Stem-loop Research Articles

Structural Characterization of the Cotranscriptional Folding of the Thiamin Pyrophosphate Sensing thiC Riboswitch in Escherichia coli.

Riboswitches are RNA-regulating elements that mostly rely on structural changes to modulate gene expression at various levels. Recent studies have revealed that riboswitches may control several regulatory mechanisms cotranscriptionally, i.e., during the transcription elongation of the riboswitch or early in the coding region of the regulated gene. Here, we study the structure of the nascent thiamin pyrophosphate (TPP)-sensing thiC riboswitch in Escherichia coli by using biochemical and enzymatic conventional probing approaches. Our chemical (in-line and lead probing) and enzymatic (nucleases S1, A, T1, and RNase H) probing data provide a comprehensive model of how TPP binding modulates the structure of the thiC riboswitch. Furthermore, by using transcriptional roadblocks along the riboswitch sequence, we find that a certain portion of nascent RNA is needed to sense TPP that coincides with the formation of the P5 stem loop. Together, our data suggest that conventional techniques may readily be used to study cotranscriptional folding of nascent RNAs.

Deciphering the Binding of 5' Stem Loop RNA to the La Domain of Human LARP6.

La-related protein 6 regulates the highly organized biosynthesis of type I procollagen polypeptides and affects proper assembly of procollagen peptides into heterotrimers of type I procollagen. LARP6-mediated regulation of collagen biosynthesis is mediated through interaction with the 5' stem loop motif found in type I and III collagen mRNA. Recent studies highlight the involvement of HsLARP6 in fibroproliferative diseases and its potential as a target for therapeutic intervention. The intrinsic instability of the La domain of HsLARP6 hampers studies probing the molecular basis of biologically- and disease-relevant structure-function relationship, particularly when high concentrations are required. This work provides detailed procedures to produce milligram amounts of RNase-free and functional La domain of HsLARP6. Furthermore, we investigated the effect of the construct length as well as RNA binding on protein stability. N- and C-terminal extensions greatly impact stability based on interactions with the core domain and modulation of the pI. When in complex with its cognate 5'SL RNA, the La domain shows unprecedented stability compared to the aggregation-prone unbound state. The protein-RNA complex remains stable for at least 50x longer than the unbound state, under identical conditions, likely due to a global change in conformational plasticity upon RNA binding. These results provide a foundation for further studies of the molecular recognition of 5'SL by HsLARP6 as well as a platform for refining potential antifibrotic therapeutics.

DNAzyme and controllable cholesterol stacking DNA machine integrates dual-target recognition CTCs enable homogeneous liquid biopsy of breast cancer

Although circulating tumor cells (CTCs) have demonstrated considerable importance in liquid biopsy, their detection is limited by low concentrations and complex sample components. Herein, we developed a homogeneous, simple, and high-sensitivity strategy targeting breast cancer cells. This method was based on a non-immunological stepwise centrifugation preprocessing approach to isolate CTCs from whole blood. Precise quantification is achieved through the specific binding of aptamers to the overexpressed mucin 1 (MUC1) and human epidermal growth factor receptor 2 (HER2) proteins of breast cancer cells. Subsequently, DNAzyme cleavage and parallel catalytic hairpin assembly (CHA) reactions on the cholesterol-stacking DNA machine were initiated, which opened the hairpin structures T-Hg2+-T and C–Ag+-C, enabling multiple amplifications. This leads to the fluorescence signal reduction from Hg2+-specific carbon dots (CDs) and CdTe quantum dots (QDs) by released ions. This strategy demonstrated a detection performance with a limit of detection (LOD) of 3 cells/mL and a linear range of 5–100 cells/mL. 42 clinical samples have been validated, confirming their consistency with clinical imaging, pathology findings and the folate receptor (FR)-PCR kit results, exhibiting desirable specificity of 100% and sensitivity of 80.6%. These results highlight the promising applicability of our method for diagnosing and monitoring breast cancer.

