Cytoplasmic Mechanisms of Recognition and Defense of Microbial Nucleic Acids.

Microbial nucleic acids are major signatures of invading pathogens, and their recognition by various host pattern recognition receptors (PRRs) represents the first step toward an efficient innate immune response to clear the pathogens. The nucleic acid-sensing PRRs are localized at the plasma membrane, the cytosol, and/or various cellular organelles. Sensing of nucleic acids and signaling by PRRs involve recruitment of distinct signaling components, and PRRs are intensively regulated by cellular organelle trafficking. PRR-mediated innate immune responses are also heavily regulated by posttranslational modifications, including phosphorylation, polyubiquitination, sumoylation, and glutamylation. In this review, we focus on our current understanding of recognition of microbial nucleic acid by PRRs, particularly on their regulation by organelle trafficking and posttranslational modifications. We also discuss how sensing of self nucleic acids and dysregulation of PRR-mediated signaling lead to serious human diseases.

Role of the biomolecular energy gap in protein design, structure, and evolution.

The folding of natural biopolymers into unique three-dimensional structures that determine their function is remarkable considering the vast number of alternative states and requires a large gap in the energy of the functional state compared to the many alternatives. This Perspective explores the implications of this energy gap for computing the structures of naturally occurring biopolymers, designing proteins with new structures and functions, and optimally integrating experiment and computation in these endeavors. Possible parallels between the generation of functional molecules in computational design and natural evolution are highlighted.

RNA Guide Complementarity Prevents Self-Targeting in Type VI CRISPR Systems.

All immune systems use precise target recognition to interrogate foreign invaders. During CRISPR-Cas immunity, prokaryotes capture short spacer sequences from infecting viruses and insert them into the CRISPR array. Transcription and processing of the CRISPR locus generate small RNAs containing the spacer and repeat sequences that guide Cas nucleases to cleave a complementary protospacer in the invading nucleic acids. In most CRISPR systems, sequences flanking the protospacer drastically affect cleavage. Here, we investigated the target requirements of the recently discovered RNA-targeting type VI-A CRISPR-Cas system in its natural host, Listeria seeligeri. We discovered that target RNAs with extended complementarity between the protospacer flanking sequence and the repeat sequence of the guide RNA are not cleaved by the type VI-A nuclease Cas13, neither in vivo nor in vitro. These findings establish fundamental rules for the design of Cas13-based technologies and provide a mechanism for preventing self-targeting in type VI-A systems.

The Nucleic Acid Knowledgebase: a new portal for 3D structural information about nucleic acids.

The Nucleic Acid Knowledgebase (nakb.org) is a new data resource, updated weekly, for experimentally determined 3D structures containing DNA and/or RNA nucleic acid polymers and their biological assemblies. NAKB indexes nucleic acid-containing structures derived from all major structure determination methods (X-ray, NMR and EM), including all held by the Protein Data Bank (PDB). As the planned successor to the Nucleic Acid Database (NDB), NAKB's design preserves all functionality of the NDB and provides novel nucleic acid-centric content, including structural and functional annotations, as well as annotations from and links to external resources. A variety of custom interactive tools have been developed to enable rapid exploration and drill-down of NAKB's content.

A novel nucleic acid linker for multi-gene expression enhances plant and animal synthetic biology.

The production of compact vectors for gene stacking is hindered by a lack of effective linkers. Here, we report that a 26-nt nucleic acid linker, NAL1, from the fungus Glarea lozoyensis and its truncated derivatives could connect two genes as a bicistron, enabling independent translation in a maize protoplast transient expression system and human 293 T cells. The optimized 9-nt NAL10 linker was then used to connect four genes driven by a bidirectional promoter; this combination was successfully used to reconstruct the astaxanthin biosynthesis pathway in transgenic maize. The short and efficient nucleic acid linker NAL10 can be widely used in multi-gene expression and synthetic biology in animals and plants.

Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides.

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids, such as RNA transcription, translation, splicing and degradation, or DNA replication and repair, is impaired by the presence of the CPPs. Interestingly, the effects of (PR)n are fully mimicked by protamine, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated with the onset of ALS.

Assessment of nucleic acid extraction protocols for antibiotic resistance genes (ARGs) quantification in aircraft wastewater.

