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 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.

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.

scholarly journals Idiosyncratic cleavage and ligation activity of individual hammerhead ribozymes and core sequence variants thereof

2007 ◽  
Vol 388 (7) ◽  
pp. 737-741 ◽  
Author(s):  
Rita Przybilski ◽  
Christian Hammann
Keyword(s):  
Small Rna ◽  
Plant Pathogens ◽  
Hammerhead Ribozyme ◽  
Structure And Function ◽  
Sequence Variants ◽  
Hammerhead Ribozymes ◽  
Satellite Dnas ◽  
Cleavage Reactions ◽  
The Individual ◽  
And Function

AbstractThe hammerhead ribozyme is a small RNA endonuclease found in sub-viral plant pathogens, in transcripts from certain animal satellite DNAs and encoded at distinct loci ofArabidopsis thaliana. Kinetic analyses of tertiary stabilised ribozymes from peach latent mosaic viroid (PLMVd),Schistosoma mansoniandA. thalianarevealed a ten-fold difference in cleavage rates. Core nucleotide variations affected cleavage reactions least in theA. thalianaribozyme, and most in theS. mansoniribozyme. The reverse ligation reaction was catalysed efficiently by the PLMVd andA. thalianaribozymes. The different behaviour of the individual hammerhead ribozymes is discussed in terms of structure and function.

scholarly journals Multiple Long-read Sequencing Survey of Herpes Simplex Virus Lytic Transcriptome

2019 ◽  
Author(s):  
Dóra Tombácz ◽  
Zsolt Balázs ◽  
Gábor Gulyás ◽  
Zsolt Csabai ◽  
Miklós Boldogkoi ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Single Molecule ◽  
Rna Molecules ◽  
Oxford Nanopore ◽  
Dna Strands ◽  
Long Read ◽  
Simplex Virus ◽  
Transcriptional Start Sites ◽  
Hsv 1

ABSTRACTLong-read sequencing (LRS) has become increasingly important in RNA research due to its strength in resolving complex transcriptomic architectures. In this regard, currently two LRS platforms have demonstrated adequate performance: the Single Molecule Real-Time Sequencing by Pacific Biosciences (PacBio) and the nanopore sequencing by Oxford Nanopore Technologies (ONT). Even though these techniques produce lower coverage and are more error prone than short-read sequencing, they continue to be more successful in identifying transcript isoforms including polycistronic and multi-spliced RNA molecules, as well as transcript overlaps. Recent reports have successfully applied LRS for the investigation of the transcriptome of viruses belonging to various families. These studies have substantially increased the number of previously known viral RNA molecules. In this work, we used the Sequel and MinION technique from PacBio and ONT, respectively, to characterize the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). In most samples, we analyzed the poly(A) fraction of the transcriptome, but we also performed random oligonucleotide-based sequencing. Besides cDNA sequencing, we also carried out native RNA sequencing. Our investigations identified more than 160 previously undetected transcripts, including coding and non-coding RNAs, multi-splice transcripts, as well as polycistronic and complex transcripts. Furthermore, we determined previously unsubstantiated transcriptional start sites, polyadenylation sites, and splice sites. A large number of novel transcriptional overlaps were also detected. Random-primed sequencing revealed that each convergent gene pair produces non-polyadenylated read-through RNAs overlapping the partner genes. Furthermore, we identified novel replication-associated transcripts overlapping the HSV-1 replication origins, and novel LAT variants with very long 5’ regions, which are co-terminal with the LAT-0.7kb transcript. Overall, our results demonstrated that the HSV-1 transcripts form an extremely complex pattern of overlaps, and that entire viral genome is transcriptionally active. In most viral genes, if not in all, both DNA strands are expressed.

scholarly journals Biological Functions of Natural Antisense Transcripts

2017 ◽  
Vol 63 (4) ◽  
Author(s):  
Wojciech Rosikiewicz ◽  
Izabela Makałowska
Keyword(s):  
Biological Significance ◽  
Transcriptional Level ◽  
Antisense Transcripts ◽  
Genomic Locus ◽  
Natural Antisense Transcripts ◽  
Rna Molecules ◽  
Current State ◽  
Dna Strands ◽  
Regulatory Functions ◽  
Rna Formation

Natural antisense transcripts (NATs) are RNA molecules that originate from opposite DNA strands of the same genomic locus (cis-NAT) or unlinked genomic loci (trans-NAT). NATs may play various regulatory functions at the transcriptional level via transcriptional interference. NATs may also regulate gene expression levels post-transcriptionally via induction of epigenetic changes or double-stranded RNA formation, which may lead to endogenous RNA interference, RNA editing or RNA masking. The true biological significance of the natural antisense transcripts remains controversial despite many years of research. Here, we summarize the current state of knowledge and discuss the sense-antisense overlap regulatory mechanisms and their potential.