Determinants of the interaction between the 5′-leader of HIV-1 genome and human lysyl-tRNA synthetase in reverse transcription primer release process

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) initiates from the 3′ end of human tRNALys3. The primer tRNALys3 is selectively packaged into the virus in the form of a complex with human lysyl-tRNA synthetase (LysRS). To facilitate reverse transcription initiation, part of the 5′ leader (5′L) of HIV-1 genomic RNA (gRNA) evolves a tRNA anticodon-like element (TLE), which binds LysRS and releases tRNALys3 for primer annealing and reverse transcription initiation. Although TLE has been identified as a key element in 5′L responsible for LysRS binding, how the conformations and various hairpin structures of 5′L regulate 5′L-LysRS interaction is not fully understood. Here, these factors have been individually investigated using direct and competitive fluorescence anisotropy binding experiments. Our data showed that the conformation of 5′L significantly influences its binding affinity with LysRS. The 5′L conformation favoring gRNA dimerization and packaging exhibits much weaker binding affinity with LysRS compared to the alternative 5′L conformation that is not selected for packaging. Additionally, dimerization of 5′L impairs LysRS-5′L interaction. Furthermore, among various regions of 5′L, both the primer binding site/TLE domain and the stem-loop 3 are important for LysRS interaction, whereas the dimerization initiation site and the splicing donor plays a minor role. In contrast, the presence of the transacting responsive and the polyadenylation signal hairpins slightly inhibit LysRS binding. These findings reveal that the conformation and various regions of the 5′L of HIV-1 genome regulate its interaction with human LysRS and the reverse transcription primer release process.

CRISPR/Cas12a-mediated DNA-AgNC label-free logical gate for multiple microRNAs' assay.

CRISPR/Cas system has been widely applied in the assay of disease-related nucleic acids. However, it is still challenging to use CRISPR/Cas system to detect multiple nucleic acids at the same time. Herein, we combined the preponderance of DNA logic circuit, label-free, and CRISPR/Cas technology to construct a label-free "AND" logical gate for multiple microRNAs detectionwith high specificity and sensitivity. With the simultaneous input of miRNA-155 and miRNA-141, the logic gate starts, and the activation chain of Cas12a is destroyed; thus, the activity is inhibited and the fluorescence of the signal probe ssDNA-AgNCs is turned on. The detection limit of this method for simultaneous quantitative detection of double target is 84 fmol/L (S/N = 3). In this "AND" logic gate, it is only necessary for the design of a simple DNA hairpin probe, which is inexpensive and easy, and sincethis method involves only one signal output, the data processing is very simple. What is more important, in this strategy two types of microRNAs can be monitored simultaneously by only using CRISPR/Cas12a and a type of crRNA, which offers a new design concept for the exploitation of single CRISPR/Cas system for multiple nucleic acid assays.

The 5'-terminal stem-loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH.

We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5'-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5'- and 3'-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data. We show that the internal loop is the major binding site for fragments of low molecular weight. This internal loop of SL1 can be stabilized by an A12-C28 interaction that promotes the transient formation of an A+•C base pair. As a consequence, the pKa of the internal loop adenosine A12 is shifted to 5.8, compared to a pKa of 3.63 of free adenosine. Furthermore, applying a recently developed pH-differential mutational profiling (PD-MaP) approach, we not only recapitulated our NMR findings of SL1 but also unveiled multiple sites potentially sensitive to pH across the 5'-UTR of SARS-CoV-2.

Conformational dynamics of the hepatitis C virus 3'X RNA.

The 3' end of the hepatitis C virus genome is terminated by a highly conserved, 98 nt sequence called 3'X. This untranslated structural element is thought to regulate several essential RNA-dependent processes associated with infection. 3'X has two proposed conformations comprised of either three or two stem-loop structures that result from the different base-pairing interactions within the first 55 nt. Here, we used single-molecule Förster resonance energy transfer spectroscopy to monitor the conformational status of fluorescently labeled constructs that isolate this region of the RNA (3'X55). We observed that 3'X55 can adopt both proposed conformations and the relative abundance of them can be modulated by either solution conditions or nucleotide deletions. Furthermore, interconversion between the two conformations takes place over the course of several hours. The simultaneous existence of two slowly interconverting conformations may help prime individual copies of the viral genome for either viral protein or RNA synthesis, thereby minimizing conflicts between these two competing processes.

Mutations in the Receptor Binding Domain of Severe Acute Respiratory Coronavirus-2 Omicron Variant Spike Protein Significantly Stabilizes Its Conformation.

