Browse
Submit Data
Databases
API
Help

Dataset Information

6 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A programmable omnipotent Argonaute nuclease from mesophilic bacteria Kurthia massiliensis.

ABSTRACT: Argonaute (Ago) proteins are conserved nucleic acid-guided proteins present in all domains of life. Eukaryotic Argonaute proteins (eAgos) are key players in RNA interference pathways and function as RNA-guided RNA endonucleases at physiological temperatures. Although eAgos are considered to evolve from prokaryotic Argonaute proteins (pAgos), previously studied pAgos were unable to catalyze RNA-guided RNA cleavage at physiological temperatures. Here, we describe a distinctive pAgo from mesophilic bacteria Kurthia massiliensis (KmAgo). KmAgo utilizes DNA guides to cleave single-stranded DNA (ssDNA) and RNA targets with high activity. KmAgo also utilizes RNA guides to cleave ssDNA and RNA targets at moderate temperatures. We show that KmAgo can use 5' phosphorylated DNA guides as small as 9-mers to cut ssDNA and RNA, like Clostridium butyricum Ago. Small DNA binding confers remarkable thermostability on KmAgo, and we can suppress the guide-independent plasmid processing activity of empty KmAgo by elevating the DNA guide loaded temperature. Moreover, KmAgo performs programmable cleavage of double-stranded DNA and highly structured RNA at 37°C. Therefore, KmAgo can be regarded as a DNA-guided programmable omnipotent nuclease for cleaving most types of nucleic acids efficiently. This study broadens our understanding of Ago proteins and could expand the pAgo-based DNA and RNA manipulation toolbox.

SUBMITTER: Liu Y

PROVIDER: S-EPMC7897485 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

Project description:Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic Argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique pAgo nuclease, KmAgo, from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo binds 16-20 nt long 5’-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acid. Target RNA cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.

2021-03-02 | GSE168010 | GEO

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis.

Project description:Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique pAgo nuclease, KmAgo, from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo binds 16-20 nt long 5'-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acids. Target RNA cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.

| S-EPMC8053121 | biostudies-literature

A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

Project description:A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

| PRJNA705755 | ENA

Characterization of Argonaute nucleases from mesophilic bacteria Paenibacillus borealis and Brevibacillus laterosporus

Project description: Not available

| S-EPMC10992608 | biostudies-literature

Non-contiguous finished genome sequence and description of Kurthia massiliensis sp. nov.

Project description:Kurthia massiliensis strain JC30(T) sp. nov. is the type strain of K. massiliensis sp. nov., a new species within the genus Kurthia. This strain, whose genome is described here, was isolated from the fecal flora of a healthy patient. K. massiliensis is a Gram-positive aerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,199,090 bp long genome contains 3,240 protein-coding genes and 86 RNA genes, including between 3 and 4 rRNA genes.

| S-EPMC3569394 | biostudies-literature

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea.

Project description:Argonaute (Ago) proteins are key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleavage of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures, which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In comparison with previously studied pAgos, CbAgo and LrAgo do not show strong preferences for the 5'-nucleotide in guide DNA and can use not only 5'-phosphorylated but also 5'-hydroxyl DNA guides. Both CbAgo and LrAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3'-guide region. Both pAgos can perform programmable endonucleolytic cleavage of double-stranded DNA substrates, showing enhanced activity at AT-rich regions and at elevated temperatures. The biochemical characterization of mesophilic pAgo proteins paves the way for their use for DNA manipulations both in vitro and in vivo.

| S-EPMC6582412 | biostudies-literature

DNA interference by a mesophilic Argonaute protein, CbcAgo.

Project description:Background: The search for putative enzymes that can facilitate gene editing has recently focused its attention on Argonaute proteins from prokaryotes (pAgos). Though they are structural homologues of human Argonaute protein, which uses RNA guides to interfere with RNA targets, pAgos use ssDNA guides to identify and, in many cases, cut a complementary DNA target. Thermophilic pAgos from Thermus thermophilus, Pyrococcus furiosus and Methanocaldococcus jasmanii have been identified and thoroughly studied, but their thermoactivity makes them of little use in mesophilic systems such as mammalian cells. Methods: Here we search for and identify CbcAgo, a prokaryotic Argonaute protein from a mesophilic bacterium, and characterize in vitro its DNA interference activity. Results: CbcAgo efficiently uses 5'P-ssDNA guides as small as 11-mers to cut ssDNA targets, requires divalent cations (preferentially, Mn 2+) and has a maximum activity between 37 and 42 °C, remaining active up to 55 °C. Nicking activity on supercoiled dsDNA was shown. However, no efficient double-strand breaking activity could be demonstrated. Conclusions: CbcAgo can use gDNA guides as small as 11 nucleotides long to cut complementary ssDNA targets at 37ºC, making it a promising starting point for the development of new gene editing tools for mammalian cells.

| S-EPMC6961421 | biostudies-literature

Mesophilic Argonaute-based isothermal detection of SARS-CoV-2.

Project description:Coronavirus disease (COVID-19), caused by SARS-CoV-2 infection and its mutations, has spread rapidly all over the world and still requires sensitive detection to distinguish mutations. CRISPR-based diagnosis has been regarded as a next-generation detection method; however, it has some limitations, such as the need for specific recognition sequences and multiple enzymes for multiplex detection. Therefore, research on the exploration and development of novel nucleases helps to promote specific and sensitive diagnoses. Prokaryotic Argonaute (Ago) proteins exert directed nuclease activity that can target any sequence. Recently, thermophilic Agos have been developed as new detection techniques achieving multiplexity for multiple targets using a single enzyme, as well as accurate recognition of single-base differential sequences. In this study, to overcome the requirement for high reaction temperature of thermophilic Ago-based methods, we expanded the mining of mesophilic Agos to achieve CRISPR-like isothermal detection, named mesophilic Ago-based isothermal detection method (MAIDEN). The principle of MAIDEN uses mesophilic Ago cleavage combined with reverse transcription, which can provide single-strand DNA as a substrate and allow cleavage of fluorescence probes to sense SARS-CoV-2 at moderate temperature. We first mined and optimized the mesophilic Ago and the fluorescence reporter system and then selected a compatible reverse transcription reaction. Furthermore, we optimized MAIDEN into a one-step reaction that can detect SARS-CoV-2 RNA at the nanomolar concentration at a constant temperature of 42°C within 60 min. Therefore, MAIDEN shows advantageous portability and easy-to-implement operation, avoiding the possibility of open-lid contamination. Our study was the first attempt to demonstrate that mesophilic Agos can be harnessed as diagnostic tools, and MAIDEN was easily extended to detect other pathogens in a rapid and efficient manner.

| S-EPMC9363790 | biostudies-literature

Kurthia massiliensis strain:JC30

Project description:Kurthia sp. JC30 Genome Sequencing

| PRJEA70831 | ENA

Programmable RNA recognition using a CRISPR-associated Argonaute.

Project description: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.

| S-EPMC5879674 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data