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

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Huarong Dong ◽  
Fei Huang ◽  
Xiang Guo ◽  
Xiaoyi Xu ◽  
Qian Liu ◽  
...  
Keyword(s):  
Small Molecule ◽  
Dna Cleavage ◽  
Low Cost ◽  
Nuclease Activity ◽  
Nucleic Acid Detection ◽  
Mesophilic Bacteria ◽  
Brevibacillus Laterosporus ◽  
Double Stranded Dna ◽  
Small Molecule Detection

AbstractThermophilic Argonaute proteins (Agos) have been shown to utilize small DNA guides for cleaving complementary DNA in vitro, which shows great potential for nucleic acid detection. In this study, we explored mesophilic Agos for the detection of small molecule by cooperating with allosteric transcription factors (aTFs). Two Agos from mesophilic bacteria, Paenibacillus borealis (PbAgo) and Brevibacillus laterosporus (BlAgo), showed nuclease activity for single-stranded DNA at moderate temperatures (37 °C) by using 5′-phosphorylated and 5′-hydroxylated DNA guides. Both Agos perform programmable cleavage of double-stranded DNA, especially in AT-rich regions of plasmid. Furthermore, we developed a simple and low-cost p-hydroxybenzoic acid detection method based on DNA-guided DNA cleavage of Agos and the allosteric effect of HosA, which expands the potential application of small molecule detection by Agos.

scholarly journals A YoeB toxin cleaves both RNA and DNA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Julia McGillick ◽  
Jessica R. Ames ◽  
Tamiko Murphy ◽  
Christina R. Bourne
Keyword(s):  
Dna Cleavage ◽  
Divalent Cations ◽  
Nuclease Activity ◽  
Dose Dependence ◽  
Cleavage Activity ◽  
Double Stranded Dna ◽  
Dna Cleavage Activity ◽  
Evolutionarily Conserved ◽  
Toxin Protein ◽  
Rna And Dna

AbstractType II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.

scholarly journals Recombinant pseudorabies virus DNase exhibits a RecBCD-like catalytic function

1998 ◽  
Vol 330 (1) ◽  
pp. 55-59 ◽  
Author(s):  
Chien-Yun HSIANG ◽  
Tin-Yun HO ◽  
Chin-Hui HSIANG ◽  
Tien-Jye CHANG
Keyword(s):  
Pseudorabies Virus ◽  
Nuclease Activity ◽  
Alkaline Ph ◽  
E Coli ◽  
Catalytic Function ◽  
Double Stranded Dna ◽  
Recombinant Pseudorabies Virus ◽  
Absolute Requirement ◽  
Na Ions

The pseudorabies virus (PRV) DNase gene has previously been mapped within the PRV genome. To characterize further the enzymic properties of PRV DNase, this enzyme was expressed in Escherichia coli with the use of a pET expression vector. The protein was purified to homogeneity and assayed for nuclease activity in vitro. Recombinant PRV DNase exhibited an alkaline pH preference and an absolute requirement for Mg2+ ions that could not be replaced by Ca2+ and Na+ ions. Further studies showed that PRV DNase exhibited endonuclease, 5ʹ-exonuclease and 3ʹ-exonuclease activities in both single-stranded and double-stranded DNA. This activity occurred randomly and no significant base preference was demonstrated. The multiple biochemical activities of PRV DNase are similar to the activities of Neurospora crassa endo-exonuclease and E. coli RecBCD, two additional enzymes that are involved in recombination. Taken together, the similarity of action between N. crassa endo-exonuclease, E. coli RecBCD, and PRV DNase suggests that PRV DNase might have a role in the process of recombination that occurs during PRV infection.

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

scholarly journals Rational Design and Identification of Harmine-Inspired, N-Heterocyclic DYRK1A Inhibitors Employing a Functional Genomic in vivo Drosophila Model System

2021 ◽  
Author(s):  
Brandon Ashfeld ◽  
Francisco Huizar ◽  
Harrison Hill ◽  
Jeremiah Zartman ◽  
Emily Bacher ◽  
...  
Keyword(s):  
Small Molecule ◽  
Rational Design ◽  
Kinase Inhibitors ◽  
Low Cost ◽  
Neuronal Cell ◽  
Model System ◽  
In Vivo Model ◽  
Functional Genomic

Deregulation of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) plays a significant role in developmental brain defects, early-onset neurodegeneration, neuronal cell loss, and dementia. Herein, we report the discovery of three new classes of <i>N</i>-heterocyclic DYRK1A inhibitors based on the potent, yet toxic kinase inhibitors, harmine and harmol. An initial in vitro evaluation of the small molecule collection assembled revealed that the core heterocyclic motifs benzofuranones, oxindoles, and pyrrolones, showed statistically significant DYRK1A inhibition. Further, the utilization of a low cost, high-throughput functional genomic in vivo model system to identify small molecule inhibitors that normalize DYRK1A overexpression phenotypes is described. This in vivo assay confirmed the in vitro results, and the resulting correspondence validates generated classes as architectural motifs that serve as potential DYRK1A inhibitors. Further expansion and analysis of these core compound structures will allow discovery of safe, more effective chemical inhibitors of DYRK1A to ameliorate phenotypes caused by DYRK1A overexpression.

