RNA Linkage Research Articles

DNAzyme-based faithful probing and pulldown to identify candidate biomarkers of low abundance.

Biomarker discovery is essential for the understanding, diagnosis, targeted therapy and prognosis assessment of malignant diseases. However, it remains a huge challenge due to the lack of sensitive methods to identify disease-specific rare molecules. Here we present MORAC, molecular recognition based on affinity and catalysis, which enables the effective identification of candidate biomarkers with low abundance. MORAC relies on a class of DNAzymes, each cleaving a sole RNA linkage embedded in their DNA chain upon specifically sensing a complex system with no prior knowledge of the system's molecular content. We show that signal amplification from catalysis ensures the DNAzymes high sensitivity (for target probing); meanwhile, a simple RNA-to-DNA mutation can shut down their RNA cleavage ability and turn them into a pure affinity tool (for target pulldown). Using MORAC, we identify previously unknown, low-abundance candidate biomarkers with clear clinical value, including apolipoprotein L6 in breast cancer and seryl-tRNA synthetase 1 in polyps preceding colon cancer.

Rapid hepatitis C virus point-of-care RNA testing and treatment at an integrated supervised consumption service in Toronto, Canada: a prospective, observational cohort study

Despite high burden of Hepatitis C (HCV) among people who inject drugs, significant barriers to care persist. The aim of this study was to evaluate the provision of rapid, low-barrier point-of-care (POC) HCV RNA testing and linkage to care among clients of a supervised consumption service (SCS) located within a community health centre in Toronto, Canada. Secondary aims included measuring HCV RNA prevalence at baseline, HCV incidence during follow-up and exploring factors associated with HCV RNA positivity and treatment uptake. Participants were enrolled in a prospective, observational cohort from August 13, 2018 to September 30, 2021. Those with positive HCV RNA tests were offered immediate referral to onsite treatment. Those with negative results were offered repeat testing every three months for up to four visits. HCV incidence was estimated as the number of incident HCV infections per 100 person-years at risk, among those HCV RNA negative at baseline who returned for ≥1 follow-up visit. Missing data were reported when present. 128 participants were enrolled with four later removed due to ineligibility. At baseline, 54 of 124 eligible participants (43.5%) tested HCV RNA positive. HCV incidence was 35.1 cases per 100 person-years (95% CI: 18.9-65.3) with a cumulative incidence of 38.3% at 15 months of follow-up. Among participants HCV RNA positive at baseline or follow-up (n=64), 67.2% (n=43) were linked to HCV care and treatment was initiated among 67.4% (n=29/43). High HCV RNA prevalence and incidence demonstrate that the SCS serves a high-risk population for HCV. Testing acceptance was high, as was treatment engagement. POC HCV RNA testing positions SCSs as an important point of HCV care access. HCV Micro-Elimination Grant, Gilead Sciences Canada; in-kind support from Cepheid.

Copy number variation of B1 controls awn length in wheat

Wheat awns contribute to photosynthesis and grain production. In this study, an F2 population and F2:3 families from a cross between the awned line 7D12 and the Chinese awnless variety Shiyou 20 (SY20) were used to identify loci associated with awn length. Bulked-segregant RNA sequencing and linkage mapping identified a single dominant locus in a 0.3 cM interval on chromosome 5AL. Five genes were in the interval, including the recently cloned awn inhibitor B1. Although a single copy of the B1 gene was detected in 7D12, SY20 carried five copies of the gene. Increased copy number of B1 in SY20 enhanced gene expression. Based on sequence variation among the promoter regions of five B1 gene copies in SY20, two dominant markers were developed and found to cosegregate with B1 in a population of 931 wheat accessions. All 77 awnless accessions harbored sequence variations in the B1 promoter regions similar to those of SY20 and thus carried multiple copies of the gene, whereas 15 randomly selected awned wheats carried only one copy. These results suggest that an increase in copy number of the B1 gene is associated with inhibition of awn length.

