific to LC-MS/MS . AnalysiLC-MS/MS B, as welynthesis . TheprotRPC10 appeared associated with 2–4 ribosomes in the WT strain  (RPC10-6M) is observed at higher levels in this fraction. This increase may emerge from the shift in the transcript from the monosome fraction, which shows lower levels of RPC10-6M transcript. Remarkably, ThrRS’s most significant protein partner is RPP1B B. RPP1B raction) C. ImportThr (AGU) levels in WT and RPC10-6M mutants revealed a decrease in tRNAThr levels. However, other tRNAs (tRNALeu) or 5S ribosomal RNA are not significantly changed  through binding a tRNA-like element within its 5′ UTR element [In this work, we present indications for the presence of an ASL-like element within o assays . Notablyo assays  tRNAThr  element ,25. Yet, element , suggestRPC10 mRNA ribosomal association and a reduction in RPC10 protein levels, consistent with reduced ribosome transit time along the RPC10-coding region  [Binding through RNA structural mimicry was revealed years ago for the nd ThrRS ,24,29,30ociation ,32. RNA ociation . A well-mimicry) ,34. WhenThr levels are lower than necessary, a free ThrRS binds RPC10 mRNA and through RPP1B affects ribosomes movement to increase RPC10 protein levels. Increased RPC10 translation supports RNA polymerase III cytosolic assembly and activates transcription of tRNAThr to restore its proper levels , an S288C-derivative laboratory strain  and rotated at 4 °C for 2 h. Samples were washed four times and eluted by 0.2 M Glycine buffer (pH 2.5). Cross-linking was reversed by heating at 65 °C for 2 h in reverse cross-linking buffer, and RNA was precipitated after phenol:choloroform extraction.RIP was essentially as described in . BrieflyS. cerevisiae version R64–2-1 genome using RNA STAR version 2.6.0b-2. Only uniquely mapped reads were counted for genes using HTSeq-count package version 0.6.1.RNA samples were subjected to RNA-seq at the Technion Genome Center. Libraries from the Input and Bound RIP samples were prepared using a TruSeq RNA Library Prep Kit v2  according to the manufacturer’s instructions. All samples were sequenced on Illumina platform, yielding 20 to 30 million reads per sample. Reads were mapped to the S288c Homologous recombination by CRISPR/Cas9 was performed as described in . Cells i2, 0.5 mM dithiothreitol, 100 μg/mL cycloheximide, 0.24 U/μL RiboLock RNase Inhibitor , 1% Triton X-100). The lysate was cleared and centrifuged for 15 min at 9000× g at 4 °C, and the supernatant was loaded onto a 12 mL 10–50% linear sucrose gradient. Gradients were centrifuged in a SW41 rotor  at 35,000 rpm for 160 min, and the entire gradient was fractionated into four fractions. RNA was extracted from each fraction by the addition of an equal volume of 8 M guanidinium HCl and two volumes of 100% ethanol, incubated overnight at −20 °C, and centrifuged at 13,000 rpm for 30 min. Pellets were washed with 80% cold ethanol, and 1 µg of RNA sample was subjected to RT-qPCR with the indicated oligos  following the manufacturer’s instructions using primers for the indicated tRNA as described in [−(ΔCt).tRNA RT-qPCR was performed as described in . Brieflyribed in   and rotated at 4 °C for 2 h. Samples were washed four times in Buffer C (20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 0.5% NP40, 0.5 mM EDTA, 0.5 mM DTT, 0.01 U/μL RiboLock RNase Inhibitor ). Then, samples were washed three times in PBS and eluted with 0.2 M Glycine buffer (pH 2.5).A strain expressing GFP-tagged ThrRS (YA1545) and an untagged control (YA1) (250 mL) were grown in YPD to mid-logarithmic phase and subjected to cross-linking by the addition of formaldehyde  for 10 min at room temp. Cross-linking was terminated with 0.125 M glycine for 3 min, and cells were lysed using Bead Beater in Buffer B (20 mM Tris-HCl (pH 7.5), 140 mM NaCl, 0.1% NP40, 0.5 mM EDTA, 1 mM DTT, 2 mM PMSF, 10 μg/mL Leupeptin, 14 μg/mL Pepstatin, and 0.02 U/×L RQ1 RNase-free DNase ). The lysate was cleared by centrifugation at 10,600ρ Saccharomyces cerevisiae proteome database. Enriched proteins were calculated as the ratio between the LFQ Intensity of the protein in the ThrRS-tagged GFP and untagged ThrRS samples. Enrichment of proteins for the GFP tagged ThrRS against untagged was set to fold enrichment >2, adjusted p-value < 0.05  were digested by trypsin, analyzed by LC-MS/MS on Q-Exactive , and identified by MaxQuant_2.0.1.0 software against the e < 0.05 .