scholarly journals Cas12a-Capture: a novel, low-cost, and scalable method for targeted sequencing

2020 ◽  
Author(s):  
Taylor L. Mighell ◽  
Andrew Nishida ◽  
Brendan L. O’Connell ◽  
Caitlin V. Miller ◽  
Sally Grindstaff ◽  
...  
Keyword(s):  
Low Cost ◽  
Targeted Sequencing ◽  
High Input ◽  
Selective Enrichment ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Fold Enrichment ◽  
Gc Bias ◽  
Dna Template

AbstractTargeted sequencing remains a valuable technique for clinical and research applications. However, many existing technologies suffer from pervasive GC sequence content bias, high input DNA requirements, and high cost for custom panels. We have developed Cas12a-Capture, a low-cost and highly scalable method for targeted sequencing. The method utilizes preprogramed guide RNAs to direct CRISPR-Cas12a cleavage of double stranded DNA in vitro and then takes advantage of the resulting four to five nucleotide overhangs for selective ligation with a custom sequencing adapter. Addition of a second sequencing adapter and enrichment for ligation products generates a targeted sequence library. We first performed a pilot experiment with 7,176 guides targeting 3.5 megabases of DNA. Using these data, we modeled the sequence determinants of Cas12a-Capture efficiency, then designed an optimized set of 11,438 guides targeting 3.0 megabases. The optimized guide set achieves an average 64-fold enrichment of targeted regions with minimal GC bias. Cas12a-Capture variant calls had strong concordance with Illumina Platinum Genome calls, especially for SNVs, which could be improved by applying basic variant quality heuristics. We believe Cas12a-Capture has a wide variety of potential clinical and research applications and is amendable for selective enrichment for any double stranded DNA template or genome.

scholarly journals Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage

2021 ◽  
Vol 118 (49) ◽  
pp. e2113747118
Author(s):  
Heyjin Son ◽  
Jaeil Park ◽  
Injoo Hwang ◽  
Youngri Jung ◽  
Sangsu Bae ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Resonance Energy ◽  
Distal Region ◽  
Catalytic Site ◽  
Nucleic Acid Detection ◽  
Double Stranded Dna ◽  
Target Strand ◽  
Target Dna ◽  
Conformational Rearrangements

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)–distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

scholarly journals Rational Design and Identification of Harmine-Inspired, N-Heterocyclic DYRK1A Inhibitors Employing a Functional Genomic in vivo Drosophila Model System

2021 ◽  
Author(s):  
Brandon Ashfeld ◽  
Francisco Huizar ◽  
Harrison Hill ◽  
Jeremiah Zartman ◽  
Emily Bacher ◽  
...  
Keyword(s):  
Small Molecule ◽  
Rational Design ◽  
Kinase Inhibitors ◽  
Low Cost ◽  
Neuronal Cell ◽  
Model System ◽  
In Vivo Model ◽  
Functional Genomic

Deregulation of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) plays a significant role in developmental brain defects, early-onset neurodegeneration, neuronal cell loss, and dementia. Herein, we report the discovery of three new classes of <i>N</i>-heterocyclic DYRK1A inhibitors based on the potent, yet toxic kinase inhibitors, harmine and harmol. An initial in vitro evaluation of the small molecule collection assembled revealed that the core heterocyclic motifs benzofuranones, oxindoles, and pyrrolones, showed statistically significant DYRK1A inhibition. Further, the utilization of a low cost, high-throughput functional genomic in vivo model system to identify small molecule inhibitors that normalize DYRK1A overexpression phenotypes is described. This in vivo assay confirmed the in vitro results, and the resulting correspondence validates generated classes as architectural motifs that serve as potential DYRK1A inhibitors. Further expansion and analysis of these core compound structures will allow discovery of safe, more effective chemical inhibitors of DYRK1A to ameliorate phenotypes caused by DYRK1A overexpression.

scholarly journals Argonaute with stepwise endonuclease activity promotes specific and multiplex nucleic acid detection

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Guanhua Xun ◽  
Qian Liu ◽  
Yuesheng Chong ◽  
Xiang Guo ◽  
Zhonglei Li ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Molecular Mechanisms ◽  
Pyrococcus Furiosus ◽  
Nucleic Acid Detection ◽  
Endonuclease Activity ◽  
Medical Sciences ◽  
Gene Detection ◽  
Double Stranded Dna ◽  
Canonical Pair ◽  
Nucleotide Resolution

