The Experts below are selected from a list of 1539 Experts worldwide ranked by ideXlab platform
Scott K. Silverman - One of the best experts on this subject based on the ideXlab platform.
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DNA-catalyzed sequence-specific hydrolysis of DNA
2016Co-Authors: Amit Sachdeva, Scott K. SilvermanAbstract:Deoxyribozymes (DNA catalysts) have been reported for cleavage of RNA phosphodiester linkages, but cleaving peptide or DNA phosphodiester linkages is much more challenging. Using in vitro selection, here we identified Deoxyribozymes that sequence-specifically hydrolyze DNA with multiple turnover and rate enhancement of 108 (possibly as high as 1014). The new DNA catalysts require both Mn2+ and Zn2+, which is intriguing because many natural DNA nucleases are bimetallic protein enzymes. The first Deoxyribozyme (DNA enzyme) was identified by in vitro selection in 1994 and cleaves an RNA phosphodiester bond1. Since that time, DNA has been shown to catalyze many reactions, including several preparatively useful transformations2. RNA cleavage is the most studied DNA-catalyzed reaction3, and Deoxyribozymes with practical cleavage abilities for nearly all RNA dinucleotide target sequences have been developed4,5. However, DNA-catalyzed hydrolysis of other biologically relevant bonds, such as amide (peptide) or DNA phosphodiester linkages, has not been reported. Non-site-selective Cu2+-dependent oxidative cleavage of DNA has been described6. These reactions appear t
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DNA Catalysis of a Normally Disfavored RNA Hydrolysis Reaction
2016Co-Authors: Darren J. Parker, Ying Xiao, John M. Aguilar, Scott K. SilvermanAbstract:ABSTRACT: We recently used in vitro selection to identify many Deoxyribozymes that catalyze DNA phosphodiester bond hydrolysis and create 5′-phosphate and 3′-hydroxyl termini. Alternatively, numerous deoxy-ribozymes have been identified for catalysis of RNA cleavage by 2′-hydroxyl transesterification, forming 2′,3′-cyclic phosphate and 5′-hydroxyl termini. In this study, we investigated the ability of DNA to catalyze RNA cleavage by hydrolysis rather than transesterification, although normally the hydrolysis reaction is substantially disfavored relative to transesterification. Via a series of in vitro selection experiments, we found that reselection of a DNA-hydrolyzing Deoxyribozyme leads either to trans-esterification or hydrolysis, depending on exclusion or inclusion of a stringent selection pressure for hydrolysis
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DNA Catalysts with Tyrosine Kinase Activity
2016Co-Authors: Shannon M. Walsh, Amit Sachdeva, Scott K. SilvermanAbstract:ABSTRACT: We show that DNA catalysts (deoxyribo-zymes, DNA enzymes) can phosphorylate tyrosine residues of peptides. Using in vitro selection, we identified Deoxyribozymes that transfer the γ-phosphoryl group from a 5′-triphosphorylated donor (a pppRNA oligonucleotide or GTP) to the tyrosine hydroxyl acceptor of a tethered hexapeptide. Tyrosine kinase Deoxyribozymes that use pppRNA were identified from each of N30, N40, and N50 random-sequence pools. Each Deoxyribozyme requires Zn2+, and most additionally require Mn2+. The deoxy-ribozymes have little or no selectivity for the amino acid identities near the tyrosine, but they are highly selective for phosphorylating tyrosine rather than serine. Analogous GTP-dependent DNA catalysts were identified and found to have apparent Km(GTP) as low as ∼20 μM. Thes
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DNA-Catalyzed Amide Hydrolysis
Journal of the American Chemical Society, 2016Co-Authors: Cong Zhou, Joshua L. Avins, Paul C. Klauser, Benjamin M. Brandsen, Scott K. SilvermanAbstract:DNA catalysts (Deoxyribozymes) for a variety of reactions have been identified by in vitro selection. However, for certain reactions this identification has not been achieved. One important example is DNA-catalyzed amide hydrolysis, for which a previous selection experiment instead led to DNA-catalyzed DNA phosphodiester hydrolysis. Subsequent efforts in which the selection strategy deliberately avoided phosphodiester hydrolysis led to DNA-catalyzed ester and aromatic amide hydrolysis, but aliphatic amide hydrolysis has been elusive. In the present study, we show that including modified nucleotides that bear protein-like functional groups (any one of primary amino, carboxyl, or primary hydroxyl) enables identification of amide-hydrolyzing Deoxyribozymes. In one case, the same Deoxyribozyme sequence without the modifications still retains substantial catalytic activity. Overall, these findings establish the utility of introducing protein-like functional groups into Deoxyribozymes for identifying new cataly...