The Omicron variant and its sub-lineages are the only current circulating SARS-CoV-2 viruses worldwide. In this study, the conformational stability of the isolated Receptor Binding Domain (RBD) of Omicron's spike protein is examined in detail. The parent Omicron lineage has over ten mutations in the ACE2 binding region of the RBD that are specifically associated with its β hairpin loop domain. It is demonstrated through biophysical molecular computations that the mutations in the β hairpin loop domain significantly increase the intra-protein interaction energies of intra-loop and loop-RBD interactions. The interaction energy increases include the formation of new hydrogen bonds in the β hairpin loop domain that help stabilize this critical ACE2 binding region. Our results also agree with recent experiments on the stability of Omicron's core β barrel domain, outside of its loop domain, and help demonstrate the overall conformational stability of the Omicron RBD. It is further shown here through dynamic simulations that the unbound state of the Omicron RBD remains closely aligned with the bound state configuration, which was not observed for the wild-type RBD. Overall, these studies demonstrate the significantly increased conformational stability of Omicron over its wild-type configuration and raise a number of questions on whether conformational stability could be a positive selection feature of SARS-CoV-2 viral mutational changes.

Nanoparticle delivery of Tat synergizes with classical latency reversal agents to express HIV antigen targets.

Limited cellular levels of the HIV transcriptional activator Tat are one contributor to proviral latency that might be targeted in HIV cure strategies. We recently demonstrated that lipid nanoparticles containing HIV tat mRNA induce HIV expression in primary CD4 T cells. Here, we sought to further characterize tat mRNA in the context of several benchmark latency reversal agents (LRAs), including inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-Terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi). tat mRNA reversed latency across several different cell line models of HIV latency, an effect dependent on the TAR hairpin loop. Synergistic enhancement of tat mRNA activity was observed with IAPi, HDACi, and BETi, albeit to variable degrees. In primary CD4 T cells from durably suppressed people with HIV, tat mRNA profoundly increased the frequencies of elongated, multiply-spliced, and polyadenylated HIV transcripts, while having a lesser impact on TAR transcript frequencies. tat mRNAs alone resulted in variable HIV p24 protein induction across donors. However, tat mRNA in combination with IAPi, BETi, or HDACi markedly enhanced HIV RNA and protein expression without overt cytotoxicity or cellular activation. Notably, combination regimens approached or in some cases exceeded the latency reversal activity of maximal mitogenic T cell stimulation. Higher levels of tat mRNA-driven HIV p24 induction were observed in donors with larger mitogen-inducible HIV reservoirs, and expression increased with prolonged exposure time. Combination LRA strategies employing both small molecule inhibitors and Tat delivered to CD4 T cells are a promising approach to effectively target the HIV reservoir.

A highly sensitive fluorescence sensor for tobacco mosaic virus RNA based on DSN cycle and ARGET ATRP double signal amplification

AbstractEarly and sensitive detection of tobacco mosaic virus (TMV) is of great significance for improving crop yield and protecting germplasm resources. Herein, we constructed a novel fluorescence sensor to detect TMV RNA (tRNA) through double strand specific nuclease (DSN) cycle and activator regenerative electron transfer atom transfer radical polymerization (ARGET ATRP) dual signal amplification strategy. The hairpin DNA complementarily paired with tRNA was used as a recognition unit to specifically capture tRNA. By the double‐stranded DNA hydrolyzed with DSN, tRNA is released to open more hairpin DNA, and more complementary DNA (cDNA) is bound to the surface of the magnetic beads (MBs) to achieve the first amplification. After binding with the initiator, the cDNA employed ARGET ATRP to attach more fluorescent signal molecules to the surface of MBs, thus achieving the second signal amplification. Under the optimal experimental conditions, the logarithm of fluorescence intensity versus tRNA concentration showed a good linear relationship in the range of 0.01–100 pM, with a detection limit of 1.03 fM. The limit of detection (LOD) was calculated according to LOD = 3 N/S. Besides, the sensor showed good reproducibility and stability, which present provided new method for early and highly sensitive detection for plant viruses.

Hairpin DNA-Based Nanomaterials for Tumor Targeting and Synergistic Therapy.

While nanoplatform-based cancer theranostics have been researched and investigated for many years, enhancing antitumor efficacy and reducing toxic side effects is still an essential problem. We exploited nanoparticle coordination between ferric (Fe2+) ions and telomerase-targeting hairpin DNA structures to encapsulate doxorubicin (DOX) and fabricated Fe2+-DNA@DOX nanoparticles (BDDF NPs). This work studied the NIR fluorescence imaging and pharmacokinetic studies targeting the ability and biodistribution of BDDF NPs. In vitro and vivo studies investigated the nano formula's toxicity, imaging, and synergistic therapeutic effects. The enhanced permeability and retention (EPR) effect and tumor targeting resulted in prolonged blood circulation times and high tumor accumulation. Significantly, BDDF NPs could reduce DOX-mediated cardiac toxicity by improving the antioxidation ability of cardiomyocytes based on the different telomerase activities and iron dependency in normal and tumor cells. The synergistic treatment efficacy is enhanced through Fe2+-mediated ferroptosis and the β-catenin/p53 pathway and improved the tumor inhibition rate. Harpin DNA-based nanoplatforms demonstrated prolonged blood circulation, tumor drug accumulation via telomerase-targeting, and synergistic therapy to improve antitumor drug efficacy. Our work sheds new light on nanomaterials for future synergistic chemotherapy.