We compared RNA extraction protocols to establish a reproducible and effective pancreatic RNA extraction method to obtain high RNA integrity number (RIN) values from healthy and streptozotocin (STZ)-induced diabetic rats for gene expression analyses. Different methods were tested focusing on RNase activity inhibition using RNAlater (Qiagen) pre-harvest of the pancreatic tissue, and extracted RNA quality and concentration were analyzed using NanoDrop spectrophotometer, Agilent Bioanalyzer, and RT-PCR. Inclusion of several pre- and post-excision modifications in the RNeasy Mini Kit (Qiagen) protocol resulted in RIN values more than two-fold higher compared to those using the standard protocol. Additionally, RT-PCR amplification of the housekeeping gene, β-actin, revealed no differences in extracted RNA quality from healthy and STZ-induced diabetic rats. We compared and developed a more effective and reproducible pancreatic RNA extraction method from healthy and diabetic rats, which resulted in RNA of superior quality and integrity and is suitable for complex molecular investigations.Advanced differential gene expression analysis requires high-quality RNA. However, isolating intact pancreatic RNA is challenging due to abundant pancreatic ribonucleases, which limits efficient downstream gene expression analysis. RNA Advanced differential gene expression analysis requires high-quality RNA. Although RNA extraction from various tissues and cells is a straightforward process, isolating RNA with a high RNA integrity number (RIN) from the pancreas is challenging, mainly because the pancreas is a dual-functioning gland comprising an endocrine and exocrine acinar portion that is rich in digestive enzymes, including ribonuclease (RNase) . During Isolation methods for pancreatic RNA are inconsistent in their reproducibility and unstandardized, although several studies have reported the use of commercially available extraction kits based on guanidinium thiocyanate-phenol-chloroform in combination with silica column-based solid extraction methods  Table 1Table 1. Healthy male Sprague-Dawley rats  were of about 6–7 weeks of age (weighing approximately 160–180 g each) at the start of the experiment. The rats were housed in the Animal Care Facility under regular ambient conditions  before and during the study and handled according to the Instructions Guide for the Care and Use of Laboratory Animals . Type 1 Following anesthesia (0.2 mL/100 g) with a mixture of ketamine  and xylazine , 18 normal rats (NR) and six diabetic rats (DR) were prepped for pancreas pre-treatment and collection.later  (later (Qiagen) infusion site and clamping of the sphincter of Oddi were performed as previously described [later (Qiagen), the perfusion technique was tested by infusing India ink , which showed a very good distribution in the pancreas  was located and traced to its joining point with the pancreatic duct  A. To ensGermany) A,B. The escribed  and resupancreas C.All dissection and surgical tools were sterilized and washed with RNaseZAP  prior to use for tissue collection. Extreme caution was taken when detaching the pancreas from the attached tissues to prevent rupture and subsequent release of ribonucleases. A small piece of tissue (<50 mg) was excised from the perfused pancreas and immediately placed in 5 mL of ice-cold QIAzol  contained in a sterile 50-mL centrifuge tube immersed in ice.g at 4 °C to remove any unhomogenized lysate, as previously described [g at 4 °C to separate the upper aqueous phase (~500 µL) containing the RNA. Then, the aqueous phase was transferred to a sterile