scholarly journals Programmable RNA recognition using a CRISPR-associated Argonaute

2018 ◽  
Vol 115 (13) ◽  
pp. 3368-3373 ◽  
Author(s):  
Audrone Lapinaite ◽  
Jennifer A. Doudna ◽  
Jamie H. D. Cate
Keyword(s):  
Complex Mixture ◽  
Seed Region ◽  
Rna Recognition ◽  
Single Nucleotide ◽  
Guide Rnas ◽  
Rna Targets ◽  
Rna Biology ◽  
Rna Targeting ◽  
Domains Of Life

Argonaute proteins (Agos) are present in all domains of life. Although the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (clustered regularly interspaced short palindromic repeats) loci and use noncanonical 5′-hydroxylated guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2′-deoxyuridine (BrdU) as the 5′ nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute–gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5′-BrdU-modified gRNAs, we mapped the seed region of the gRNA and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the sixth and seventh positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific adenosine-to-inosine–edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5′-BrdU-modified gRNAs can be used as a highly specific RNA-targeting platform to probe RNA biology.

scholarly journals Programmable RNA recognition using a CRISPR-associated Argonaute

2017 ◽  
Author(s):  
Audrone Lapinaite ◽  
Jennifer A. Doudna ◽  
Jamie H. D. Cate
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
Complex Mixture ◽  
Seed Region ◽  
Single Nucleotide ◽  
Guide Rna ◽  
Argonaute Proteins ◽  
Wide Range ◽  
Domains Of Life

ABSTRACTArgonaute proteins (Agos) are present in all domains of life. While the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci and use non-canonical 5’-hydroxyled guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2′-deoxyuridine (BrdU) as the 5’ nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5’-BrdU modified gRNAs, we mapped the seed region of the gRNA, and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the 6th and 7th positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific Adenosine to Inosine edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5’-BrdU modified gRNAs can be used as a highly-specific RNA-targeting platform to probe RNA biology.SIGNIFICANCEArgonaute proteins are present in bacteria, archaea and eukaryotes. They play an important role in a wide range of biological processes, from transcriptional and translational gene expression regulation to defense against viruses and silencing of mobile genetic elements. Here we present mechanistic insights into the interactions of the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) with its guide RNA (gRNA) and RNA substrates. By modifying the 5’-nucleotide of the gRNA, we demonstrate that MpAgo-gRNA complexes (RNPs) are easily programmable, have high affinity to fully complementary RNA substrates, and can discriminate by over 300 fold between substrates that differ by only a single nucleotide. These MpAgo RNPs should be useful for probing endogenous RNAs in living cells.

scholarly journals Modified RNAs as potential drug targets.

1998 ◽  
Vol 45 (1) ◽  
pp. 13-18 ◽  
Author(s):  
R Guenther ◽  
B Forrest ◽  
W Newman ◽  
A Małkiewicz ◽  
P F Agris
Keyword(s):  
Drug Targets ◽  
Model Systems ◽  
Rna Recognition ◽  
Magnesium Ions ◽  
Yeast Trna ◽  
Rna Targets ◽  
Therapeutic Mechanism ◽  
Potential Drug Targets

Bleomycin (BLM) is a natural antibiotic that is effective in treatment of selected cancers. Although the exact therapeutic mechanism of bleomycin is not known, its target is thought to be a nucleic acid. Besides cleaving DNA, in vitro, Fe-bleomycin cleaves the anticodon of yeast tRNA(Phe) specifically. Using CD and fluorescence spectroscopy we have found that apo-bleomycin binds to synthetic RNA analogs of the anticodon of yeast tRNA(Phe) with an affinity similar to that previously reported for DNA. In order to understand BLM's selectivity, the role magnesium ions play in RNA recognition should be explained. Many RNA substrates for Fe-BLM, including yeast tRNA(Phe), are not cleaved by the drug when the Mg2+ concentration exceeds 1 mM. Competition experiments with anticodon analogs provide insight into the role of magnesium ions in RNA recognition by BLM. These simple modified RNAs may be useful as model systems for investigating BLM/RNA recognition and development of highly selective drugs toward RNA targets.

DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM

2020 ◽  
Author(s):  
M.A. Tyumentseva ◽  
◽  
A.I. Tyumentsev ◽  
V.G. Akimkin ◽  
◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Health Monitoring ◽  
Biological Samples ◽  
Proviral Dna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Low Concentrations ◽  
Immunodeficiency Virus ◽  
Hiv 1

For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications

2019 ◽  
Vol 26 (30) ◽  
pp. 5609-5624
Author(s):  
Dijana Saftić ◽  
Željka Ban ◽  
Josipa Matić ◽  
Lidija-Marija Tumirv ◽  
Ivo Piantanida
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Biomedical Applications ◽  
Protein Targets ◽  
New Class ◽  
Rna Targets ◽  
Covalently Linked ◽  
Structural Aspects

: Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder – nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

scholarly journals Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yang Zhang ◽  
Tuan M. Nguyen ◽  
Xiao-Ou Zhang ◽  
Limei Wang ◽  
Tin Phan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
High Throughput ◽  
Biological Effect ◽  
Human Hepatocellular Carcinoma ◽  
Guide Rnas ◽  
Rna Targeting ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Screening Library ◽  
High Throughput Study

AbstractShort hairpin RNAs (shRNAs) are used to deplete circRNAs by targeting back-splicing junction (BSJ) sites. However, frequent discrepancies exist between shRNA-mediated circRNA knockdown and the corresponding biological effect, querying their robustness. By leveraging CRISPR/Cas13d tool and optimizing the strategy for designing single-guide RNAs against circRNA BSJ sites, we markedly enhance specificity of circRNA silencing. This specificity is validated in parallel screenings by shRNA and CRISPR/Cas13d libraries. Using a CRISPR/Cas13d screening library targeting > 2500 human hepatocellular carcinoma-related circRNAs, we subsequently identify a subset of sorafenib-resistant circRNAs. Thus, CRISPR/Cas13d represents an effective approach for high-throughput study of functional circRNAs.

scholarly journals Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

2021 ◽  
Vol 22 (13) ◽  
pp. 7099
Author(s):  
Pradeep Kumar Kopparapu ◽  
Meghshree Deshmukh ◽  
Zhicheng Hu ◽  
Majd Mohammad ◽  
Marco Maugeri ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Responses ◽  
Surface Proteins ◽  
Host Cells ◽  
Immune Stimulation ◽  
Domains Of Life ◽  
Major Surface ◽  
Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals Somatic variants for seed and fruit set in grapevine

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Laura Costantini ◽  
Paula Moreno-Sanz ◽  
Chinedu Charles Nwafor ◽  
Silvia Lorenzi ◽  
Annarita Marrano ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Fruit Set ◽  
Climatic Factors ◽  
Embryo Sac ◽  
Wine Industry ◽  
Nucleotide Polymorphisms ◽  
Wine Quality ◽  
Single Nucleotide ◽  
Germplasm Collections

Abstract Background Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like seedlessness, berry and bunch size, cluster compactness and berry skin to pulp ratio. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections. Results We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on seed and fruit set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. Moreover, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. Five missense single nucleotide polymorphisms were identified between Corinto Nero and Sangiovese from transcriptomic data. Conclusions Our observations allowed us to attribute a seedlessness type to some variants for which it was not documented in the literature. Interestingly, the VvAGL11 mutation responsible for Sultanina stenospermocarpy was also discovered in a seedless mutant of Gouais Blanc. We suggest that Corinto Nero parthenocarpy is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. The single nucleotide polymorphisms identified between Sangiovese and Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype.

scholarly journals Association between HOX Transcript Antisense RNA Single-Nucleotide Variants and Recurrent Implantation Failure