Quantifying Microsecond Solution-Phase Conformational Dynamics of a DNA Hairpin at the Single-Molecule Level.

Quantifying the rapid conformational dynamics of biological systems is fundamental to understanding the mechanism. However, biomolecules are complex, often containing static and dynamic heterogeneity, thus motivating the use of single-molecule methods, particularly those that can operate in solution. In this study, we measure microsecond conformational dynamics of solution-phase DNA hairpins at the single-molecule level using an anti-Brownian electrokinetic (ABEL) trap. Different conformational states were distinguished by their fluorescence lifetimes, and kinetic parameters describing transitions between these states were determined using two-dimensional fluorescence lifetime correlation (2DFLCS) analysis. Rather than combining fluorescence signals from the entire data set ensemble, long observation times of individual molecules allowed ABEL-2DFLCS to be performed on each molecule independently, yielding the underlying distribution of the system's kinetic parameters. ABEL-2DFLCS on the DNA hairpins resolved an underlying heterogeneity of fluorescence lifetimes and provided signatures of two-state exponential dynamics with rapid (<millisecond) transition times between states without observation of the substantially stretched exponential kinetics that had been observed in previous measurements on diffusing molecules. Numerical simulations were performed to validate the accuracy of this technique and the effects the underlying heterogeneity has on the analysis. Finally, ABEL-2DFLCS was performed on a mixture of hairpins and used to resolve their kinetic data.

A versatile photoelectrochemical biosensor based on in-situ grown 2D COFs film for sensitive detection of Hg2+ and aflatoxin B1

Herein, a highly stable and low-toxic photoelectrochemical (PEC) biosensor was constructed based on two-dimensional covalent organic frameworks (COFs) film composed of tris(4-aminophenyl)amine and 1,4-phthalaldehyde connected through imine bonds (TA-P COFs) was grown in situ on the electrode surface under mild conditions for detecting mercury ion (Hg2+) and aflatoxin B1 (AFB1). Impressively, the TA-P COF film adhered to the electrode surface through in-situ growth not only accelerated the charge transfer between the interfaces, but also improved the stability of the biosensor, further improving the accuracy of detection. Accordingly, when the cyclic amplification process was triggered in the presence Hg2+ to generate a large amount of output DNA, the signal probe with abundant quencher porous carbon hollow spheres s (PCHS) was modified on the electrode surface through the output DNA for the signal “OFF” detection of Hg2+. Meanwhile, due to the specific binding of the target to the aptamer, the released hairpin DNA 2 (H2) signal probe was modified on the electrode surface through base complementary pairing to achieve the signal “ON” detection of AFB1. The suggested multifunction biosensor achieved sensitive detection of Hg2+ in the range of 10 fM–100 nM and AFB1 in the range of 10 fg mL−1–10 ng mL−1, which showed great practical application potential in both environmental and food analysis.

Engineering smart thioflavin T binders with switchable DNA topologies for constructing multiple label-free biosensors

Thioflavin T (ThT) has been used as a sensitive fluorescence probe for lighting up nucleic acids. The binding of ThT is dependent on the structural topologies of DNA sequences. To date, most oligonucleotides interact with ThT through only a single topology, and this limits the applications of ThT as a sensing agent. Here, we modulated an HIV-1 integrase aptamer, 93del, to have switchable topologies by optimizing its 3′ flanking residues to enhance its ThT-binding capacity. Our data revealed that the sequences formed stem-loop hairpins and I-motif/G-quadruplex (G4) hybrids in Na+ buffer under alkaline and acidic conditions, respectively. Both topologies could bind ThT with high affinity (Kd ∼ 0.5 μM) and dramatically enhance the fluorescent signal of ThT. However, in the presence of K+, the sequences could be assembled into interlocked G4s, which had a low affinity for ThT but a high affinity for N-methyl mesoporphyrin IX (NMM). This allowed us to build highly sensitive fluorescent K+ sensors that were insusceptible to interference from Na+. Additionally, we found that the stacking of homologous base pairs in the hairpin loops was essential for ThT recognition and could be used as an indicator in the construction of label-free molecular beacons, allowing monitoring of microRNAs effectively in human serum spiked test. By employing external stimuli to induce the transformation of these topologies, we were able to sensitively detect adenosine triphosphate (ATP) molecules or Hg2+in vitro under specific acidic conditions. Our study provides a strategy for exploiting undiscovered functions of existing aptamers.