microcentrifuge tube, and an equal volume of 70% ethanol was added to it prior to being transferred to an RNeasy spin column. The addition of 70% ethanol provides ideal binding conditions for the silica membrane of the spin column. Following two centrifugations (≥8000× g at 15 °C) for 15 s to remove the ethanol, the column was washed once with 700 µL RWT washing buffer (Qiagen) and twice with 500 µL RPE washing buffer (Qiagen). During each wash, the spin column was centrifuged at ≥8000× g at 15 °C, and the flow-through was discarded. Afterward, the column was centrifuged at 16,000× g for 1 min at 15 °C to eliminate any buffer or ethanol carryover. Total RNA was eluted in RNase-free water and stored at −80 °C until future use. All steps and modifications are summarized in Initially, pancreatic RNA was extracted using the TriPure isolation reagent . RNeasy Mini Kit  was also used following the manufacturer’s instructions and with modifications to improve RNA quality. The tissue was rapidly homogenized using a TissueRuptor Ultra-Turrax T8  for 30–60 s on ice, pausing for a few seconds every 20 s to avoid heating, until the lysate was uniformly homogeneous. An additional centrifugation step was performed for 1 min at 12,000× RNA 6000 Nano Kit using an Agilent 2100 Bioanalyzer .RNA concentration (ng/µL) and purity at ratios A260/A280 and A260/A230 were recorded using a NanoDrop 8000 spectrophotometer . RIN values were determined using an Agilent ® IV First-Strand Synthesis System for RT-PCR  was used to convert RNA to cDNA by mixing 300 ng/µL template RNA with Oligo d(T)20 primer, dNTP, and RNase-free water. RNA-primer solutions were heated at 65 °C for 5 min in an automated thermocycler , followed by incubation on ice for 1 min. This was followed by addition of the RT reaction mixture to the annealed RNA and incubation at 23 °C followed by 50–55 °C for 10 min each, and finally at 80 °C for 10 min to inactivate the reaction. Beta-actin (β-actin) gene was amplified in all samples in a reaction volume of 25 µL that included 1× PCR buffer , 3 mM MgCl2, 0.5 mM dNTP mix, 0.3 µM each of forward XAHR 17: 5′ CGGAACCGCTCATTGCC 3′ and reverse XAHR 20: 5′ ACCCACACTGTGCCCATCTA 3′ β-actin primers, 50 mU/µL Taq DNA polymerase, 17.5 µL H2O, and 0.5 µL RT template. The PCR thermal profile was as follows: initial denaturation at 94 °C for 5 min, followed by 42 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. The quality of the β-actin amplicon (289 bp) in all 13 samples was visualized and assessed on a 1% agarose gel prepared in 1× Tris/Borate/EDTA (TBE) buffer stained with 3 µL of 10 mg/mL ethidium bromide .Thirteen RNA samples from pancreatic tissue extracted using the standard and optimized RNA protocols, and one control RNA sample extracted from liver tissue using the standard protocol, were used for reverse transcription (RT)-polymerase chain reaction (PCR). The SuperScriptp < 0.05.The integrity and quality of RNA measurements are presented as mean ± standard error of the mean (SEM). The data of the different groups, samples, and protocols were compared using one-way analysis of variance (ANOVA) for RNA analysis followed by Fisher’s LSD test using GraphPad Prism version 9.2.0.332. Differences among readings were considered significant at Two methods were tested for pancreatic RNA extraction: the TriPure isolation reagent (Roche) and the RNeasy Mini Kit (Qiagen). Both methods yielded similar RIN values of 3.7 and 3.6, respectively. However, unlike the RNAeasy Mini Kit (Qiagen), the TriPure isolation reagent (Roche) repeatedly produced variable results and low-quality and degraded