Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Systematic analysis of the role of target site accessibility in the activity of DNA enzymes.
Doran G
,
Sohail M
.
???displayArticle.abstract???
We employed an approach using oligonucleotide scanning arrays and computational analysis to conduct a systematic analysis of the interaction between catalytic nucleic acids (DNA enzymes or DNAzymes) and long RNA targets. A radio-labelled transcript representing mRNA of Xenopus cyclin B5 was hybridised to an array of oligonucleotides scanning the first 120 nucleotides of the coding region to assess the ability of the immobilised oligonucleotides to form heteroduplexes with the target. The hybridisation revealed oligonucleotides showing varying levels of signal intensities along the length of the array, reflecting on the variable accessibility of the corresponding complementary regions in the target RNA. Deoxyribozymes targeting a number of these regions were selected and tested for their ability to cleave the target RNA. The mRNA cleavage observed indicates that indeed target accessibility was an important component in the activity of deoxyribozymes and that it was important that at least one of the two binding arms was complementary to an accessible site. Computational analysis suggested that intra-molecular folding of deoxyribozymes into stable structures may also negatively contribute to their activity. 10-23 type deoxyribozymes generally appeared more active than 8-17 type and it was possible to predict deoxyribozymes with high cleavage efficiency using scanning array hybridization and computational analysis as guides. The data presented here therefore have implications on designing effective DNA enzymes.
Figure 1. A and B. Moving window of deoxyribozyme complementarity with 16-mer oligonucleotides in the array, with two
possible modes of interaction. C. Histograms showing accessibility profile of the cyclin B5 mRNA in the first 120 nucleotides at 16-mer oligonucleotide lengths. It also shows binding sites of the two guide arms, X and Z, of the various deoxyribozymes in the histogram. D. Identity of the various deoxyribozymes in the cyclin B5 sequence.
Figure 2. A. Cleavage of B5 RNA by the various deoxyribozymes. Two fragments produced by DR36-43 are marked
with arrows. DR29N and DR36N has a single mutation in the catalytic domain, making them inactive (scR = scrambled
right arm; scL = scrambled left arm). B. Quantification of cleavage by deoxyribozymes. Quantification for scrambled
deoxyribozymes is not shown since they did not produce any cleavage.
Figure 3. A. Graphical representations of Tm calculations for deoxyribozyme binding arms. B. Computer folding of B5 RNA in the presence of deoxyribozyme oligonucleotides. The numbers show relative stability of the structures. C. RNAstructure analysis of DR36-43 fold.
Figure 4. Autoradiograph of deoxyribozyme cleavage assay in the presence of an effector (F2) oligonucleotide. In the presence of F2 DR80 showed enhanced activity. DR59 cleavage was blocked and that the activity of DR36 only increased slightly, in line with the observation on the array (Sohail et al, 2005).
Figure 5. Autoradiograph showing cleavage activity of DR36 against full length cyclin B5 mRNA in the presence (RnH) and absence (C) of RNase H. Other control lanes represent full-length cyclin B5 mRNA incubated for 2 hr at 37°C in the presence or absence of RNase H. RNase H mapping reaction contained ∼25 fmol 32P-end-labelled transcript, 10 mM MgCl2, 50 mM KCl, 50 mM Tris-HCl pH 7.4, 1 mM DTT, 0.5 U RNase H, 0.5 U RNase inhibitor (Promega) and 100pmol of DNAzyme) in a total volume of 10 μl. The reactions were incubated at 30°C for 1hr and terminated by addition of 10 μl of formamide gel loading dye buffer. A 5 μl aliquot was analysed on a 8% (w/v) denaturing polyacrylamide gel.
Beale,
Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system.
2003, Pubmed
Beale,
Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system.
2003,
Pubmed
Bohula,
The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript.
2003,
Pubmed
Breaker,
Catalytic DNA: in training and seeking employment.
1999,
Pubmed
Breaker,
DNA enzymes.
1997,
Pubmed
Breaker,
Emergence of a replicating species from an in vitro RNA evolution reaction.
1994,
Pubmed
Cairns,
Nucleic acid mutation analysis using catalytic DNA.
2000,
Pubmed
Cairns,
Optimisation of the 10-23 DNAzyme-substrate pairing interactions enhanced RNA cleavage activity at purine-cytosine target sites.
2003,
Pubmed
Carmi,
Cleaving DNA with DNA.