This study evaluated ten nucleic acid extraction protocols (EP1 to EP10) for measuring five endogenous antibiotic resistance genes (ARGs) in four aircraft wastewater samples (AWW1 to AWW4). The targeted ARGs, including blaCTX-M, blaNDM-1, ermB, qnrS, and tetA, encompassed highly and minimally abundant ARGs. TetA and ermB were consistently detected across four aircraft wastewater samples using the DNeasy Blood and Tissue Kit and the AllPrep PowerViral DNA/RNA kit. QnrS displayed high detection rates with specific extraction protocols and aliquot volumes. Concentrations of ARGs varied across aircraft wastewater samples, with differing extraction protocols influencing quantitative results. The concentrations of tetA, ermB, and qnrS in AWW1 were distinct, while AWW2 to AWW4 exhibited a broader range for tetA, ermB, qnrS, blaCTX-M, and blaNDM-1. EP1 consistently produced the highest concentrations for several ARGs. Collective data analysis revealed varying ARG concentrations across the ten extraction protocols, suggesting the importance of careful extraction protocol selection in ARG monitoring in aircraft wastewater samples. Based on the results, we suggest that a small sample volume (as low as 0.2 mL) may be sufficient for ARG characterization in aircraft wastewater samples. The findings also emphasize the need for considering toilet paper removal without compromising nucleic acid extraction efficiency. The study highlights promising prospects for aircraft wastewater monitoring of ARGs, calling for further investigation into the import and spread of unique ARGs through transport hubs.

Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 13 (Cas13) has been rapidly developed for nucleic-acid-based diagnostics by using its characteristic collateral activity. Despite the recent progress in optimizing the Cas13 system for the detection of nucleic acids, engineering Cas13 protein with enhanced collateral activity has been challenging, mostly because of its complex structural dynamics. Here we successfully employed a novel strategy to engineer the Leptotrichia wadei (Lwa)Cas13a by inserting different RNA-binding domains into a unique active-site-proximal loop within its higher eukaryotes and prokaryotes nucleotide-binding domain. Two LwaCas13a variants showed enhanced collateral activity and improved sensitivity over the wild type in various buffer conditions. By combining with an electrochemical method, our variants detected the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification. Our engineered LwaCas13a enzymes with enhanced collateral activity are ready to be integrated into other Cas13a-based platforms for ultrasensitive detection of nucleic acids.

Nucleic acid degradation as barrier to gene delivery: a guide to understand and overcome nuclease activity.

Gene therapy is on its way to revolutionize the treatment of both inherited and acquired diseases, by transferring nucleic acids to correct a disease-causing gene in the target cells of patients. In the fight against infectious diseases, mRNA-based therapeutics have proven to be a viable strategy in the recent Covid-19 pandemic. Although a growing number of gene therapies have been approved, the success rate is limited when compared to the large number of preclinical and clinical trials that have been/are being performed. In this review, we highlight some of the hurdles which gene therapies encounter after administration into the human body, with a focus on nucleic acid degradation by nucleases that are extremely abundant in mammalian organs, biological fluids as well as in subcellular compartments. We overview the available strategies to reduce the biodegradation of gene therapeutics after administration, including chemical modifications of the nucleic acids, encapsulation into vectors and co-administration with nuclease inhibitors and discuss which strategies are applied for clinically approved nucleic acid therapeutics. In the final part, we discuss the currently available methods and techniques to qualify and quantify the integrity of nucleic acids, with their own strengths and limitations.

Terminal Chemical Modifications of crRNAs Enable Improvement in the Performance of CRISPR-Cas for Point-of-Care Nucleic Acid Detection.

CRISPR-Cas systems, harnessing their precise nucleic acid recognition via CRISPR RNA (crRNA), offer promise for the accurate testing of nucleic acids in the field. However, the inherent susceptibility of crRNA to degradation poses challenges for accurate detection in low-resource settings. Here, we utilized the chemically modified crRNA for the CRISPR-Cas-based assay (CM-CRISPR). We found that the extension and chemical modification to crRNA significantly enhanced the trans-cleavage activity of LbCas12a. The chemically modified crRNA was resistant to degradation, and CM-CRISPR showed superior detection capability in complex environments. CM-CRISPR could be combined with recombinase polymerase amplification (RPA) and applied in a droplet digital platform, enabling attomolar-level sensitivity. We also developed a portable and automated device for a digital CRISPR assay, which is amenable to point-of-care testing (POCT). The extraction-free procedure was integrated with this assay to streamline the workflow, and clinical samples were successfully detected. This work finds a simple and efficient way to improve the performance of CRISPR-Cas and develops a portable platform for POCT, representing a significant advance toward practical applications of CRISPR-based diagnostics.