RNA (A260/A230 of 1.00 ± 0.13). Although RNA extracted using the RNeasy Mini Kit (Qiagen) resulted in improved quality and purity, the RIN value remained low at 3.6. Therefore, it is not recommended for downstream applications, such as microarray analysis, where the RIN should ideally be >8. Therefore, to increase the RIN value, protocols reported in previous studies were tested to optimize RNA extraction, along with some modifications in the RNA extraction protocol of the RNeasy Mini Kit (Qiagen).later (Qiagen) significantly increased the RIN values (p < 0.01) in protocol C (7.3 ± 0.5) as compared to that in the standard protocol A (3.6 ± 0.8) (later (Qiagen) perfusion also significantly increased the RIN values in optimized protocol D (8.1 ± 0.1) as compared to that in the standard protocol (p < 0.001) (n = 12) extracted using the optimized method had strong 18S and 28S peaks  . Additio6 ± 0.8)  prior to< 0.001) B. Furthe< 0.001) A. All RN8S peaks  as well 8S peaks .n = 6) and diabetic rats (n = 6) using our optimized method. However, differences in RNA concentrations were observed between the two groups: RNA extracted from healthy rats had a higher concentration (455.0 ± 83.2 ng/µL) than that extracted from diabetic rats (295.1 ± 64.8 ng/µL)  . Moreove8 ng/µL) . These rβ-actin. A significant increase in the band intensity of β-actin was observed in pancreatic RNA samples extracted from healthy rats using our optimized protocol  prior to placing it in ice-cold QIAzol. In the protocol developed by Mullin et al. [While the procedure involving perfusion and clamping of the duodenum was similar to Mullin et al. , we adden et al. , the volThe second modification was a centrifugation step performed after homogenizing the tissue with the lysis reagent, which was adapted from Azevedo-Pouly et al.  who repoThe concentration of RNA extracted from healthy and diabetic rats showed some variation in our improved protocol A,B. Althβ-actin gene; the differences in its amplification reflected the effect of the modifications on the quality of the pancreatic RNA extracted. Following comparison with the control rat liver RNA that was extracted using the standard RNeasy Mini Kit (Qiagen) (The cDNA synthesized from pancreatic RNA isolated using different extraction protocols was used in RT-PCR to amplify the housekeeping (Qiagen) , lane 8 Following our protocol, we were able to approximately double the RIN value of the extracted pancreatic RNA using the RNeasy Mini Kit (Qiagen) without any modifications. The improved RNA extraction protocol is reproducible and effective despite the additional cost of the QIAzol step, which is necessary to inhibit the high level of endogenous pancreatic RNase activity. All modifications to the standard protocol performed in this study were compared to those reported in other studies from which some steps were adapted .Our optimized pancreatic RNA extraction protocol using the RNeasy Mini Kit (Qiagen) significantly increased RIN values as compared to using the TriPure isolation reagent (Roche) and the RNeasy Mini Kit (Qiagen) without modifications and is a reliable and reproducible technique for isolating pancreatic RNA of superior quality and integrity. This is supported by the analysis of the quality of extracted RNA as well as the amplicons generated using RT-PCR of cDNA synthesized from the extracted RNA. Pancreatic RNA of high quality is essential for complex downstream gene expression studies in healthy and diabetic rat models. RNA isolated in this study is suitable for use in microarray analysis and RNA-Seq by next-generation sequencing.


