scholarly journals Measurement of Kinetics of Hammerhead Ribozyme Cleavage Reactions using Toehold Mediated Strand Displacement

2020 ◽  
Author(s):  
Jay Bhakti Kapadia ◽  
Nawwaf Kharma ◽  
Alen Nellikulam Davis ◽  
Nicolas Kamel ◽  
Jonathan Perreault
Keyword(s):  
Hammerhead Ribozyme ◽  
Polyacrylamide Gels ◽  
Strand Displacement ◽  
Rna Molecules ◽  
Starting Point ◽  
Dna Strands ◽  
Regular Sampling ◽  
Cleavage Reactions ◽  
Labeled Rna ◽  
Kinetics Of

ABSTRACTThis paper presents a probe comprising a fluorophore and a quencher, enabling measurement of hammerhead ribozyme cleavage reactions, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labelled with a fluorophore at its 5’-end, while the other strand is labelled with a quencher at its 3’-end. These two DNA strands are perfectly complementary, but with a 3’-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the kinetics of ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

Toehold mediated strand displacement to measure released product from self-cleaving ribozymes

RNA ◽  
2021 ◽  
pp. rna.078823.121
Author(s):  
Jay Bhakti Kapadia ◽  
Nawwaf Kharma ◽  
Alen Nellikulam Davis ◽  
Nicolas Kamel ◽  
Jonathan Perreault
Keyword(s):  
Hammerhead Ribozyme ◽  
Polyacrylamide Gels ◽  
Strand Displacement ◽  
Rna Molecules ◽  
Starting Point ◽  
Dna Strands ◽  
Regular Sampling ◽  
Method Of Measurement ◽  
Cleavage Reactions ◽  
Labeled Rna

This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labelled with a fluorophore at its 5′-end, while the other strand is labelled with a quencher at its 3′-end. These two DNA strands are perfectly complementary, but with a 3′-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

scholarly journals Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids

2017 ◽  
Vol 121 (12) ◽  
pp. 2594-2602 ◽  
Author(s):  
Xiaoping Olson ◽  
Shohei Kotani ◽  
Bernard Yurke ◽  
Elton Graugnard ◽  
William L. Hughes
Keyword(s):  
Nucleic Acids ◽  
Strand Displacement ◽  
Dna Strand Displacement ◽  
Locked Nucleic Acids ◽  
Dna Strand ◽  
Kinetics Of

scholarly journals Kinetics of Toehold-Mediated DNA Strand Displacement Depend on FeII4L4 Tetrahedron Concentration

Nano Letters ◽  
2021 ◽  
Vol 21 (3) ◽  
pp. 1368-1374
Author(s):  
Jinbo Zhu ◽  
Filip Bošković ◽  
Bao-Nguyen T. Nguyen ◽  
Jonathan R. Nitschke ◽  
Ulrich F. Keyser
Keyword(s):  
Strand Displacement ◽  
Dna Strand Displacement ◽  
Dna Strand ◽  
Kinetics Of

Fast Cleavage Kinetics of a Natural Hammerhead Ribozyme

2004 ◽  
Vol 126 (35) ◽  
pp. 10848-10849 ◽  
Author(s):  
Marella D. Canny ◽  
Fiona M. Jucker ◽  
Elizabeth Kellogg ◽  
Anastasia Khvorova ◽  
Sumedha D. Jayasena ◽  
...  
Keyword(s):  
Hammerhead Ribozyme ◽  
Kinetics Of

scholarly journals Viroids: unusual small pathogenic RNAs.

2004 ◽  
Vol 51 (3) ◽  
pp. 587-607 ◽  
Author(s):  
Anna Góra-Sochacka
Keyword(s):  
Secondary Structure ◽  
Hammerhead Ribozyme ◽  
Direct Interaction ◽  
Plant Diseases ◽  
Plant Host ◽  
Rolling Circle ◽  
Rna Molecules ◽  
Viroid Replication ◽  
Plant Genes ◽  
Rolling Circle Mechanism

Viroids are small (about 300 nucleotides), single-stranded, circular, non-encapsidated pathogenic RNA molecules. They do not code for proteins and thus depend on plant host enzymes for their replication and other functions. They induce plant diseases by direct interaction with host factors but the mechanism of pathogenicity is still unknown. They can alter the expression of selected plant genes important for growth and development. Viroids belong to two families, the Avsunviroidae and the Pospiviroidae. Viroids of the Avsunviroidae family adopt a branched or quasi rod-like secondary structure in their native state. Members of the Pospiviroidae family adopt a rod-like secondary structure. In such native structures five structural/functional domains have been identified: central (C), pathogenicity, variable and two terminal domains. The central conserved region (CCR) within the C domain characterizes viroids of the Pospiviroidae. Specific secondary structures of this region play an important role in viroid replication and processing. Viroids of the Avsunviroidae family lack a CCR but possess self-cleaving properties by forming hammerhead ribozyme structures; they accumulate and replicate in chloroplasts, whereas members of the Pospiviroidae family have a nuclear localization. Viroid replication occurs via a rolling circle mechanism using either a symmetric or asymmetric pathway in three steps, RNA transcription, processing and ligation.

scholarly journals Fragmentation of Plasmid DNA Produced by Gamma Radiation: A Theoretical Approach

2012 ◽  
Vol 2012 ◽  
pp. 1-6
Author(s):  
R. A. S. Silva ◽  
J. D. T. Arruda-Neto ◽  
L. Nieto
Keyword(s):  
Plasmid Dna ◽  
Fragment Size ◽  
Power Laws ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Starting Point ◽  
Dna Strands ◽  
High Let ◽  
Fragment Distribution ◽  
Dna Strand

Breaks in DNA, resulting in fragmented parts, can be produced by ionizing radiation which, in turn, is the starting point in the search for novel physical aspects of DNA strands. Double-strand breaks in particular cause disruption of the DNA strand, splitting it into several fragments. In order to study effects produced by radiation in plasmid DNA, a new simple mechanical model for this molecule is proposed. In this model, a Morse-like potential and a high-LET component are used to describe the DNA-radiation interaction. Two power laws, used to fit results of the model, suggest that, firstly, distribution of fragment size is nonextensive and, secondly, that a transition phase is present in the DNA fragment distribution pattern.