A novel single-base mutation in CaSGR1 confers the stay-green phenotype in pepper

The stay-green trait is of considerable importance in extending the shelf life of green pepper fruit (Capsicum annuum L.) and in enhancing the appearance of ornamental plants. The study revealed the genetic and regulatory mechanisms of the stay-green trait in pepper, which will aid in the selection of ornamental pepper varieties. In this study, a pepper mutant with stay-green fruit named TNX348 was identified from a germplasm resource bank. Two segregating populations were constructed using the stay-green mutant TNX348 and then used in bulked segregant analysis combined with RNA sequencing and linkage analyses. The causal gene of the stay-green trait was mapped to an approximately 131-kb region, and a senescence-induced chloroplast protein gene, CaSGR1 (Capana01g000359), was identified as a candidate gene. Sequencing analysis revealed a G→A single-base mutation of CaSGR1 in TNX348 that led to early termination of translation. Based on the single-base mutation, a single nucleotide polymorphism (SNP) marker co-segregating with the stay-green trait was developed. Furthermore, in transcriptome analysis, expression patterns of 11 hormone transduction-related transcription factors, such as abscisic acid-insensitive (ABI), abscisic acid-responsive element-binding factor (ABF), and NAC transcription factor, were similar or opposite to that of CaSGR1. The results indicated that the transcription factors might mediate chlorophyll degradation by regulating the expression of CaSGR1.

Tanshinone I restrains osteosarcoma progression by regulating circ_0000376/miR-432-5p/BCL2 axis

Circular RNAs (circRNAs) have been identified as important regulators in cancer progression. Nevertheless, little is known about the biological function of circ_0000376 in the progression of osteosarcoma (OS). Cell viability, colony formation ability, apoptosis, and motility were analyzed by Cell Counting Kit-8 assay, colony formation assay, flow cytometry, and transwell assays. Cellular glycolytic metabolism was analyzed using commercial kits. RT-qPCR and Western blot assay were performed to analyze RNA and protein expression in OS tissues and cells. Starbase software was used to establish circRNA-microRNA (miRNA)-messenger RNA linkage, and intermolecular interaction was verified by dual-luciferase reporter assay. Xenograft tumor assay was conducted to analyze the effects of Tanshinone I (Tan I) and circ_0000376 on xenograft tumor growth in vivo. Tan I treatment suppressed the viability, migration, invasion, and glycolysis and triggered the apoptosis of OS cells. Tan I treatment markedly down-regulated circ_0000376 expression in OS cells. The addition of circ_0000376 plasmid largely rescued the malignant behaviors of OS cells upon Tan I exposure. Circ_0000376 interacted with miR-432-5p in OS cells. Circ_0000376 overexpression-mediated protective effects in Tan I-induced OS cells were partly attenuated by the accumulation of miR-432-5p. miR-432-5p bound to the 3' untranslated region (3'UTR) of B-cell leukemia/lymphoma 2 (BCL2) in OS cells. miR-432-5p interference-induced effects in Tan I-treated OS cells were partly overturned by the silence of BCL2. Circ_0000376 can act as miR-432-5p sponge to up-regulate BCL2 expression in OS cells. Circ_0000376 silencing contributed to the anti-tumor effect of Tan I on the growth of xenograft tumors in vivo. Tan I exerted an anti-tumor role in OS progression by targeting circ_0000376/miR-432-5p/BCL2 axis.

A Feasibility Study to Increase Chronic Hepatitis C Virus RNA Testing and Linkage to Care among Clients Attending Homeless Services in Amsterdam, The Netherlands.

People who inject drugs (PWID) are disproportionately affected by hepatitis C virus (HCV) infections and are frequently homeless. To improve HCV case finding in these individuals, we examined the feasibility of rapid HCV RNA testing in homeless services in Amsterdam. In 2020, we provided a comprehensive service to homeless facilities, which included workshops on HCV for personnel, a “hepatitis ambassador” at each facility, a rapid, onsite HCV RNA fingerstick test service, and assistance with linkage to care. Risk factors for HCV RNA-positive status were examined using Bayesian logistic regression. Of the 152 participants enrolled, 150 (87% men; median age: 47 years) accepted rapid HCV testing. Seven tested HCV RNA positive (4.7%, 95%CrI = 1.31–8.09; 7/150). Of these, five (71%) were linked to care, of whom four (57%, 4/7) initiated treatment and one (14%, 1/7) delayed treatment due to a drug–drug interaction. Of these four people, two completed treatment (50%), of whom one (25%) achieved sustained virologic response after 12 weeks. HCV RNA-positive individuals were more likely to originate from Eastern Europe (posterior-odds ratio (OR) = 3.59 (95% credible interval (CrI) = 1.27–10.04)) and to inject drugs (ever: posterior-OR = 3.89 (95% CrI = 1.37–11.09); recent: posterior-OR = 3.94 (95% CrI = 1.29–11.71)). We identified HCV RNA-positive individuals and linkage to care was relatively high. Screening in homeless services with rapid testing is feasible and could improve HCV case finding for PWID who do not regularly attend primary care or other harm reduction services for people who use drugs.

Single-round deoxyribozyme discovery.