The on-going global pandemic of COVID-19 is caused by SARS-CoV-2, which features a proofreading mechanism to facilitate the replication of its large RNA genome. The 3′-to-5′ exoribonuclease (ExoN) activity of SARS-CoV-2 non-structural protein 14 (nsp14) removes nucleotides misincorporated during RNA synthesis by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and thereby compromises the efficacy of antiviral nucleoside/nucleotide analogues. Here we show biochemically that SARS-CoV-2 nsp14 can excise the natural antiviral chain-terminating nucleotide, 3′-deoxy-3′,4′-didehydro-cytidine 5′-monophosphate (ddhCMP), incorporated by RdRp at the 3′ end of an RNA strand. Nsp14 ExoN processes an RNA strand terminated with ddhCMP more efficiently than that with a non-physiological chain terminator 3′-deoxy-cytidine monophosphate (3′-dCMP), whereas RdRp is more susceptible to chain termination by 3′-dCTP than ddhCTP. These results suggest that nsp14 ExoN could play a role in protecting SARS-CoV-2 from ddhCTP, which is produced as part of the innate immune response against viral infections, and that the SARS-CoV-2 enzymes may have adapted to minimize the antiviral effect of ddhCTP. SARS-CoV-2 replication depends on the exoribonuclease (ExoN) activity of non-structural protein 14 (nsp14), which provides proofreading during viral RNA synthesis and antagonism against host innate immune responses ,4,5. In Nucleoside analogues are widely used as antiviral drugs. Besides remdesivir mentioned above, this class of drugs include AZT targeting HIV-1 reverse transcriptase, sofosbuvir targeting hepatitis C virus RNA polymerase, and ganciclovir/aciclovir targeting herpes virus DNA polymerases ,17,18. TWhile the chain-terminating and mutagenic nucleoside analogues mentioned above are human-made drugs, recent studies have identified a novel antiviral chain-terminating nucleotide, 3′-deoxy-3′,4′-didehydro-cytidine triphosphate ddhCTP, , produceEscherichia coli and purified as reported previously [ddhCTP was synthesized as reported previously . Data deeviously . RNA oli2, 10 mM dithiothreitol, and 0.01% Tween-20. After an initial incubation at 37 °C for 25 min, the reactions were supplemented with 200 μM NTPs or water (as controls) and further incubated for 25 min to let RdRp re-extend stalled products. The reactions were stopped by the addition of formamide to 89% and heating to 95 °C for 10 min and the products were separated by gel electrophoresis on a 15% polyacrylamide TBE-Urea denaturing gel, which was scanned on a Typhoon FLA 9500 imager. For the primer extension reactions shown in https://imagej.nih.gov/ij/, accessed on 14 August 2022). For each condition, the sum of fluorescence intensities from the single and double-stranded full-length product bands was divided by the total fluorescence signal integrated over the lane to calculate the fraction of full-length product.RNA primer extension reactions shown in Exonucleolytic degradation reactions shown in We prepared SARS-CoV-2 RdRp by mixing purified nsp12, the polymerase catalytic subunit, with its key co-factors nsp7 and nsp8, which confer processivity to nsp12 ,35. The Earlier studies have shown that SARS-CoV-2 RdRp can incorporate the natural chain-terminating nucleotide ddhCTP during RNA synthesis . To confNext, we tested primer extension in the presence of ddhCTP or 3′-dCTP in addition to the normal NTPs to examine how well these chain-terminating nucleotides compete with CTP for incorporation by RdRp. For this experiment, we used the same 5′-FAM labeled primer as above, annealed to a 40-nt template strand. In a titration of 3′-dCTP concentration from 0 to 1.5 mM against a fixed concentration (15 μM) of CTP , we obseThe ExoN activity of SARS-CoV-2 nsp14 has been shown to be able to excise various nucleotide analogues including ribavirin, remdesivir, and sofosbuvir from the 3′ terminus of RNA ,26. To tThe recent discovery of a natural chain-terminating nucleotide ddhCTP, produced upon viral infections, poses an important question as to how RNA viruses may overcome this endogenous antiviral mechanism in mammalian cells . Our expCoronavirus nsp14 is a multi-functional enzyme, which contains the N-terminal ExoN domain responsible for proofreading during RNA synthesis and the C-terminal guanine N7 methyltransferase domain essential in viral mRNA capping ,37,38,39