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

2021 ◽  
Author(s):  
Ekaterina Kropocheva ◽  
Anton Kuzmenko ◽  
Alexei A. Aravin ◽  
Daria Esyunina ◽  
Andrey Kulbachinskiy
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Detection ◽  
Bacterial Cells ◽  
Dna And Rna ◽  
Rna Targets ◽  
Wide Range ◽  
Mesophilic Bacterium ◽  
Strict Specificity ◽  
Programmable Nucleases

ABSTRACTArgonaute 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 KmAgo nuclease 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 preferentially 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. Target cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA targets. 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. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.

Barcoded microbial system for high-resolution object provenance

Science ◽  
2020 ◽  
Vol 368 (6495) ◽  
pp. 1135-1140 ◽  
Author(s):  
Jason Qian ◽  
Zhi-xiang Lu ◽  
Christopher P. Mancuso ◽  
Han-Ying Jhuang ◽  
Rocío del Carmen Barajas-Ornelas ◽  
...  
Keyword(s):  
Food Safety ◽  
Nucleic Acid ◽  
High Resolution ◽  
Human Health ◽  
Nucleic Acid Detection ◽  
Single Spore ◽  
Detection Assay ◽  
Wide Range ◽  
Fundamental Challenge ◽  
Microbial System

Determining where an object has been is a fundamental challenge for human health, commerce, and food safety. Location-specific microbes in principle offer a cheap and sensitive way to determine object provenance. We created a synthetic, scalable microbial spore system that identifies object provenance in under 1 hour at meter-scale resolution and near single-spore sensitivity and can be safely introduced into and recovered from the environment. This system solves the key challenges in object provenance: persistence in the environment, scalability, rapid and facile decoding, and biocontainment. Our system is compatible with SHERLOCK, a Cas13a RNA-guided nucleic acid detection assay, facilitating its implementation in a wide range of applications.

scholarly journals Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers

2016 ◽  
Vol 60 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Pawan Jolly ◽  
Pedro Estrela ◽  
Michael Ladomery
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Rna Aptamers ◽  
Locked Nucleic Acid ◽  
Synthetic Analogue ◽  
Dna And Rna ◽  
Naturally Occurring ◽  
Wide Range ◽  
Synthetic Oligonucleotides ◽  
Shed Light

There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications.

scholarly journals Nucleic Acid Computing and its Potential to Transform Silicon-Based Technology

2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Seth G. Abels ◽  
Emil F. Khisamutdinov
Keyword(s):  
Nucleic Acid ◽  
Logic Gates ◽  
Rna Nanotechnology ◽  
Dna And Rna ◽  
Cellular Machinery ◽  
Living Organisms ◽  
Silicon Based ◽  
Molecular Computers

AbstractMolecular computers have existed on our planet for more than 3.5 billion years. Molecular computing devices, composed of biological substances such as nucleic acids, are responsible for the logical processing of a variety of inputs, creating viable outputs that are key components of the cellular machinery of all living organisms. We have begun to adopt some of the structural and functional knowledge of the cellular apparatus in order to fabricate nucleic-acid-based molecular computers in vitro and in vivo. Nucleic acid computing is directly dependent on advances in DNA and RNA nanotechnology. The field is still emerging and a number of challenges persist. Perhaps the most salient among these is how to translate a variety of nucleic-acid-based logic gates, developed by numerous research laboratories, into the realm of silicon-based computing. This mini-review provides some basic information on the advances in nucleic-acid-based computing and its potential to serve as an alternative that can revolutionize silicon-based technology.

scholarly journals Detection and Quantification of the Red Tide Dinoflagellate Karenia brevis by Real-Time Nucleic Acid Sequence-Based Amplification

2004 ◽  
Vol 70 (8) ◽  
pp. 4727-4732 ◽  
Author(s):  
Erica T. Casper ◽  
John H. Paul ◽  
Matthew C. Smith ◽  
Michael Gray
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Wildlife Conservation ◽  
Red Tide ◽  
Diagnostic Testing ◽  
Karenia Brevis ◽  
Nucleic Acid Sequence ◽  
Cell Counts ◽  
Wide Range

ABSTRACT Nucleic acid sequence-based amplification (NASBA) is an isothermal method of RNA amplification that has been previously used in clinical diagnostic testing. A real-time NASBA assay has been developed for the detection of rbcL mRNA from the red tide dinoflagellate Karenia brevis. This assay is sensitive to one K. brevis cell and 1.0 fg of in vitro transcript, with occasional detection of lower concentrations of transcript. The assay did not detect rbcL mRNA from a wide range of nontarget organisms and environmental clones, while 10 strains (all tested) of K. brevis were detected. By the use of standard curves based on time to positivity, concentrations of K. brevis in environmental samples were predicted by NASBA and classified into different levels of blooms per the Florida Fish and Wildlife Conservation Commission (FWC) system. NASBA classification matched FWC classification (based on cell counts) 72% of the time. Those samples that did not match were off by only one class. NASBA is sensitive, rapid, and effective and may be used as an additional or alternative method to detect and quantify K. brevis in the marine environment.

scholarly journals Convolutional models of RNA energetics

2018 ◽  
Author(s):  
Michelle J. Wu
Keyword(s):  
Nucleic Acid ◽  
Network Architecture ◽  
Nearest Neighbor ◽  
Current Model ◽  
Mrna Translation ◽  
Large Space ◽  
Dna And Rna ◽  
Nucleic Acid Interactions