2021 ◽  
Vol 22 (6) ◽  
pp. 3021
Author(s):  
Jeong Yong Lee ◽  
Eun Hee Ahn ◽  
Hyeon Woo Park ◽  
Ji Hyang Kim ◽  
Young Ran Kim ◽  
...  
Keyword(s):  
Antisense Rna ◽  
Coagulation Factors ◽  
Healthy Controls ◽  
Implantation Failure ◽  
Single Nucleotide Variants ◽  
Recurrent Implantation Failure ◽  
Single Nucleotide ◽  
Vitro Fertilization ◽  
And Control

Recurrent implantation failure (RIF) refers to the occurrence of more than two failed in vitro fertilization–embryo transfers (IVF-ETs) in the same individual. RIF can occur for many reasons, including embryo characteristics, immunological factors, and coagulation factors. Genetics can also contribute to RIF, with some single-nucleotide variants (SNVs) reported to be associated with RIF occurrence. We examined SNVs in a long non-coding RNA, homeobox (HOX) transcript antisense RNA (HOTAIR), which is known to affect cancer development. HOTAIR regulates epigenetic outcomes through histone modifications and chromatin remodeling. We recruited 155 female RIF patients and 330 healthy controls, and genotyped HOTAIR SNVs, including rs4759314, rs920778, rs7958904, and rs1899663, in all participants. Differences in these SNVs were compared between the patient and control groups. We identified significant differences in the occurrence of heterozygous genotypes and the dominant expression model for the rs1899663 and rs7958904 SNVs between RIF patients and control subjects. These HOTAIR variants were associated with serum hemoglobin (Hgb), luteinizing hormone (LH), total cholesterol (T. chol), and blood urea nitrogen (BUN) levels, as assessed by analysis of variance (ANOVA). We analyzed the four HOTAIR SNVs and found significant differences in haplotype patterns between RIF patients and healthy controls. The results of this study showed that HOTAIR is not only associated with the development of cancer but also with pregnancy-associated diseases. This study represents the first report showing that HOTAIR is correlated with RIF.

scholarly journals Single-nucleotide control of tRNA folding cooperativity under near-cellular conditions

2019 ◽  
Vol 116 (46) ◽  
pp. 23075-23082
Author(s):  
Kathleen A. Leamy ◽  
Ryota Yamagami ◽  
Neela H. Yennawar ◽  
Philip C. Bevilacqua
Keyword(s):  
Rna Folding ◽  
3D Structure ◽  
Single Nucleotide ◽  
Transfer Rnas ◽  
E Coli ◽  
Posttranscriptional Processing ◽  
Structural Levels ◽  
Trna Folding ◽  
Precursor Transcript

RNA folding is often studied by renaturing full-length RNA in vitro and tracking folding transitions. However, the intracellular transcript folds as it emerges from the RNA polymerase. Here, we investigate the folding pathways and stability of numerous late-transcriptional intermediates of yeast and Escherichia coli transfer RNAs (tRNAs). Transfer RNA is a highly regulated functional RNA that undergoes multiple steps of posttranscriptional processing and is found in very different lengths during its lifetime in the cell. The precursor transcript is extended on both the 5′ and 3′ ends of the cloverleaf core, and these extensions get trimmed before addition of the 3′-CCA and aminoacylation. We studied the thermodynamics and structures of the precursor tRNA and of late-transcriptional intermediates of the cloverleaf structure. We examined RNA folding at both the secondary and tertiary structural levels using multiple biochemical and biophysical approaches. Our findings suggest that perhaps nature has selected for a single-base addition to control folding to the functional 3D structure. In near-cellular conditions, yeast tRNAPhe and E. coli tRNAAla transcripts fold in a single, cooperative transition only when nearly all of the nucleotides in the cloverleaf are transcribed by indirectly enhancing folding cooperativity. Furthermore, native extensions on the 5′ and 3′ ends do not interfere with cooperative core folding. This highly controlled cooperative folding has implications for recognition of tRNA by processing and modification enzymes and quality control of tRNA in cells.

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