Enhancing Endosomal Escape and Gene Regulation Activity for Spherical Nucleic Acids.

The therapeutic potential of small interfering RNAs (siRNAs) is limited by their poor stability and low cellular uptake. When formulated as spherical nucleic acids (SNAs), siRNAs are resistant to nuclease degradation and enter cells without transfection agents with enhanced activity compared to their linear counterparts; however, the gene silencing activity of SNAs is limited by endosomal entrapment, a problem that impacts many siRNA-based nanoparticle constructs. To increase cytosolic delivery, SNAs are formulated using calcium chloride (CaCl2 ) instead of the conventionally used sodium chloride (NaCl). The divalent calcium (Ca2+ ) ions remain associated with the multivalent SNA and have a higher affinity for SNAs compared to their linear counterparts. Importantly, confocal microscopy studies show a 22% decrease in the accumulation of CaCl2 -salted SNAs within the late endosomes compared to NaCl-salted SNAs, indicating increased cytosolic delivery. Consistent with this finding, CaCl2 -salted SNAs comprised of siRNA and antisense DNA all exhibit enhanced gene silencing activity (up to 20-fold), compared to NaCl-salted SNAs regardless of sequence or cell line (U87-MG and SK-OV-3) studied. Moreover, CaCl2 -salted SNA-based forced intercalation probes show improved cytosolic mRNA detection.

Pyrophosphate detection method using 5-Br-PAPS to detect nucleic acid amplification - Application to LAMP method.

Genetic testing has been increasingly used in several fields. In many applications, nucleic acid amplification technology is required. However, current methods to detect nucleic acid amplification require expensive reagents and special equipment or exhibit limited sensitivity, which hinders their use. To address this issue, this study reports an assay method for detecting occurrence of acid amplification in post-amplification samples using pyrophosphate, a highly sensitive byproduct of nucleic acid amplification. The method proposed requires two reagents and an automated analyzer. First, hydrogen peroxide is derived from pyrophosphate, an indicator of nucleic acid amplification, and the oxidizing power of hydrogen peroxide is used to produce Fe (III) from Fe (II). The specific metal chelator 5-Br-PAPS forms a complex with the trivalent iron produced, resulting in a highly sensitive coloration. The within-run reproducibility of our method (n = 20) was less than 3.67% at each concentration tested, and the detection limit was 0.075 µmol/L, sufficient for quantitative analysis. The technique described could detect pyrophosphate in a sample that was amplified using the loop-mediated isothermal amplification method after only 10 min. Therefore, the proposed method has the potential to be a new, rapid, and simple detection technique for amplified nucleic acids.

ProNAB: database for binding affinities of protein-nucleic acid complexes and their mutants.

Protein-nucleic acid interactions are involved in various biological processes such as gene expression, replication, transcription, translation and packaging. The binding affinities of protein-DNA and protein-RNA complexes are important for elucidating the mechanism of protein-nucleic acid recognition. Although experimental data on binding affinity are reported abundantly in the literature, no well-curated database is currently available for protein-nucleic acid binding affinity. We have developed a database, ProNAB, which contains more than 20 000 experimental data for the binding affinities of protein-DNA and protein-RNA complexes. Each entry provides comprehensive information on sequence and structural features of a protein, nucleic acid and its complex, experimental conditions, thermodynamic parameters such as dissociation constant (Kd), binding free energy (ΔG) and change in binding free energy upon mutation (ΔΔG), and literature information. ProNAB is cross-linked with GenBank, UniProt, PDB, ProThermDB, PROSITE, DisProt and Pubmed. It provides a user-friendly web interface with options for search, display, sorting, visualization, download and upload the data. ProNAB is freely available at https://web.iitm.ac.in/bioinfo2/pronab/ and it has potential applications such as understanding the factors influencing the affinity, development of prediction tools, binding affinity change upon mutation and design complexes with the desired affinity.

Nucleic acid ligands act as a PAM and agonist depending on the intrinsic ligand binding state of P2RY2.