AbstractNucleic acid molecular biology and synthetic biology are undergoing rapid advances with the emergence of designer riboswitches controlling living cells, CRISPR/Cas9-based genome editing, high-throughput RNA-based silencing, and reengineering of mRNA translation. Many of these efforts require the design of nucleic acid interactions, which relies on accurate models for DNA and RNA energetics. Existing models utilize nearest neighbor rules, which were parameterized through careful optical melting measurements. However, these relatively simple rules often fail to quantitatively account for the biophysical behavior of molecules even in vitro, let alone in vivo. This is due to the limited experimental throughput of optical melting experiments and the infinitely large space of possible motifs that can be formed. Here, we present a convolutional neural network architecture to model the energies of nucleic acid motifs, allowing for learning of representations of physical interactions that generalize to arbitrary unmeasured motifs. First, we used existing parameterizations of motif energies to train the model and demonstrate that our model is expressive enough to recapitulate the current model. Then, through training on optical melting datasets from the literature, we have shown that the model can accurately predict the thermodynamics of hairpins containing unmeasured motifs. This work demonstrates the utility of convolutional models for capturing the thermodynamic parameters that underlie nucleic acid interactions.

scholarly journals Detection of rifampin- and ciprofloxacin-resistant Mycobacterium tuberculosis by using species-specific assays for precursor rRNA.

1996 ◽  
Vol 40 (8) ◽  
pp. 1790-1795 ◽  
Author(s):  
G A Cangelosi ◽  
W H Brabant ◽  
T B Britschgi ◽  
C K Wallis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Lower Limit ◽  
Cultured Cells ◽  
Rrna Synthesis ◽  
Bacterial Cells ◽  
Mycobacterium Species ◽  
Antibiotic Resistant ◽  
Species Specific

rRNA precursor (pre-rRNA) molecules carry terminal stems which are removed during rRNA synthesis to form the mature rRNA subunits. Their abundance in bacterial cells can be markedly affected by antibiotics which directly or indirectly inhibit RNA synthesis. We evaluated the feasibility of rapidly detecting antibiotic-resistant Mycobacterium tuberculosis strains by measuring the effects of brief in vitro antibiotic exposure on mycobacterial pre-rRNA. By hybridizing extracted M. tuberculosis nucleic acid with radiolabeled nucleic acid probes specific for pre-16S rRNA stem sequences, we detected clear responses to rifampin and ciprofloxacin within 24 and 48 h, respectively, of exposure of cultured cells to these drugs. Detectable pre-rRNA was depleted in susceptible cells but remained abundant in resistant cells. In contrast, no measurable responses to isoniazid or ethambutol were observed. Probes for pre-rRNA were specific for the M. tuberculosis complex when tested against a panel of eight Mycobacterium species and 48 other bacteria. After 24 h of incubation with rifampin, resistant M. tuberculosis strains were detectable in a reverse transcriptase PCR assay for pre-rRNA with a calculated lower limit of sensitivity of approximately 10(2) cells. Susceptible cells were negative in this assay at over 500 times the calculated lower limit of sensitivity. This general approach may prove useful for rapidly testing the susceptibility of slowly growing Mycobacterium species to the rifamycin and fluoroquinolone drugs and, with possible modifications, to other drugs as well.

scholarly journals A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

PLoS ONE ◽  
2019 ◽  
Vol 14 (12) ◽  
pp. e0220091 ◽  
Author(s):  
Florian Katzmeier ◽  
Lukas Aufinger ◽  
Aurore Dupin ◽  
Jorge Quintero ◽  
Matthias Lenz ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Low Cost ◽  
In Vitro Transcription ◽  
Nucleic Acid Detection

Synthesis of α-(R)-/γ-(S)-Dimethyl Substituted Peptide Nucleic Acid Submonomer Using Mitsunobu Reaction

2019 ◽  
Vol 16 (5) ◽  
pp. 437-446
Author(s):  
Ahmed S. Abdelbaky ◽  
Ivan A. Prokhorov ◽  
Igor P. Smirnov ◽  
Kristina M. Koroleva ◽  
Vitaliy I. Shvets ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Solid Phase ◽  
Genetic Diseases ◽  
Mitsunobu Reaction ◽  
Phase Synthesis ◽  
Dna And Rna ◽  
Pna Synthesis ◽  
Biomedical Technologies

One of the major challenges facing modern biochemical and biomedical technologies are finding molecular tools for diagnosis and detection of genetic diseases. In this connection, several classes of oligonucleotides have been developed that can recognize and bind to DNA and RNA with high affinity and sequence selectivity and withstand enzymatic degradation by proteases and nucleases; however, few can traverse the cell membrane on their own. One such promising class of nucleic acid mimics developed in the last two decades which showed good results in vitro, are the peptide nucleic acids (PNAs). New chiral α- and γ-peptide Nucleic Acid (PNA) submonomer with methyl substituents in pseudopeptide backbone were synthesized via Mitsunobu reaction. The α-(R)-/γ-(S)-configuration of the chiral centres will ensure the preorganization of the PNA oligomer into a right-handed helix. The results obtained showed that Boc/Fmoc-submonomer compatible with Boc-protocol PNAs solid-phase synthesis on an MBHA resin. We synthesized simple and efficient α-R-, γ-S-disubstituted PNA submonomer based on L-Ala and D-Ala with the construction of the intermediate pseudopeptide moiety by Mitsunobu reaction for subsequent use in the Boc-Protocol of solid phase PNA synthesis.

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