Newly formed mRNA lacking polyadenylic acid enters the cytoplasm and the polyribosomes but has a shorter half-life in the absence of polyadenylic acid

1982 ◽  
Vol 2 (5) ◽  
pp. 517-525
Author(s):  
M Zeevi ◽  
J R Nevins ◽  
J E Darnell
Keyword(s):  
Half Life ◽  
Exit Time ◽  
Adenovirus Type ◽  
Rna Molecules ◽  
Polyadenylic Acid ◽  
Initial Appearance ◽  
Labeled Rna ◽  
And Control ◽  
Adenovirus Type 2

Labeled adenovirus type 2 nuclear RNA molecules from cells treated with 3'-deoxyadenosine (3'dA) were earlier reported to lack polyadenylic acid [poly(A)], but to be correctly spliced in the nucleus (M. Zeevi et al., Cell 26:39-46, 1981). We have now found that the shortened mRNA molecules, lacking poly(A), can also be found in the cytoplasm of 3'dA-treated cells in association with the polyribosomes. In addition, the accumulation of labeled, nuclear adenovirus-specific RNA complementary to early regions 1a, 1b, and 2 of the adenovirus genome was approximately equal in 3'dA-treated and control cells. At the initial appearance of newly labeled adenovirus type 2 RNA (10 min) in the cytoplasm, there was one-half as much labeled RNA in 3'dA-treated cells as in the control. However, control cells accumulated additional mRNA in the cytoplasm very rapidly in the first 40 min of labeling, whereas the 3'dA-treated cells did not. Therefore, it appears that the correctly spliced, poly(A)- mRNA molecules that are labeled in the presence of 3'dA can be transported from the nucleus with nearly the same frequency and the same exit time as in control cells and can be translated in the cytoplasm but have a much shorter half-life than the poly(A)+ mRNA molecules from control infected cells. From these results it is suggested that the role of poly(A) may be entirely to increase the longevity of cytoplasmic mRNA.

scholarly journals Less haste, less waste: on recycling and its limits in strand displacement systems

2012 ◽  
Vol 2 (4) ◽  
pp. 512-521 ◽  
Author(s):  
Anne Condon ◽  
Alan J. Hu ◽  
Ján Maňuch ◽  
Chris Thachuk
Keyword(s):  
Chemical Reaction ◽  
Polynomial Function ◽  
Gray Code ◽  
Strand Displacement ◽  
Reaction Systems ◽  
Chemical Reaction Systems ◽  
Binary Counter ◽  
Dna Strand Displacement ◽  
Dna Strands ◽  
Dna Strand

We study the potential for molecule recycling in chemical reaction systems and their DNA strand displacement realizations. Recycling happens when a product of one reaction is a reactant in a later reaction. Recycling has the benefits of reducing consumption, or waste, of molecules and of avoiding fuel depletion. We present a binary counter that recycles molecules efficiently while incurring just a moderate slowdown compared with alternative counters that do not recycle strands. This counter is an n -bit binary reflecting Gray code counter that advances through 2 n states. In the strand displacement realization of this counter, the waste—total number of nucleotides of the DNA strands consumed—is polynomial in n , the number of bits of the counter, while the waste of alternative counters grows exponentially in n . We also show that our n -bit counter fails to work correctly when many ( Θ ( n )) copies of the species that represent the bits of the counter are present initially. The proof applies more generally to show that in chemical reaction systems where all but one reactant of each reaction are catalysts, computations longer than a polynomial function of the size of the system are not possible when there are polynomially many copies of the system present.

scholarly journals RNA 3’-terminal phosphate cyclases and cyclase-like proteins

2016 ◽  
Vol 62 (3) ◽  
pp. 327-334
Author(s):  
Witold Filipowicz
Keyword(s):  
Rna Cleavage ◽  
Rna Molecules ◽  
Metabolic Reactions ◽  
Cyclic Phosphate ◽  
Cleavage Reactions

RNA molecules bearing terminal 2’,3’-cyclic phosphate are quite common in nature. For example, 2’,3’-cyclic phosphate termini are produced during RNA cleavage by many en-doribonucleases either as intermediates or final products. Many RNA-based nucleases (ribo-zymes) also generate cyclic phosphate termini. However, cleavage reactions are not the only way in which RNAs bearing cyclic phosphate ends are produced. They can also be generated by RNA 3’-terminal phosphate cyclases (RtcA), a family of enzymes conserved in eukaryotes, bacteria, and archaea. These enzymes catalyze the ATP-dependent conversion of the 3’-phos-phate to a 2’,3’-cyclic phosphodiester at the end of RNA. In this article, I review knowledge about the biochemistry and structure of RNA 3’-phosphate cyclases and also proteins of the RNA cyclase-like (Rcl1) family, and discuss their documented or possible roles in different RNA metabolic reactions.

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