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Systematic Evaluation of the Dependence of Deoxyribozyme Catalysis on Random Region Length
2016Co-Authors: Tania E. Velez, Ying Xiao, Jaydeep Singh, Emily C. Allen, On Yi Wong, Madhavaiah Chandra, Sarah C. Kwon, Scott K. SilvermanAbstract:Functional nucleic acids are DNA and RNA aptamers that bind targets, or they are Deoxyribozymes and ribozymes that have catalytic activity. These functional DNA and RNA sequences can be identified from random-sequence pools by in vitro selection, which requires choosing the length of the random region. Shorter random regions allow more complete coverage of sequence space but may not permit the structural complexity necessary for binding or catalysis. In contrast, longer random regions are sampled incompletely but may allow adoption of more complicated structures that enable function. In this study, we systematically examined random region length (N20 through N60) for two particular Deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified Deoxyribozymes using only N40 regions. In the case of DNA cleavage, here we found that shorter N20 and N30 regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via β-elimination, whereas longer N50 and N60 regions did not lead to catalytically active DNA sequences. Follow-up selections with N20, N30, and N40 regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N30 and N60 regions provided catalytically active sequences, whereas N20 was unsuccessful, and the N40 Deoxyribozymes were functionally superior (in terms of rate and yield) to N30 and N60. Collectively, the results indicate that with future in vitro selection experiments for DNA and RNA catalysts, and by extension for aptamers, random region length should be an important experimental variable
Yasuhide Okumoto - One of the best experts on this subject based on the ideXlab platform.
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Development of a short Ca2+-dependent Deoxyribozyme with RNA cleavage activity
2016Co-Authors: Naoki Sugimoto, Yasuhide OkumotoAbstract:We developed a short Ca^+.dependent Deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA cleavage reaction between rA and rU. The second-order rate constant of this short Deoxyribozyme is 1.7 x 10 ^ M~l min~l at 37 °C, and this value is very similar to that of the Deoxyribozyme (dGGCTAGC-TACAACGA) in the presence of Ca 2 +
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Novel biomaterials derived from Deoxyribozyme and NAPzyme
Macromolecular Symposia, 2003Co-Authors: Yasuhide Okumoto, Hiroyoshi Fujiki, Junji Kawakami, Shoji Nakashima, Shu-ichi Nakano, Tatsuo Ohmichi, Daisuke Miyoshi, Naoki SugimotoAbstract:We report the potential of a small Ca 2+ -dependent Deoxyribozyme as a novel biomaterial to distinguish RNA foldings. It is found that an immobilized Deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The RNA cleavage reaction is also detected using the Deoxyribozyme SPR sensor chip. Furthermore, we develop a novel NAPzyme (nucleic acid peptide deoxyribozyrne) with its RNA cleavage function in the absence of divalent metal ions.
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Factors that Contribute to Efficient Catalytic Activity of a Small Ca2+-Dependent Deoxyribozyme in Relation to Its RNA Cleavage Function†
Biochemistry, 2003Co-Authors: Yasuhide Okumoto, Yoshiatsu Tanabe, Naoki SugimotoAbstract:Recently, we found a small Ca2+-dependent Deoxyribozyme (unmodified), d(GCCTGGCAG1G2C3T4A5C6A7A8C9G10A11GTCCCT), with cleavage activity for its RNA substrate, r(AGGGACA↓UGCCAGGC) (↓ denotes the RNA cleavage site), in the presence of Ca2+ and developed a functional SPR sensor chip with this Deoxyribozyme [Okumoto, Y., Ohmichi, T., and Sugimoto, N. (2002) Biochemistry 41, 2769−2773]. In the study presented here, to clarify the factors contributing to the efficient catalytic activity of the unmodified Deoxyribozyme, RNA cleavage reactions were carried out using 24 mutant Deoxyribozymes containing one unnatural DNA nucleotide, such as dI (2‘-deoxyinosine), 7-deaza-dG, 2-aminopurine, 7-deaza-dA, 2-amino-dA, dm5C (5-methyl-2‘-deoxycytosine), or dPC (5-propynyl-2‘-deoxycytosine). The Km values (Michaelis constants) with the mutants that lacked N7 and O6 of G1 and O6 of G2 were 4.5 and 6.6 times that of the unmodified one, respectively. The kcat value (cleavage rate constant) with the mutants that lacked O6 of G1...