G protein-coupled receptors (GPCRs) play diverse roles in physiological processes, and hence the ligands to modulate GPCRs have served as important molecules in biological and pharmacological approaches. However, the exploration of novel ligands for GPCR still remains an arduous challenge. In this study, we report a method for the discovery of nucleic acid ligands against GPCRs by an advanced RNA aptamer screening technology that employs a virus-like particle (VLP), exposing the GPCR of interest. An array of biochemical analyses coupled with a cell-based assay revealed that one of the aptamers raised against purinergic receptor P2Y2 (P2RY2), a GPCR, exhibits an activation potency to unliganded receptor and prohibits a further receptor activation by endogenous ligand, behaving like a partial agonist. However, the aptamer enhances the activity of intrinsic ligand-binding P2RY2, thereby acting as a positive allosteric modulator (PAM) to liganded receptor. Our findings demonstrate that the nucleic acid aptamer conditionally exerts PAM and agonist effects on GPCRs, depending on their intrinsic ligand binding state. These results indicate the validity of our VLP-based aptamer screening targeting GPCR and reemphasize the great potential of nucleic acid ligands for exploring the GPCR activation mechanism and therapeutic applications.

Soil metabolome response to whole-ecosystem warming at the Spruce and Peatland Responses under Changing Environments experiment.

In this study, a suite of complementary environmental geochemical analyses, including NMR and gas chromatography-mass spectrometry (GC-MS) analyses of central metabolites, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of secondary metabolites, and lipidomics, was used to investigate the influence of organic matter (OM) quality on the heterotrophic microbial mechanisms controlling peatland CO2, CH4, and CO2:CH4 porewater production ratios in response to climate warming. Our investigations leverage the Spruce and Peatland Responses under Changing Environments (SPRUCE) experiment, where air and peat warming were combined in a whole-ecosystem warming treatment. We hypothesized that warming would enhance the production of plant-derived metabolites, resulting in increased labile OM inputs to the surface peat, thereby enhancing microbial activity and greenhouse gas production. Because shallow peat is most susceptible to enhanced warming, increases in labile OM inputs to the surface, in particular, are likely to result in significant changes to CO2 and CH4 dynamics and methanogenic pathways. In support of this hypothesis, significant correlations were observed between metabolites and temperature consistent with increased availability of labile substrates, which may stimulate more rapid turnover of microbial proteins. An increase in the abundance of methanogenic genes in response to the increase in the abundance of labile substrates was accompanied by a shift toward acetoclastic and methylotrophic methanogenesis. Our results suggest that as peatland vegetation trends toward increasing vascular plant cover with warming, we can expect a concomitant shift toward increasingly methanogenic conditions and amplified climate-peatland feedbacks.

CRISPR/Cas systems for in situ imaging of intracellular nucleic acids: Concepts and applications.

Accurate and precise localization of intracellular nucleic acids is crucial for regulating genetic information transcription and diagnosing diseases. Although intracellular nucleic acid imaging methods are available for various cell types, their widespread utilization is impeded by the intricate nature of the process and its exorbitant cost. Recently, numerous intracellular nucleic acid labeling techniques based on clustered regularly interspaced short palindromic repeats (CRISPR) have been established due to their modularity, flexibility, and specificity. In this work, we present various CRISPR methods that are currently employed for visualizing intracellular genomic sequences and RNA, based on their detection principles and application scenarios. Furthermore, we discuss the advantages and drawbacks of the existing CRISPR imaging methods, as well as future research directions. We anticipate that with continued refinement, more advanced CRISPR-based imaging techniques can be developed to better elucidate the localization and dynamics of intracellular nucleic acids, thereby providing a powerful tool for molecular biology research and clinical molecular pathology diagnosis.

Directed Evolution: Methodologies and Applications.

Directed evolution aims to expedite the natural evolution process of biological molecules and systems in a test tube through iterative rounds of gene diversifications and library screening/selection. It has become one of the most powerful and widespread tools for engineering improved or novel functions in proteins, metabolic pathways, and even whole genomes. This review describes the commonly used gene diversification strategies, screening/selection methods, and recently developed continuous evolution strategies for directed evolution. Moreover, we highlight some representative applications of directed evolution in engineering nucleic acids, proteins, pathways, genetic circuits, viruses, and whole cells. Finally, we discuss the challenges and future perspectives in directed evolution.