Artificial evolution experiments typically use libraries of ∼1015 sequences and require multiple rounds of selection to identify rare variants with a desired activity. Based on the simple structures of some aptamers and nucleic acid enzymes, we hypothesized that functional motifs could be isolated from significantly smaller libraries in a single round of selection followed by high-throughput sequencing. To test this idea, we investigated the catalytic potential of DNA architectures in which twelve or fifteen randomized positions were embedded in a scaffold present in all library members. After incubating in either the presence or absence of lead (which promotes the nonenzymatic cleavage of RNA), library members that cleaved themselves at an RNA linkage were purified by PAGE and characterized by high-throughput sequencing. These selections yielded deoxyribozymes with activities 8- to 30-fold lower than those previously isolated under similar conditions from libraries containing 1014 different sequences, indicating that the disadvantage of using a less diverse pool can be surprisingly small. It was also possible to elucidate the sequence requirements and secondary structures of deoxyribozymes without performing additional experiments. Due to its relative simplicity, we anticipate that this approach will accelerate the discovery of new catalytic DNA and RNA motifs.

Genome-wide landscape of mRNAs, microRNAs, lncRNAs, and circRNAs in hemorrhagic shock-induced ALI/ARDS in rats.

Hemorrhagic shock (HS) can develop into multiple organ dysfunction syndrome, among which acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) usually lead to poor outcomes. The underlying molecular mechanisms of HS-induced ALI/ARDS remain unclear. This study sought to investigate gene expression profiles and predict competing endogenous RNA (ceRNA) regulatory networks in an HS-induced ALI/ARDS preclinical model. Sprague Dawley rats were subjected to a fixed volume of hemorrhage (HS, 40% estimated total blood volume) or not (sham) randomly. After 8 hours of observation, left lung tissue was harvested to evaluate lung injury. Right lung was collected for RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed and the long noncoding RNA (lncRNA)/circular RNA (circRNA)-microRNA (miRNA)-messenger RNA (mRNA) linkages were predicted using the ceRNA theory. Quantitative real-time polymerase chain reaction was used to validate the RNA sequencing findings. Hemorrhagic shock lungs showed noticeable ALI/ARDS features, and 437 mRNAs, 31 miRNAs, 734 lncRNAs, and 29 circRNAs were differentially expressed. In Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, the differentially expressed transcripts were enriched in the following terms: the metabolic pathways, signal transduction pathways, necroptosis, DNA damage recognition and repair, inflammatory cell migration and chemotaxis, the NOD-like receptor signaling pathway, the Janus kinase/signal transducer and activator of transcription signaling pathway, the mitogen-activated protein kinase signaling pathway, the phosphatidylinositol-3-kinase/protein kinase B signaling pathway, and so on. Also, this study identified lncRNA-miRNA-mRNA linkages with 12 lncRNAs, 5 miRNAs, 15 mRNAs, and circRNA-miRNA-mRNA linkages with 10 circRNAs, 16 miRNAs, 39 mRNAs. These networks might play important regulatory roles. This is the first high-throughput analysis of gene expression profiles in HS-induced ALI/ARDS. It shows that metabolism, cell signaling, DNA damage and repair, and necroptosis-related RNAs altered, and inflammatory response-associated RNAs and pathways have pivotal roles in HS-induced ALI/ARDS progression. It also prompts some important RNAs and regulatory networks for future research. Basic science article.

A P catalyst for stereogenic P(III)

Nucleotide Synthesis Phosphodiesters, the key linkage in DNA, RNA, and bioactive oligonucleotide and cyclic nucleotides, are typically synthesized from phosphoramidite precursors. Phosphorothioate linkages, which are more stable, can also be produced this way but have the complication of chirality at the P center, which is synthetically problematic, especially for multiple linkages. Featherston et al. found that two different chiral phosphoric acid catalysts provide stereodivergent synthesis of a stereogenic phosphite intermediate that can be oxidatively modified to the desired stereopure nucleotide derivative. They used this approach in the synthesis of a phosphorothioate derivative of the cyclic dinucleotide 2′,3′-cyclic guanosine monophosphate adenosine monophosphate, which is an important human immune signaling molecule. Science , this issue p. [702][1] [1]: /lookup/doi/10.1126/science.abf4359

Catalytic asymmetric and stereodivergent oligonucleotide synthesis.