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Development of a short Ca2+-dependent Deoxyribozyme with RNA cleavage activity
Nucleic Acids Symposium Series, 1999Co-Authors: Naoki Sugimoto, Yasuhide OkumotoAbstract:We developed a short Ca^+.dependent Deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA cleavage reaction between rA and rU. The second-order rate constant of this short Deoxyribozyme is 1.7 x 10^ M~l min~l at 37 °C, and this value is very similar to that of the Deoxyribozyme (dGGCTAGCTACAACGA) in the presence of Ca 2 + . INTRODUCTION Metal ions play the crucial roles in the catalytic activity of all ribozymes (1), because these ribozymes are considered as metalloenzymes (1,2). The ribozymes have a catalytic loop with a metal binding site. The deletion of nucleotides from the catalytic loop would show higher activity or unique property in the case of the small ribozyme (3). An Deoxyribozyme that was derived from a random library by in vitro selection is one of the DNA enzymes that requires Mg^+ and catalyzed site-specific RNA cleavage reaction (4). The original Deoxyribozyme, dGCCTGGCAGiG2C3T4A5T6G7C8 A9C1 oA 11A12C13G14A15GTCCCT, binds to the RNA substrate, rAGGGACA^lUGCCAGGC, and acts as a enzyme in the presence of Ca2+ or Mg2+ so that it cleaves the RNA substrate at one site of rApiU in the asymmetric internal loop (4). The catalytic efficiency of the original Deoxyribozyme in the presence of Mg2+ is very similar to that of Ca2+. In this study, we developed a short metal iondependent Deoxyribozyme with site-specific RNA cleavage activity. MATERIALS AND METHODS Materials The RNA and DNA oligonucleotides used here were synthesized chemically on a solid support using phosphoramidite procedures and purified by HPLC as described previously (5). The blocked unnatural nucleotide monomers were purchased from Glen Research. The RNA substrate was 5'-end labeled with [y-32p] ATP by the T4 ploynucleotide kinase method. Cleavage Reactions Kinetic measurements were done under multipleturnover conditions in a buffer containing 50 mM Tris-HCl and 25 mM divalent metal ions (pH 8.0) at 37 °C. After cleavage reactions were stopped, the cleavage products and the RNA substrates were separated by electrophoresis on 20% polyacrylamide denaturing gels. The RNA cleavage yields were determined by quantition of radioactivity in the bands of the 5'-end labeled products and the RNA substrate with a Bio-Image Analyzer model BAS 2000 (Fuji Film, Tokyo). Km and &cat values were calculated from EadieHofstee plots. RESULTS AND DISCUSSION The catalytic efficiency (&cat/^m value) of the original Deoxyribozyme in the presence 282 Nucleic Acids Symposium Series No. 42
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Development of a short Ca2+-dependent Deoxyribozyme with RNA cleavage activity.
Nucleic acids symposium series, 1999Co-Authors: Naoki Sugimoto, Yasuhide OkumotoAbstract:We developed a short Ca2+-dependent Deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA cleavage reaction between rA and rU. The second-order rate constant of this short Deoxyribozyme is 1.7 x 10(7) M(-1) min(-1) at 37 degrees C, and this value is very similar to that of the Deoxyribozyme (dGGCTAGCTACAACGA) in the presence of Ca2+.
Naoki Sugimoto - One of the best experts on this subject based on the ideXlab platform.
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Development of a short Ca2+-dependent Deoxyribozyme with RNA cleavage activity
2016Co-Authors: Naoki Sugimoto, Yasuhide OkumotoAbstract:We developed a short Ca^+.dependent Deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA cleavage reaction between rA and rU. The second-order rate constant of this short Deoxyribozyme is 1.7 x 10 ^ M~l min~l at 37 °C, and this value is very similar to that of the Deoxyribozyme (dGGCTAGC-TACAACGA) in the presence of Ca 2 +
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Novel biomaterials derived from Deoxyribozyme and NAPzyme
Macromolecular Symposia, 2003Co-Authors: Yasuhide Okumoto, Hiroyoshi Fujiki, Junji Kawakami, Shoji Nakashima, Shu-ichi Nakano, Tatsuo Ohmichi, Daisuke Miyoshi, Naoki SugimotoAbstract:We report the potential of a small Ca 2+ -dependent Deoxyribozyme as a novel biomaterial to distinguish RNA foldings. It is found that an immobilized Deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The RNA cleavage reaction is also detected using the Deoxyribozyme SPR sensor chip. Furthermore, we develop a novel NAPzyme (nucleic acid peptide deoxyribozyrne) with its RNA cleavage function in the absence of divalent metal ions.
