scholarly journals 5’ modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

2020 ◽  
Author(s):  
Grace Mullally ◽  
Kara van Aelst ◽  
Mohsin M. Naqvi ◽  
Fiona M. Diffin ◽  
Tautvydas Karvelis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Rna Aptamers ◽  
Loop Formation ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Rnp Complex ◽  
Target Binding ◽  
The Impact

A key aim in exploiting CRISPR-Cas is the engineering of gRNA to introduce additional functionalities, ranging from small nucleotide changes that increase efficiency of on-target binding to the inclusion of large functional RNA aptamers and ribonucleoproteins (RNPs. Interactions between gRNA and Cas9 are crucial for RNP complex assembly but several distinct regions of the gRNA are amenable to modification. Using a library of modified gRNAs, we used in vitro ensemble and single-molecule assays to assess the impact of RNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that R-loop formation and DNA cleavage activity are essentially unaffected by gRNA modifications of the Upper Stem, first Hairpin and 3’ end. In contrast, 5’ additions of only two or three nucleotides reduced R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such gRNA modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5’ end supported formation of a stable ~9 bp R-loop that could not activate DNA cleavage. These observations will assist in successful gRNA design.

scholarly journals 5′ modifications to CRISPR–Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

2020 ◽  
Vol 48 (12) ◽  
pp. 6811-6823 ◽  
Author(s):  
Grace Mullally ◽  
Kara van Aelst ◽  
Mohsin M Naqvi ◽  
Fiona M Diffin ◽  
Tautvydas Karvelis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Rna Aptamers ◽  
Loop Formation ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Rnp Complex ◽  
Target Binding ◽  
The Impact

Abstract A key aim in exploiting CRISPR–Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9–gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3′ end. In contrast, we found that 5′ additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5′ end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.

scholarly journals Interplay between DNA sequence and negative superhelicity drives R-loop structures

2019 ◽  
Vol 116 (13) ◽  
pp. 6260-6269 ◽  
Author(s):  
Robert Stolz ◽  
Shaheen Sulthana ◽  
Stella R. Hartono ◽  
Maika Malig ◽  
Craig J. Benham ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Single Molecule ◽  
Energy State ◽  
In Vitro Transcription ◽  
Loop Formation ◽  
Base Pairing ◽  
Theoretical Predictions ◽  
Nucleic Acid Structures ◽  
The Impact

R-loops are abundant three-stranded nucleic-acid structures that formin cisduring transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms—junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription.

DNA-cleavage activity of the iron(II) complex with optically active ligands, meta- and para-xylyl-linked N’,N’-dipyridylmethyl-cyclohexane-1,2-diamine

2021 ◽  
Vol 36 ◽  
pp. 127834
Author(s):  
Koichi Kato ◽  
Yoshimi Ichimaru ◽  
Yoshinori Okuno ◽  
Yoshihiro Yamaguchi ◽  
Wanchun Jin ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Optically Active ◽  
Cleavage Activity ◽  
Dna Cleavage Activity

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 Correction to Copper(II) Complexes of l-Arginine as Netropsin Mimics Showing DNA Cleavage Activity in Red Light

2019 ◽  
Vol 58 (19) ◽  
pp. 13502-13503
Author(s):  
Ashis K. Patra ◽  
Tuhin Bhowmick ◽  
Sovan Roy ◽  
Suryanarayanarao Ramakumar ◽  
Akhil R. Chakravarty
Keyword(s):  
Dna Cleavage ◽  
Red Light ◽  
Cleavage Activity ◽  
Dna Cleavage Activity

Cytotoxic copper (II) mixed ligand complexes: Crystal structure and DNA cleavage activity

2011 ◽  
Vol 46 (9) ◽  
pp. 4537-4547 ◽  
Author(s):  
Verasuntharam M. Manikandamathavan ◽  
Royapuram P. Parameswari ◽  
Thomas Weyhermüller ◽  
Hannah R. Vasanthi ◽  
Balachandran Unni Nair
Keyword(s):  
Crystal Structure ◽  
Dna Cleavage ◽  
Mixed Ligand ◽  
Mixed Ligand Complexes ◽  
Cleavage Activity ◽  
Dna Cleavage Activity

scholarly journals Effects of Pathological Mutations on the Prion-Like Polymerisation of MyD88

2018 ◽  
Author(s):  
Ailís O’Carroll ◽  
Brieuc Chauvin ◽  
James Brown ◽  
Ava Meagher ◽  
Joanne Coyle ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Full Length ◽  
Binary Response ◽  
Death Domain ◽  
Tir Domain ◽  
The Impact

AbstractA novel concept has emerged whereby the higher-order self-assembly of proteins provides a simple and robust mechanism for signal amplification. This appears to be a universal signalling mechanism within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns need to trigger a strong, binary response within cells. Previously, multiple structural studies have been limited to single domains, expressed and assembled at high protein concentrations. We therefore set out to develop new in vitro strategies to characterise the behaviour of full-length proteins at physiological levels. In this study we focus on the adaptor protein MyD88, which contains two domains with different self-assembly properties: a TIR domain that can polymerise similarly to the TIR domain of Mal, and a Death Domain that has been shown to oligomerise with helical symmetry in the Myddosome complex. To visualize the behaviour of full-length MyD88 without purification steps, we use single-molecule fluorescence coupled to eukaryotic cell-free protein expression. These experiments demonstrate that at low protein concentration, only full-length MyD88 forms prion-like polymers. We also demonstrate that the metastability of MyD88 polymerisation creates the perfect binary response required in innate signalling: the system is silenced at normal concentrations but upstream signalling creates a “seed” that triggers polymerisation and amplification of the response. These findings pushed us to re-interpret the role of polymerisation in MyD88-related diseases and we studied the impact of disease-associated point mutations L93P, R196C and L252P/L265P at the molecular level. We discovered that all mutations completely block the ability of MyD88 to polymerise. We also confirm that L252P, a gain-of-function mutation, allows the MyD88 mutant to form extremely stable oligomers, even when expressed at low nanomolar concentrations. Thus, our results are consistent with and greatly add to the findings on the Myddosomes digital ‘all-or-none’ responses and the behaviour of the oncogenic mutation of MyD88.

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