We report the catalytic stereocontrolled synthesis of dinucleotides. We have demonstrated, for the first time to our knowledge, that chiral phosphoric acid (CPA) catalysts control the formation of stereogenic phosphorous centers during phosphoramidite transfer. Unprecedented levels of diastereodivergence have also been demonstrated, enabling access to either phosphite diastereomer. Two different CPA scaffolds have proven to be essential for achieving stereodivergence: peptide-embedded phosphothreonine-derived CPAs, which reinforce and amplify the inherent substrate preference, and C2-symmetric BINOL-derived CPAs, which completely overturn this stereochemical preference. The presently reported catalytic method does not require stoichiometric activators or chiral auxiliaries and enables asymmetric catalysis with readily available phosphoramidites. The method was applied to the stereocontrolled synthesis of diastereomeric dinucleotides as well as cyclic dinucleotides, which are of broad interest in immuno-oncology as agonists of the stimulator of interferon genes (STING) pathway.

Aromatic Interactions Drive the Coupled Folding and Binding of the Intrinsically Disordered Sesbania mosaic Virus VPg Protein.

The plant Sesbania mosaic virus [a (+)-ssRNA sobemovirus] VPg protein is intrinsically disordered in solution. For the virus life cycle, the VPg protein is essential for replication and for polyprotein processing that is carried out by a virus-encoded protease. The nuclear magnetic resonance (NMR)-derived tertiary structure of the protease-bound VPg shows it to have a novel tertiary structure with an α-β-β-β topology. The quaternary structure of the high-affinity protease-VPg complex (≈27 kDa) has been determined using HADDOCK protocols with NMR (residual dipolar coupling, dihedral angle, and nuclear Overhauser enhancement) restraints and mutagenesis data as inputs. The geometry of the complex is in excellent agreement with long-range orientational restraints such as residual dipolar couplings and ring-current shifts. A "vein" of aromatic residues on the protease surface is pivotal for the folding of VPg via intermolecular edge-to-face π···π stacking between Trp271 and Trp368 of the protease and VPg, respectively, and for the CH···π interactions between Leu361 of VPg and Trp271 of the protease. The structure of the protease-VPg complex provides a molecular framework for predicting sites of important posttranslational modifications such as RNA linkage and phosphorylation and a better understanding of the coupled folding upon binding of intrinsically disordered proteins. The structural data presented here augment the limited structural data available on viral proteins, given their propensity for structural disorder.

Highly sensitive detection of Hg2+ using molybdenum disulfide-DNA sensors

Mercury ion (Hg2+) is one of the heavy metal ions that cause significant physiological damage to an organism even in trace amount. To facilitate Hg2+ detection, a thiol-modified DNA was used as helper sequence to hybridize with a DNA modified with FAM (Carboxyfluorescein) and containing phosphorothioate (PS)-modified RNA linkages that can be cleaved by Hg2+, because thiol group can covalently bond with gold particles. The hybridized DNA sequence was immobilized onto the surface of MoS2@AuNPs (Molybdenum disulfide-gold particle) to form a MoS2-DNA sensors for detecting Hg2+. The results showed that the detection limit in this method was 9.41 nM. Moreover, when the immobilization process was conducted under freezing conditions, the detection limit was further increased to 4.58 nM, lower than the recommended maximum contaminant level in drinking water (9.9 nM).

Rapid HCV RNA Testing and LInkage to Care

Rapid HCV RNA Testing and LInkage to Care

Hybrid DNA/RNA nanostructures with 2'-5' linkages.

Nucleic acid nanostructures with different chemical compositions have shown utility in biological applications as they provide additional assembly parameters and enhanced stability. The naturally occurring 2'-5' linkage in RNA is thought to be a prebiotic analogue and has potential use in antisense therapeutics. Here, we report the first instance of DNA/RNA motifs containing 2'-5' linkages. We synthesized and incorporated RNA strands with 2'-5' linkages into different DNA motifs with varying number of branch points (a duplex, four arm junction, double crossover motif and tensegrity triangle motif). Using experimental characterization and molecular dynamics simulations, we show that hybrid DNA/RNA nanostructures can accommodate interspersed 2'-5' linkages with relatively minor effect on the formation of these structures. Further, the modified nanostructures showed improved resistance to ribonuclease cleavage, indicating their potential use in the construction of robust drug delivery vehicles with prolonged stability in physiological conditions.

Improving hepatitis C screening and diagnosis in patients born between 1945 and 1965 in a safety-net primary care clinic

Individuals born between 1945–1965 represent 81% of all persons chronically infected with hepatitis C virus (HCV) in the USA and are largely unaware of their positive status. The baseline HCV...