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Factors that Contribute to Efficient Catalytic Activity of a Small Ca2+-Dependent Deoxyribozyme in Relation to Its RNA Cleavage Function†
Biochemistry, 2003Co-Authors: Yasuhide Okumoto, Yoshiatsu Tanabe, Naoki SugimotoAbstract:Recently, we found a small Ca2+-dependent Deoxyribozyme (unmodified), d(GCCTGGCAG1G2C3T4A5C6A7A8C9G10A11GTCCCT), with cleavage activity for its RNA substrate, r(AGGGACA↓UGCCAGGC) (↓ denotes the RNA cleavage site), in the presence of Ca2+ and developed a functional SPR sensor chip with this Deoxyribozyme [Okumoto, Y., Ohmichi, T., and Sugimoto, N. (2002) Biochemistry 41, 2769−2773]. In the study presented here, to clarify the factors contributing to the efficient catalytic activity of the unmodified Deoxyribozyme, RNA cleavage reactions were carried out using 24 mutant Deoxyribozymes containing one unnatural DNA nucleotide, such as dI (2‘-deoxyinosine), 7-deaza-dG, 2-aminopurine, 7-deaza-dA, 2-amino-dA, dm5C (5-methyl-2‘-deoxycytosine), or dPC (5-propynyl-2‘-deoxycytosine). The Km values (Michaelis constants) with the mutants that lacked N7 and O6 of G1 and O6 of G2 were 4.5 and 6.6 times that of the unmodified one, respectively. The kcat value (cleavage rate constant) with the mutants that lacked O6 of G1...
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Development of a short Ca2+-dependent Deoxyribozyme with RNA cleavage activity
Nucleic Acids Symposium Series, 1999Co-Authors: Naoki Sugimoto, Yasuhide OkumotoAbstract:We developed a short Ca^+.dependent Deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA cleavage reaction between rA and rU. The second-order rate constant of this short Deoxyribozyme is 1.7 x 10^ M~l min~l at 37 °C, and this value is very similar to that of the Deoxyribozyme (dGGCTAGCTACAACGA) in the presence of Ca 2 + . INTRODUCTION Metal ions play the crucial roles in the catalytic activity of all ribozymes (1), because these ribozymes are considered as metalloenzymes (1,2). The ribozymes have a catalytic loop with a metal binding site. The deletion of nucleotides from the catalytic loop would show higher activity or unique property in the case of the small ribozyme (3). An Deoxyribozyme that was derived from a random library by in vitro selection is one of the DNA enzymes that requires Mg^+ and catalyzed site-specific RNA cleavage reaction (4). The original Deoxyribozyme, dGCCTGGCAGiG2C3T4A5T6G7C8 A9C1 oA 11A12C13G14A15GTCCCT, binds to the RNA substrate, rAGGGACA^lUGCCAGGC, and acts as a enzyme in the presence of Ca2+ or Mg2+ so that it cleaves the RNA substrate at one site of rApiU in the asymmetric internal loop (4). The catalytic efficiency of the original Deoxyribozyme in the presence of Mg2+ is very similar to that of Ca2+. In this study, we developed a short metal iondependent Deoxyribozyme with site-specific RNA cleavage activity. MATERIALS AND METHODS Materials The RNA and DNA oligonucleotides used here were synthesized chemically on a solid support using phosphoramidite procedures and purified by HPLC as described previously (5). The blocked unnatural nucleotide monomers were purchased from Glen Research. The RNA substrate was 5'-end labeled with [y-32p] ATP by the T4 ploynucleotide kinase method. Cleavage Reactions Kinetic measurements were done under multipleturnover conditions in a buffer containing 50 mM Tris-HCl and 25 mM divalent metal ions (pH 8.0) at 37 °C. After cleavage reactions were stopped, the cleavage products and the RNA substrates were separated by electrophoresis on 20% polyacrylamide denaturing gels. The RNA cleavage yields were determined by quantition of radioactivity in the bands of the 5'-end labeled products and the RNA substrate with a Bio-Image Analyzer model BAS 2000 (Fuji Film, Tokyo). Km and &cat values were calculated from EadieHofstee plots. RESULTS AND DISCUSSION The catalytic efficiency (&cat/^m value) of the original Deoxyribozyme in the presence 282 Nucleic Acids Symposium Series No. 42
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Development of a short Ca2+-dependent Deoxyribozyme with RNA cleavage activity.