1998,
Pubmed
Carmi,
Characterization of a DNA-cleaving deoxyribozyme.
2001,
Pubmed
Cuenoud,
A DNA metalloenzyme with DNA ligase activity.
1995,
Pubmed
Emilsson,
Deoxyribozymes: new activities and new applications.
2002,
Pubmed
Fahmy,
Suppression of vascular permeability and inflammation by targeting of the transcription factor c-Jun.
2006,
Pubmed
Feldman,
A new and efficient DNA enzyme for the sequence-specific cleavage of RNA.
2001,
Pubmed
Flynn-Charlebois,
In vitro evolution of an RNA-cleaving DNA enzyme into an RNA ligase switches the selectivity from 3'-5' to 2'-5'.
2003,
Pubmed
Hjiantoniou,
DNazyme-mediated cleavage of Twist transcripts and increase in cellular apoptosis.
2003,
Pubmed
Kurreck,
Comparative study of DNA enzymes and ribozymes against the same full-length messenger RNA of the vanilloid receptor subtype I.
2002,
Pubmed
Levy,
Selection of deoxyribozyme ligases that catalyze the formation of an unnatural internucleotide linkage.
2001,
Pubmed
Li,
In vitro selection of kinase and ligase deoxyribozymes.
2001,
Pubmed
Li,
Capping DNA with DNA.
2000,
Pubmed
Mathews,
Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure.
2004,
Pubmed
Mei,
An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling.
2003,
Pubmed
Mir,
Determining the influence of structure on hybridization using oligonucleotide arrays.
1999,
Pubmed
Okumoto,
Immobilized small deoxyribozyme to distinguish RNA secondary structures.
2002,
Pubmed
Ota,
Effects of helical structures formed by the binding arms of DNAzymes and their substrates on catalytic activity.
1998,
Pubmed
Petch,
Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.
2003,
Pubmed
Ricca,
Optimization and generality of a small deoxyribozyme that ligates RNA.
2003,
Pubmed
Santoro,
A general purpose RNA-cleaving DNA enzyme.
1997,
Pubmed
Schmidt,
Application of locked nucleic acids to improve aptamer in vivo stability and targeting function.
2004,
Pubmed
Schubert,
Gaining target access for deoxyribozymes.
2004,
Pubmed
Schubert,
RNA cleaving '10-23' DNAzymes with enhanced stability and activity.
2003,
Pubmed
Sohail,
Structural rearrangements in RNA on the binding of an antisense oligonucleotide: implications for the study of intra-molecular RNA interactions and the design of cooperatively acting antisense reagents with enhanced efficacy.
2005,
Pubmed
Sohail,
Hybridization of antisense reagents to RNA.
2000,
Pubmed
Sohail,
The folding of large RNAs studied by hybridization to arrays of complementary oligonucleotides.
1999,
Pubmed
Sohail,
Antisense oligonucleotides selected by hybridisation to scanning arrays are effective reagents in vivo.
2001,
Pubmed
,
Xenbase
Sohail,
A simple and cost-effective method for producing small interfering RNAs with high efficacy.
2003,
Pubmed
Southern,
Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids.
1994,
Pubmed
Sriram,
In vitro-selected RNA cleaving DNA enzymes from a combinatorial library are potent inhibitors of HIV-1 gene expression.
2000,
Pubmed
Stojanovic,
Homogeneous assays based on deoxyribozyme catalysis.
2000,
Pubmed
Toyoda,
Inhibition of influenza virus replication in cultured cells by RNA-cleaving DNA enzyme.
2000,
Pubmed
Travascio,
A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites.
1999,
Pubmed
Wang,
Sequence diversity, metal specificity, and catalytic proficiency of metal-dependent phosphorylating DNA enzymes.
2002,
Pubmed
Wang,
A general approach for the use of oligonucleotide effectors to regulate the catalysis of RNA-cleaving ribozymes and DNAzymes.
2002,
Pubmed
Wu,
Properties of cloned and expressed human RNase H1.
1999,
Pubmed
Yen,
Sequence-specific cleavage of Huntingtin mRNA by catalytic DNA.
1999,
Pubmed
Zhang,
Squamous cell carcinoma growth in mice and in culture is regulated by c-Jun and its control of matrix metalloproteinase-2 and -9 expression.
2006,
Pubmed
Zhang,
Inhibition of infection of incoming HIV-1 virus by RNA-cleaving DNA enzyme.
1999,
Pubmed