AbstractArgonaute proteins (Agos) from thermophiles function as endonucleases via guide-target base-pairing cleavage for host defense. Since guides play a key role in regulating the catalytic specificity of Agos, elucidating its underlying molecular mechanisms would promote the application of Agos in the medical sciences. Here, we reveal that an Ago from Pyrococcus furiosus (PfAgo) showed a stepwise endonuclease activity, which was demonstrated through a double-stranded DNA cleavage directed by a single guide DNA (gDNA) rather than a canonical pair of gDNAs. We validated that the cleavage products with 5'-phosphorylated ends can be used as a new guide to induce a new round of cleavage. Based on the reprogrammable capacity of Ago’s stepwise activity, we established a rapid and specific platform for unambiguous multiplex gene detection, termed Renewed-gDNA Assisted DNA cleavage by Argonaute (RADAR). Combined with a pre-amplification step, RADAR achieved sensitivity at the femtomolar level and specificity with at least a di-nucleotide resolution. Furthermore, RADAR simultaneously discriminated among multiple target sequences simply by corresponding multiple guides. We successfully distinguished four human papillomavirus serotypes from patient samples in a single reaction. Our technique, based on the unique properties of Ago, provides a versatile and sensitive method for molecular diagnosis.

scholarly journals A YoeB toxin from A. tumefaciens has metal-dependent DNA cleaving activity

2019 ◽  
Author(s):  
Julia McGillick ◽  
Jessica R. Ames ◽  
Tamiko Murphy ◽  
Eswar Reddem ◽  
Christina R. Bourne
Keyword(s):  
Dna Cleavage ◽  
Potential Role ◽  
Fold Increase ◽  
Nuclease Activity ◽  
Dose Dependence ◽  
Bacterial Cells ◽  
Toxin Concentration ◽  
E Coli ◽  
Dna Cleaving

ABSTRACTToxin-antitoxin (TA) systems, including YoeB-YefM, are important mediators of bacterial physiological changes. Agrobacterium tumefaciens YoeB and YefM are similar to that from E. coli, and interact as a tight heterotetramer with a KD of 653 pM. We have verified that AtYoeB can perform both ribosome-dependent and –independent RNA cleavage. We have also characterized a newly described metal-dependent and pH-sensitive DNA cleaving ability. We note that this DNA cleaving ability is observed at toxin concentrations as low as 150 nM. The dose-dependence of in vitro ribosome-independent RNA and metal-dependent DNA cleavage is equivalent, and requires a ten-fold increase in toxin concentration as opposed to in the presence of the ribosome. The toxin concentration inside bacterial cells is unknown and according to current models, should increase upon activation of YoeB through degradation of the YefM antitoxin. The discovery of general nuclease activity by AtYoeB, and perhaps other YoeB toxins, offers an opportunity to explore the plasticity of this protein fold and its potential role in the evolution of nucleases.

scholarly journals Further evidence for redox activation of the plasmid – dirhenium(III) complexes interactions

2020 ◽  
Keyword(s):  
Dna Cleavage ◽  
Nuclease Activity ◽  
Cluster Compounds ◽  
Organic Radical ◽  
Mobility Shift ◽  
Anticancer Properties ◽  
Dna Cleaving ◽  
Supercoiled Form ◽  
Redox Activation

The DNA-interactions in vitro are still necessary investigations for determination of the possible anticancer properties of the compounds, candidates for application in cancer therapy. The aim of the present work was to realize if the interaction of cis-dicarboxylates of dirhenium(III), with pivalato- (I), isobutirato- (II) and adamanthyl- (III) ligands cleaves the plasmid in the same manner and what is the influence of the ligands on this process. For experiments we used the prokaryotic plasmid which is good model to analyze DNA-cleaving ability of different substances that exists in supercoiled conformation and turns to nicked and linear forms. It was shown that gradual conversion of the supercoiled Form I to a mixture of supercoiled (Form I) and nicked (Form II) DNA takes place and increasing amounts of Form II are produced with higher concentrations of I–III under increasing of concentration that showed the DNA-cleaving abilities of all investigated dirhenium complexes. This process was taking place with different intensity in the range I ˃ II ˃ III, that demonstrates the influence of the organic radical on the cleaving activity of the dirhenium(III) complexes. Under hydrogen peroxide conditions, I and II showed close results, demonstrating more intensive process of cleaving, including formation of the linear plasmid (Form III) under higher concentration, witnessing about redox-activation of the DNA-cleaving reaction. Cleaving activity of III was approximately the same in all experiments, that was demonstrated only by decreasing of the supercoiled form I and increasing of the nicked form II of the plasmid and by absolutely absence of the linear form III of the plasmid. The electrophoresis mobility shift assays showed that rhenium cluster compounds have nuclease activity and confirmed that natural DNA may be their target in the living cells. The conclusion was made that the mechanism of DNA-cleavage reaction of the dirhenium(III) complexes is multiple in which the electron donating (withdrawing) effects of the ligands and catalytic activity of the metal core should be taken in consideration.

Sign in / Sign up

Export Citation Format

Share Document