Nucleic acids symposium series, 1999Co-Authors: Naoki Sugimoto, Yasuhide OkumotoAbstract:We developed a short Ca2+-dependent Deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA cleavage reaction between rA and rU. The second-order rate constant of this short Deoxyribozyme is 1.7 x 10(7) M(-1) min(-1) at 37 degrees C, and this value is very similar to that of the Deoxyribozyme (dGGCTAGCTACAACGA) in the presence of Ca2+.
Ying Xiao - One of the best experts on this subject based on the ideXlab platform.
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DNA Catalysis of a Normally Disfavored RNA Hydrolysis Reaction
2016Co-Authors: Darren J. Parker, Ying Xiao, John M. Aguilar, Scott K. SilvermanAbstract:ABSTRACT: We recently used in vitro selection to identify many Deoxyribozymes that catalyze DNA phosphodiester bond hydrolysis and create 5′-phosphate and 3′-hydroxyl termini. Alternatively, numerous deoxy-ribozymes have been identified for catalysis of RNA cleavage by 2′-hydroxyl transesterification, forming 2′,3′-cyclic phosphate and 5′-hydroxyl termini. In this study, we investigated the ability of DNA to catalyze RNA cleavage by hydrolysis rather than transesterification, although normally the hydrolysis reaction is substantially disfavored relative to transesterification. Via a series of in vitro selection experiments, we found that reselection of a DNA-hydrolyzing Deoxyribozyme leads either to trans-esterification or hydrolysis, depending on exclusion or inclusion of a stringent selection pressure for hydrolysis
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Systematic Evaluation of the Dependence of Deoxyribozyme Catalysis on Random Region Length
2016Co-Authors: Tania E. Velez, Ying Xiao, Jaydeep Singh, Emily C. Allen, On Yi Wong, Madhavaiah Chandra, Sarah C. Kwon, Scott K. SilvermanAbstract:Functional nucleic acids are DNA and RNA aptamers that bind targets, or they are Deoxyribozymes and ribozymes that have catalytic activity. These functional DNA and RNA sequences can be identified from random-sequence pools by in vitro selection, which requires choosing the length of the random region. Shorter random regions allow more complete coverage of sequence space but may not permit the structural complexity necessary for binding or catalysis. In contrast, longer random regions are sampled incompletely but may allow adoption of more complicated structures that enable function. In this study, we systematically examined random region length (N20 through N60) for two particular Deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified Deoxyribozymes using only N40 regions. In the case of DNA cleavage, here we found that shorter N20 and N30 regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via β-elimination, whereas longer N50 and N60 regions did not lead to catalytically active DNA sequences. Follow-up selections with N20, N30, and N40 regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N30 and N60 regions provided catalytically active sequences, whereas N20 was unsuccessful, and the N40 Deoxyribozymes were functionally superior (in terms of rate and yield) to N30 and N60. Collectively, the results indicate that with future in vitro selection experiments for DNA and RNA catalysts, and by extension for aptamers, random region length should be an important experimental variable
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DNA Catalysis of a Normally Disfavored RNA Hydrolysis Reaction
2013Co-Authors: Darren J. Parker, John M. Aguilar, Ying Xiao, Scott K. SilvermanAbstract:We recently used in vitro selection to identify many Deoxyribozymes that catalyze DNA phosphodiester bond hydrolysis and create 5′-phosphate and 3′-hydroxyl termini. Alternatively, numerous Deoxyribozymes have been identified for catalysis of RNA cleavage by 2′-hydroxyl transesterification, forming 2′,3′-cyclic phosphate and 5′-hydroxyl termini. In this study, we investigated the ability of DNA to catalyze RNA cleavage by hydrolysis rather than transesterification, although normally the hydrolysis reaction is substantially disfavored relative to transesterification. Via a series of in vitro selection experiments, we found that reselection of a DNA-hydrolyzing Deoxyribozyme leads either to transesterification or hydrolysis, depending on exclusion or inclusion of a stringent selection pressure for hydrolysis. An entirely new selection starting from a random DNA pool, using an all-RNA substrate and imposing the same selection pressure, also leads to RNA hydrolysis. Collectively, these results establish experimentally that small DNA sequences have the catalytic ability to direct a chemical reaction down a disfavored pathway, even when a more favorable mechanism is readily available. Our view of DNA catalysis is therefore expanded beyond merely increasing the rates of reactions that would have occurred more slowly without the catalyst
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Systematic evaluation of the dependence of Deoxyribozyme catalysis on random region length.