Determining translocation orientations of nucleic acid helicases.

Helicase enzymes translocate along an RNA or DNA template with a defined polarity to unwind, separate, or remodel duplex strands for a variety of genome maintenance processes. Helicase mutations are commonly associated with a variety of diseases including aging, cancer, and neurodegeneration. Biochemical characterization of these enzymes has provided a wealth of information on the kinetics of unwinding and substrate preferences, and several high-resolution structures of helicases alone and bound to oligonucleotides have been solved. Together, they provide mechanistic insights into the structural translocation and unwinding orientations of helicases. However, these insights rely on structural inferences derived from static snapshots. Instead, continued efforts should be made to combine structure and kinetics to better define active translocation orientations of helicases. This review explores many of the biochemical and biophysical methods utilized to map helicase binding orientation to DNA or RNA substrates and includes several time-dependent methods to unequivocally map the active translocation orientation of these enzymes to better define the active leading and trailing faces.

A guide to accelerated direct digital counting of single nucleic acid molecules by FRET-based intramolecular kinetic fingerprinting.

Cell-free nucleic acids (cfNAs) such as short non-coding microRNA (miRNA) and circulating tumor DNA (ctDNA) that reside in bodily fluids have emerged as potential cancer biomarkers. Methods for the rapid, highly specific, and sensitive monitoring of cfNAs in biofluids have, therefore, become increasingly attractive as clinical diagnosis tools. As a next generation technology, we provide a practical guide for an amplification-free, single molecule Förster resonance energy transfer (smFRET)-based kinetic fingerprinting approach termed intramolecular single molecule recognition through equilibrium Poisson sampling, or iSiMREPS, for the rapid detection and counting of miRNA and mutant ctDNA with virtually unlimited specificity and single molecule sensitivity. iSiMREPS utilizes a pair of fluorescent detection probes, wherein one probe immobilizes the target molecules on the surface, and the other probe transiently and reversibly binds to the target to generate characteristic time-resolved fingerprints as smFRET signal that are detected in a total internal reflection fluorescence microscope. Analysis of these kinetic fingerprints enables near-perfect discrimination between specific binding to target molecules and nonspecific background binding. By accelerating kinetic fingerprinting using the denaturant formamide and reducing background signals by removing target-less probes from the surface via toehold-mediated strand displacement, iSiMREPS has been demonstrated to count miR-141 and EGFR exon 19 deletion ctDNA molecules with a limit of detection (LOD) of ~1 and 3 fM, respectively, as well as mutant allele fractions as low as 0.0001%, during a standard acquisition time of only ~10 s per field of view. In this review, we provide a detailed roadmap for implementing iSiMREPS more broadly in research and clinical diagnostics, combining rapid analysis, high specificity, and high sensitivity.

Analysis of the epidemiological trends of enterovirus A in Asia and Europe.

BACKGROUND: Enteroviruses have been in massive, cyclical epidemics worldwide. An in-depth understanding of the international epidemiological characteristics of Enterovirus A (EVA) is critical to determining its clinical significance and total disease burden. Although much research has been conducted on EVA epidemiology, there is still a lack of a comprehensive overview of EVA epidemiological characteristics and trends. OBJECTIVE: EVA nucleic acid sequences from the NCBI virus database were used to summarize the epidemic time (based on the time of specimen collection), spatial and serotype distribution of EVA, and to analyze EVA isolated from cerebrospinal fluid specimens. METHODS: EVA sequences were searched in NCBI Virus by keyword ("Enterovirus A″ or "EVA") to screen sequences released before December 2021 and sort them to analyze EVA by year, geographic region and serotype prevalence. RESULTS: The results found 23,041 retrieved nucleic acid sequences with precise collection dates and geographical regions as of December 2021, with Asia accounting for 87%, Europe for 11% and Africa and the Americas for only 2%. Overall, EV-A71, CVA6 and CVA16 are a few of the main prevalent serotypes; and the prevalence characteristics of the different serotypes change over time from place to place. CONCLUSION: The prevalence of different serotypes of EVA varies considerably over time and space, and we focused on analysing the epidemiological characteristics of EVAs in Asia and Europe and EVAs that invade the nervous system. This study will likely provide important clues for prevention, control and future research in virological surveillance, disease management and vaccine development.