Editorial

Editorial

RNA-Cleaving DNAzymes: Old Catalysts with New Tricks for Intracellular and In Vivo Applications

DNAzymes are catalytically active DNA molecules that are normally isolated through in vitro selection methods, among which RNA-cleaving DNAzymes that catalyze the cleavage of a single RNA linkage embedded within a DNA strand are the most studied group of this DNA enzyme family. Recent advances in DNA nanotechnology and engineering have generated many RNA-cleaving DNAzymes with unique recognition and catalytic properties. Over the past decade, numerous RNA-cleaving, DNAzymes-based functional probes have been introduced into many research areas, such as in vitro diagnostics, intracellular imaging, and in vivo therapeutics. This review focus on the fundamental insight into RNA-Cleaving DNAzymes and technical tricks for their intracellular and in vivo applications, highlighting the recent progress in the clinical trial of RNA-Cleaving DNAzymes with selected examples. The challenges and opportunities for the future translation of RNA-cleaving DNAzymes for biomedicine are also discussed.

Daniel McGillivray Brown. 3 February 1923—24 April 2012

Dan Brown was a nucleic acids chemist of the highest order, beginning with pioneering work under Lord Alexander Todd in the 1950s at University of Cambridge on chemical methods for synthesis of nucleosides and nucleotides. This work helped to confirm the furanose chemical structure of the sugar in nucleosides as well as the 3′-5′ phosphodiester linkage in DNA and RNA, perhaps the most well thought of achievement of his career. Later, as a chemistry department lecturer, he established the chemical structures of glycerol monophosphoinositides as well as triphosphoinositides. Turning back to the nucleic acids in 1961, he became fascinated by the effect of mutagens on DNA. He elucidated the mechanism for the reaction of hydroxylamine on cytidine to form an initial ‘bis-adduct’ and thereafter N 6 -hydroxycytidine. Moving in 1982 to the MRC Laboratory of Molecular Biology, he developed a method to prepare single-stranded DNA probes for detection of RNA sequences and in addition worked on a novel automated device for oligonucleotide synthesis. Reverting to his interest in mutagens, he then designed and synthesized hydrogen bonding degenerate bases and developed novel P and K modified pyrimidine and purine bases respectively as transition mutagens. Finally, he synthesized the base analogue 5-nitroindole as a potential universal base, which became useful in cycle DNA sequencing, and in addition developed the concept of ‘error catastrophe’ for the ribonucleoside of the P base as an antiviral agent. The P, K and 5-nitroindole bases became the most valued chemical entities of his career to molecular biologists. His legacy to the nucleic acids includes both his significant contributions to studies of the chemical nature of DNA and RNA and their constituents as well as a variety of enabling nucleic acids chemistry methods and mechanisms of DNA mutagenicity.

Comparative spectral analysis of the extra-cell matrixes surface of heart valves before and during the process of their decullularization

There are presented the results of the application of Raman-scattering spectroscopy method (RS) for the qualitative analysis of rams' heart valves surfaces before and during their decellularization. While analyzing RS spectra, it was found that basic differences appear at wave numbers 812 cm-1, 1062 cm-1, 1340 cm-1 and 1440 cm-1, corresponding to phosphodiester linkage of RNA; OSO-3 corresponds to symmetrical stretching of glycosaminoglycans and chondroitin-6-sulfate; corresponds to deformation mode of proteins and nucleic acids (DNA); proteins, lipids. Optical analysis has shown that while analyzing decellularization on the valves surfaces, the content of glycosaminoglycans, proteins and lipids decreases; retains a high DNA content. It was found that with the aid of entered optical numbers it is possible to control the process efficiency of decellularization of the heart valves.

Site-Selective RNA Activation by Acridine-Modified Oligodeoxynucleotides in Metal-Ion Catalyzed Hydrolysis: A Comprehensive Study.

Various types of acridine were conjugated to DNA and used for site-selective RNA scission together with another unmodified DNA and a Lu(III) ion. The target phosphodiester linkage in the substrate RNA was selectively and efficiently activated, and was hydrolyzed by the free Lu(III) ion. Among the investigated 14 conjugates, the conjugate bearing 9-amino-2-isopropoxy-6-nitroacridine was the best RNA-activator. Systematic evaluation of the RNA-activating ability of the acridines showed that (1) the acridines act as an acid catalyst within the RNA activation, (2) the amino-group at the 9-position of acridine is essential to modulate the acidity of acridine, (3) the electron-withdrawing group at the 3-position further enhances the acid catalysis, and (4) the substituent at the 2-position sterically modulates the orientation of acridine-intercalation favorably for the catalysis. Moreover, it is revealed that the opposite base of acridine does not inhibit direct interaction of acridine with the target phosphodiester linkage.