ACS Combinatorial Science, 2012Co-Authors: Tania E. Velez, Ying Xiao, Jaydeep Singh, Emily C. Allen, On Yi Wong, Madhavaiah Chandra, Sarah C. Kwon, Scott K. SilvermanAbstract:Functional nucleic acids are DNA and RNA aptamers that bind targets, or they are Deoxyribozymes and ribozymes that have catalytic activity. These functional DNA and RNA sequences can be identified from random-sequence pools by in vitro selection, which requires choosing the length of the random region. Shorter random regions allow more complete coverage of sequence space but may not permit the structural complexity necessary for binding or catalysis. In contrast, longer random regions are sampled incompletely but may allow adoption of more complicated structures that enable function. In this study, we systematically examined random region length (N20 through N60) for two particular Deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified Deoxyribozymes using only N40 regions. In the case of DNA cleavage, here we found that shorter N20 and N30 regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via β-elimination, whereas longer N50 and N60 regions did not lead to catalytically active DNA sequences. Follow-up selections with N20 ,N 30, and N40 regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N30 and N60 regions provided catalytically active sequences, whereas N20 was unsuccessful, and the N40 Deoxyribozymes were functionally superior (in terms of rate and yield) to N30 and N60. Collectively, the results indicate that with future in vitro selection experiments for DNA and RNA catalysts, and by extension for aptamers, random region length should be an important experimental variable.
John D. Brennan - One of the best experts on this subject based on the ideXlab platform.
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characterizing the secondary structure and identifying functionally essential nucleotides of ph6dz1 a fluorescence signaling and rna cleaving Deoxyribozyme
Biochemistry, 2005Co-Authors: Yutu Shen, John D. BrennanAbstract:pH6DZ1 is a synthetic Deoxyribozyme that is able to couple catalysis with fluorescence signal generation. This Deoxyribozyme has the ability to cleave itself at a lone ribonucleotide that is present between a pair of deoxyribothymidines, one modified with a fluorophore (fluorescein) and the other with a quencher (DABCYL). Herein, we report on the sequence truncation and secondary structure characterization of pH6DZ1 as well as the identification of functionally important nucleotides within this Deoxyribozyme. Our data indicate that pH6DZ1 has a four-way, junction-like secondary structure comprised of four short duplexes, three hairpin loops, and three interhelical unpaired elements. Ten nucleotides, all located in two separate single-stranded regions, were identified as functionally indispensable nucleotides (complete loss of the catalytic function was obtained upon mutation). Nine nucleotides, most of which are also distributed in three single-stranded DNA elements, were identified as functionally vital nucleotides (at least a 1000-fold activity reduction was obtained upon mutation). Our study has shown that pH6DZ1 has a secondary structure that is more complex than those reported for other RNA-cleaving Deoxyribozymes. The identification of functionally important nucleotides lays the foundation for future mechanistic studies on this DNAzyme. The elucidation of the secondary structure of pH6DZ1 should facilitate the future exploration of this unique DNAzyme for the development of DNAzyme-based biosensors.
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Characterization of a fluorescence-signaling and RNA-cleaving Deoxyribozyme
Nucleic Acids Symposium Series, 2005Co-Authors: Yutu Shen, John D. BrennanAbstract:DNA enzymes (Deoxyribozymes or DNAzymes) are single-stranded DNA molecules with catalytic function. Previously, we isolated several RNA-cleaving Deoxyribozymes with fluorescence-signaling properties. These special DNA molecules are capable of cleaving an RNA linkage embedded within a DNA sequence and flanked by a pair of deoxyribothymidines modified with a fluorophore (fluorescein) and a quencher (dabcyl). Here we report on the sequence truncation and secondary structure characterization of one such Deoxyribozyme known as pH6DZ1 as well as the identification of functionally important nucleotides within this Deoxyribozyme. Our data indicates that pH6DZ1 has a four-way junction-like secondary structure comprising four short duplexes, three hairpin loops, and three inter-helical unpaired elements.