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John Paul Pezacki - One of the best experts on this subject based on the ideXlab platform.
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structural insights into interactions between viral suppressor of rna silencing Protein P19 mutants and small rnas
FEBS Open Bio, 2019Co-Authors: Dana V Foss, Nicole T Schirle, Ian J Macrae, John Paul PezackiAbstract:Viral suppressors of RNA silencing (VSRSs) are a diverse group of viral Proteins that have evolved to disrupt eukaryotic RNA silencing pathways, thereby contributing to viral pathogenicity. The P19 Protein is a VSRS that selectively binds to short interfering RNAs (siRNAs) over microRNAs (miRNAs). Mutational analysis has identified single amino acid substitutions that reverse this selectivity through new high-affinity interactions with human miR-122. Herein, we report crystal structures of complexed P19-T111S (2.6 A), P19-T111H (2.3 A) and wild-type P19 Protein (2.2 A) from the Carnation Italian ringspot virus with small interfering RNA (siRNA) ligands. Structural comparisons reveal that these mutations do not lead to major changes in P19 architecture, but instead promote subtle rearrangement of residues and solvent molecules along the P19 midline. These observations suggest P19 uses many small interactions to distinguish siRNAs from miRNAs and perturbing these interactions can create P19 variants with novel RNA-recognition properties. DATABASE: Model data are deposited in the PDB database under the accession numbers 6BJG, 6BJH and 6BJV.
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Suppressing RNA silencing with small molecules and the viral suppressor of RNA silencing Protein P19
Biochemical and biophysical research communications, 2015Co-Authors: Dana C. Danielson, Roxana Filip, Megan H. Powdrill, Shifawn O'hara, John Paul PezackiAbstract:Abstract RNA silencing is a gene regulatory and host defense mechanism whereby small RNA molecules are engaged by Argonaute (AGO) Proteins, which facilitate gene knockdown of complementary mRNA targets. Small molecule inhibitors of AGO represent a convenient method for reversing this effect and have applications in human therapy and biotechnology. Viral suppressors of RNA silencing, such as P19, can also be used to suppress the pathway. Here we assess the compatibility of these two approaches, by examining whether synthetic inhibitors of AGO would inhibit P19-siRNA interactions. We observe that aurintricarboxylic acid (ATA) is a potent inhibitor of P19's ability to bind siRNA (IC50 = 0.43 μM), oxidopamine does not inhibit P19:siRNA interactions, and suramin is a mild inhibitor of P19:siRNA interactions (IC50 = 430 μM). We observe that P19 and suramin are compatible inhibitors of RNA silencing in human hepatoma cells. Our data suggests that at least some inhibitors of AGO may be used in combination with P19 to inhibit RNA silencing at different points in the pathway.
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enhanced specificity of the viral suppressor of rna silencing Protein P19 toward sequestering of human microrna 122
Biochemistry, 2011Co-Authors: Jenny Cheng, Dana C. Danielson, John Paul Pezacki, Neda Nasheri, Ragunath SingaraveluAbstract:Tombusviruses express a 19 kDa Protein (P19) that, as a dimeric Protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The P19 Protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, Proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the P19 binding surface with the aim of creating P19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe P19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant P19, P19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type P19. Overall, our results suggest that P19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The P19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver.
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quantitative analysis of microrna in blood serum with Protein facilitated affinity capillary electrophoresis
Analytical Chemistry, 2011Co-Authors: Nasrin Khan, John Paul Pezacki, Jenny Cheng, Maxim V BerezovskiAbstract:MicroRNAs (miRNAs) are small (∼22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a Protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding Protein (SSB) and double-stranded RNA binding Protein (P19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of P19 to miRNA–RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by P19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30 000 miRNA molecules in 1 mL of serum as a potential source of naive miRNAs.
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an enzyme linked assay for the rapid quantification of micrornas based on the viral suppressor of rna silencing Protein P19
Analytical Biochemistry, 2011Co-Authors: Neda Nasheri, John Paul Pezacki, Jenny Cheng, Ragunath Singaravelu, Mark T McdermottAbstract:Abstract MicroRNAs (miRNAs) are endogenous posttranscriptional regulators found in all metazoa and play crucial roles in virtually all cellular processes. Their aberrant expression has been linked to several diseased states; therefore, techniques capable of sensitive and specific profiling of the miRNA milieu will have significant application in prognostics, diagnostics, and therapeutics. Here we present a method for rapid quantification of miRNA levels using P19, a tombusvirus-encoded suppressor of RNA interference with sequence-independent and size-selective affinity toward 19-bp RNA duplexes. We present a surface plasmon resonance (SPR)-based miRNA sensing method where RNA probes are immobilized on gold surfaces demonstrating P19’s utility in recognition of miRNA-bound probes. This allows detection of miRNAs in the low nanomolar range. To increase the sensitivity, a bead-based enzyme immunoassay was performed, and this technique displays a lower detection limit of 1 fmol and a linear dynamic range from 1 pmol to 1 fmol.