RNA Structure Analysis: A Multifaceted Approach

1999 ◽  
Author(s):  
Bruce A. Shapiro ◽  
Wojciech Kasprzak
Keyword(s):  
Nucleic Acid ◽  
Secondary Structure ◽  
Tertiary Structure ◽  
Hammerhead Ribozyme ◽  
3D Structure ◽  
Computer Prediction ◽  
Rna Molecules ◽  
Tertiary Interactions ◽  
The One ◽  
Secondary And Tertiary Structure

Genomic information (nucleic acid and amino acid sequences) completely determines the characteristics of the nucleic acid and protein molecules that express a living organism’s function. One of the greatest challenges in which computation is playing a role is the prediction of higher order structure from the one-dimensional sequence of genes. Rules for determining macromolecule folding have been continually evolving. Specifically in the case of RNA (ribonucleic acid) there are rules and computer algorithms/systems (see below) that partially predict and can help analyze the secondary and tertiary interactions of distant parts of the polymer chain. These successes are very important for determining the structural and functional characteristics of RNA in disease processes and hi the cell life cycle. It has been shown that molecules with the same function have the potential to fold into similar structures though they might differ in their primary sequences. This fact also illustrates the importance of secondary and tertiary structure in relation to function. Examples of such constancy in secondary structure exist in transfer RNAs (tRNAs), 5s RNAs, 16s RNAs, viroid RNAs, and portions of retroviruses such as HIV. The secondary and tertiary structure of tRNA Phe (Kim et al., 1974), of a hammerhead ribozyme (Pley et al., 1994), and of Tetrahymena (Cate et al., 1996a, 1996b) have been shown by their crystal structure. Currently little is known of tertiary interactions, but studies on tRNA indicate these are weaker than secondary structure interactions (Riesner and Romer, 1973; Crothers and Cole, 1978; Jaeger et al., 1989b). It is very difficult to crystallize and/or get nuclear magnetic resonance spectrum data for large RNA molecules. Therefore, a logical place to start in determining the 3D structure of RNA is computer prediction of the secondary structure. The sequence (primary structure) of an RNA molecule is relatively easy to produce. Because experimental methods for determining RNA secondary and tertiary structure (when the primary sequence folds back on itself and forms base pairs) have not kept pace with the rapid discovery of RNA molecules and their function, use of and methods for computer prediction of secondary and tertiary structures have increasingly been developed.

Sequence, Salt, Charge, and the Stability of DNA at High Pressure

1996 ◽  
Author(s):  
Robert B. Macgregor Jr ◽  
John Q. Wu
Keyword(s):  
Molar Volume ◽  
Volume Change ◽  
Salt Concentration ◽  
Electrostatic Interactions ◽  
Proposed Model ◽  
Dna Strands ◽  
Positive Volume ◽  
Ion Radius ◽  
The Stability ◽  
Kinetics Of

The effect of pressure on the helix-coil transition temperature (Tm) is reported for the double-stranded polymers poly(dA)poly(dT), poly[d(A-T)], poly[d(l-C], and poly[d(G-C] and triple-stranded poly(dA)2poly(dT). The Tm increases as a function of pressure, implying a positive volume change for the transition and leading to the conclusion that the molar volume of the coil form is larger than the molar volume of the helix. From the change in Tm as a function of pressure, molar volume changes of the transition (ΔVt) are calculated using the Clapeyron equation and calorimetrically determined enthalpies. For the doublestranded polymers, ΔVt, increases in the order poly[d(l-C] < polyt[d(A-T)] < poly(dA)-poly(dT) < polylcl(G-C)]. The value of ΔVt, for the triple-stranded to single-stranded transition of poly(dA) 2poly(dT) is larger than that of poly[d(G-C)I. The magnitude of ΔVt increases with salt concentration in all cases studied; however, the change of ΔVt with salt concentration depends on the sequence of the DNA and the number of strands involved in the transition. In the model proposed to explain the results, the overall molar volume change of the transition is a function of a negative volume change arising from changes in the electrostatic interactions of the DNA strands, and a positive volume change due to unstacking the bases. The model predicted the direction of the change in the ΔVt for several experiments. The magnitude of AVJ increases with counter ion radius, thus for polyld(A-T)], ΔVt, increases in the series Na+ , K+, Cs+, The ΔVt also increases if the charge on the phosphodiester groups is removed. The kinetics of the formation of double-stranded (dA)19(dT)19 in 50 mM NaCI are slowed approximately 14-fold at 200 MPa relative to atmospheric pressure. The implied volume of activation of +37 ml mol−l in the direction of this change is also in agreement with the proposed model. The stability of double- and triple-stranded DNA helices in water around neutral pH depends on the base composition and sequence, as well as on the ionic strength of the solution. Each of these dependencies also defines how DNA interacts with water.

scholarly journals Kinetics of DNA Strand Displacement

2019 ◽  
Vol 116 (3) ◽  
pp. 499a
Author(s):  
Alexander W. Cook ◽  
Bo Broadwater ◽  
Harold Kim
Keyword(s):  
Strand Displacement ◽  
Dna Strand Displacement ◽  
Dna Strand ◽  
Kinetics Of
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