ANNaMo: Coarse-grained modeling for folding and assembly of RNA and DNA systems.

The folding of RNA and DNA strands plays crucial roles in biological systems and bionanotechnology. However, studying these processes with high-resolution numerical models is beyond current computational capabilities due to the timescales and system sizes involved. In this article, we present a new coarse-grained model for investigating the folding dynamics of nucleic acids. Our model represents three nucleotides with a patchy particle and is parameterized using well-established nearest-neighbor models. Thanks to the reduction of degrees of freedom and to a bond-swapping mechanism, our model allows for simulations at timescales and length scales that are currently inaccessible to more detailed models. To validate the performance of our model, we conducted extensive simulations of various systems: We examined the thermodynamics of DNA hairpins, capturing their stability and structural transitions, the folding of an MMTV pseudoknot, which is a complex RNA structure involved in viral replication, and also explored the folding of an RNA tile containing a k-type pseudoknot. Finally, we evaluated the performance of the new model in reproducing the melting temperatures of oligomers and the dependence on the toehold length of the displacement rate in toehold-mediated displacement processes, a key reaction used in molecular computing. All in all, the successful reproduction of experimental data and favorable comparisons with existing coarse-grained models validate the effectiveness of the new model.

A structure-switching electrochemical aptamer sensor for mercury ions based on an ordered assembled gold nanorods-modified electrode

In this paper, an electrochemical aptamer sensor with high sensitivity and selectivity was proposed for the detection of mercury ion (Hg2+). The ordered assembled gold nanorods-modified screen-printed electrode (AuNRs/SPE) was used to improve the sensor with higher sensitivity and data reproducibility. The sensor was designed based on the specific recognition of Hg2+ with aptamer to form T-Hg2+-T binding structure. Two aptamer chains were applied in the designed sensor, aptamer S1 with terminal thiolate group was self-assembled on the AuNRs/SPE via Au–S bond, and complementary S2 labeled with ferrocene (Fc) would generate a strong redox electrical signal. The addition of Hg2+ would mediate the folding of the T-base of S1 to form a hairpin structure, resulting in the dissociation of the Fc-labeled S2 from the sensing system and a significant reduction in the redox current. The electrochemical properties of the sensor were further investigated using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The effects of aptamer reaction time, concentration, and target incubation time on the sensor response were also investigated. The results showed that the sensor had a sensitive response to Hg2+ at concentrations ranging from 1 pM to 1 μM, with a detection limit of 0.3 pM. Furthermore, the proposed sensor was applied in the determination of Hg2+ in real samples, demonstrating its potential practical value.

DNA-Functionalized Gold Nanorods for One-Step Simultaneous Determination of Multiple microRNAs Based upon Plasma-Enhanced Fluorescence

MicroRNAs (miRNAs) are endogenous, noncoding, and sequence-specific single-stranded RNA regulatory factors whose abnormal expression levels are closely related to various diseases, especially cancer, and are considered effective biomarkers for disease diagnosis and prognosis. Since the concentrations of miRNAs in the body are low and disease diagnosis by detecting single miRNAs may result in a high false positive, it is essential to construct a sensitive biosensor for the determination of multiple miRNAs. In this work, an enzyme-free signal amplification assay based on plasma-enhanced fluorescence (PEF) is reported for the one-step simultaneous determination of two miRNAs. Gold nanorods (AuNRs) were functionalized with fluorescein isothiocyanate (FAM)-tagged hairpin DNA1 (H1) and cyanine 5.5 (Cy5.5)-tagged hairpin DNA2 (H2). The fluorescence signals of FAM and Cy5.5 were quenched due to their proximity to the surface of the AuNRs. In the presence of target miRNA-21 and miRNA-155, H1 and H2 complemented miRNA-21 and miRNA-155, respectively, and the fluorescence signals of FAM and Cy5.5 were not only restored but amplified due to the plasma-enhanced fluorescence. A suitable linear range (0.1 to 10 nM) and low limits of detection for miRNA-21 (23 pM) and miRNA-155 (1.6 pM) were achieved. The developed assay for the one-step simultaneous measurement of two miRNAs is simple, fast and sensitive, exhibiting great potential for monitoring these biomarkers.

Noncanonical mutations in ribosome nascent peptide exit tunnel confer clarithromycin resistance in Mycobacterium abscessus complex

ObjectivesMycobacterium abscessus is a non-tuberculous mycobacterial pathogen that causes pulmonary and skin infections globally. Clarithromycin plays a pivotal role in treating M. abscessus infections, with resistance often leading to treatment failure. While canonical mutations in the 23S rRNA residue 2270/2271 are recognized as the primary mechanism for acquired clarithromycin resistance, resistant isolates lacking these mutations have been widely reported. This study aims to identify new mechanisms of clarithromycin resistance in M. abscessus. MethodsWe selected spontaneous resistant mutants derived from two parental strains characterized by erm(41) T28 and C28 sequevars, respectively. Whole-genome sequencing was performed on mutants lacking the 23S rRNA 2270/2271 mutations. Site-directed mutagenesis was used to confirm the resistance phenotypes of newly identified mutations. Bioinformatic analysis of publicly available genomes was conducted to evaluate the presence of these mutations in clinical isolates. The spatial localization of these mutations in the ribosome was analyzed to investigate potential mechanisms of resistance. ResultsA total of 135 resistant mutants were selected from the parental strains. Sequencing of the 78 mutants lacking the 23S rRNA 2270/2271 mutations identified mutations within the peptidyl-transferase center and hairpin loops 35, 49, and 74 of the 23S rRNA. These noncanonical mutations were identified in 57 of 1875 genomes of clinical isolates. Thirteen representative mutations were introduced into the bacterial genome, and their contributions to macrolide resistance were confirmed. The newly identified mutations all localized at the entrance of the nascent peptide exit tunnel, potentially contributing to resistance by disrupting the macrolide binding pocket. ConclusionSeveral noncanonical 23S rRNA mutations conferring clarithromycin resistance were identified. These mutations enhance our understanding of macrolide resistance in M. abscessus and could serve as important markers for diagnosing clarithromycin resistance.

M6A Methylation of Transcription Leader Sequence of SARS-CoV-2 Impacts Discontinuous Transcription of Subgenomic mRNAs.

The SARS-CoV-2 genome has been shown to be m6A methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base-paired in the short stem-loop structure 5'SL3 that opens during discontinuous transcription to form long-range RNA-RNA interactions with nascent (-)-strand transcripts at complementary TRS-body sequences. A74 can be methylated by the human METTL3/METTL14 complex in vitro. Here, we investigate its impact on the structural stability of 5'SL3 and the long-range TRS-leader:TRS-body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5'SL3 and long-range duplexes and alters their relative equilibrium populations, suggesting that the m6A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization.

Knotify_V2.0: Deciphering RNA Secondary Structures with H-Type Pseudoknots and Hairpin Loops.

Accurately predicting the pairing order of bases in RNA molecules is essential for anticipating RNA secondary structures. Consequently, this task holds significant importance in unveiling previously unknown biological processes. The urgent need to comprehend RNA structures has been accentuated by the unprecedented impact of the widespread COVID-19 pandemic. This paper presents a framework, Knotify_V2.0, which makes use of syntactic pattern recognition techniques in order to predict RNA structures, with a specific emphasis on tackling the demanding task of predicting H-type pseudoknots that encompass bulges and hairpins. By leveraging the expressive capabilities of a Context-Free Grammar (CFG), the suggested framework integrates the inherent benefits of CFG and makes use of minimum free energy and maximum base pairing criteria. This integration enables the effective management of this inherently ambiguous task. The main contribution of Knotify_V2.0 compared to earlier versions lies in its capacity to identify additional motifs like bulges and hairpins within the internal loops of the pseudoknot. Notably, the proposed methodology, Knotify_V2.0, demonstrates superior accuracy in predicting core stems compared to state-of-the-art frameworks. Knotify_V2.0 exhibited exceptional performance by accurately identifying both core base pairing that form the ground truth pseudoknot in 70% of the examined sequences. Furthermore, Knotify_V2.0 narrowed the performance gap with Knotty, which had demonstrated better performance than Knotify and even surpassed it in Recall and F1-score metrics. Knotify_V2.0 achieved a higher count of true positives (tp) and a significantly lower count of false negatives (fn) compared to Knotify, highlighting improvements in Prediction and Recall metrics, respectively. Consequently, Knotify_V2.0 achieved a higher F1-score than any other platform. The source code and comprehensive implementation details of Knotify_V2.0 are publicly available on GitHub.