Jenny Cheng - One of the best experts on this subject based on the ideXlab platform.
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enhanced specificity of the viral suppressor of rna silencing Protein P19 toward sequestering of human microrna 122
Biochemistry, 2011Co-Authors: Jenny Cheng, Dana C. Danielson, John Paul Pezacki, Neda Nasheri, Ragunath SingaraveluAbstract:Tombusviruses express a 19 kDa Protein (P19) that, as a dimeric Protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The P19 Protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, Proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the P19 binding surface with the aim of creating P19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe P19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant P19, P19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type P19. Overall, our results suggest that P19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The P19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver.
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quantitative analysis of microrna in blood serum with Protein facilitated affinity capillary electrophoresis
Analytical Chemistry, 2011Co-Authors: Nasrin Khan, John Paul Pezacki, Jenny Cheng, Maxim V BerezovskiAbstract:MicroRNAs (miRNAs) are small (∼22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a Protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding Protein (SSB) and double-stranded RNA binding Protein (P19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of P19 to miRNA–RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by P19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30 000 miRNA molecules in 1 mL of serum as a potential source of naive miRNAs.
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an enzyme linked assay for the rapid quantification of micrornas based on the viral suppressor of rna silencing Protein P19
Analytical Biochemistry, 2011Co-Authors: Neda Nasheri, John Paul Pezacki, Jenny Cheng, Ragunath Singaravelu, Mark T McdermottAbstract:Abstract MicroRNAs (miRNAs) are endogenous posttranscriptional regulators found in all metazoa and play crucial roles in virtually all cellular processes. Their aberrant expression has been linked to several diseased states; therefore, techniques capable of sensitive and specific profiling of the miRNA milieu will have significant application in prognostics, diagnostics, and therapeutics. Here we present a method for rapid quantification of miRNA levels using P19, a tombusvirus-encoded suppressor of RNA interference with sequence-independent and size-selective affinity toward 19-bp RNA duplexes. We present a surface plasmon resonance (SPR)-based miRNA sensing method where RNA probes are immobilized on gold surfaces demonstrating P19’s utility in recognition of miRNA-bound probes. This allows detection of miRNAs in the low nanomolar range. To increase the sensitivity, a bead-based enzyme immunoassay was performed, and this technique displays a lower detection limit of 1 fmol and a linear dynamic range from 1 pmol to 1 fmol.
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erratum to studies of a viral suppressor of rna silencing P19 cfp fusion Protein a fret based probe for sensing double stranded fluorophore tagged small rnas biophysical chemistry 143 2009 166 169
Biophysical Chemistry, 2009Co-Authors: Roger Koukiekolo, Selena M. Sagan, Jenny Cheng, Zygmunt J. Jakubek, John Paul PezackiAbstract:It has been brought to the author's attention that the above paper containsmissing text in the Abstract and they would like to correct this. The text was inadvertently deleted during the typesetting and editing of the proofs. On page 166, the Abstract should read as follows: Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small Protein, P19, that acts as a suppressor of RNA silencing. This Protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity. Here we demonstrate a new approach for rapidly determining the quantities of siRNA using fluorescence resonance energy transfer (FRET) between the Carnation Italian ringspot virus (CIRV) P19-CFP fusion Protein and Cy3-labeled siRNA. The CIRV P19 fusion Protein binds double-stranded siRNAs with nanomolar affinity as determined by FRET.
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studies of a viral suppressor of rna silencing P19 cfp fusion Protein a fret based probe for sensing double stranded fluorophore tagged small rnas
Biophysical Chemistry, 2009Co-Authors: Roger Koukiekolo, John Paul Pezacki, Jenny Cheng, Selena M. Sagan, Zygmunt J. JakubekAbstract:Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small Protein, P19, that acts as a suppressor of RNA silencing. This Protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity.
Selena M. Sagan - One of the best experts on this subject based on the ideXlab platform.
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erratum to studies of a viral suppressor of rna silencing P19 cfp fusion Protein a fret based probe for sensing double stranded fluorophore tagged small rnas biophysical chemistry 143 2009 166 169
Biophysical Chemistry, 2009Co-Authors: Roger Koukiekolo, Selena M. Sagan, Jenny Cheng, Zygmunt J. Jakubek, John Paul PezackiAbstract:It has been brought to the author's attention that the above paper containsmissing text in the Abstract and they would like to correct this. The text was inadvertently deleted during the typesetting and editing of the proofs. On page 166, the Abstract should read as follows: Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small Protein, P19, that acts as a suppressor of RNA silencing. This Protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity. Here we demonstrate a new approach for rapidly determining the quantities of siRNA using fluorescence resonance energy transfer (FRET) between the Carnation Italian ringspot virus (CIRV) P19-CFP fusion Protein and Cy3-labeled siRNA. The CIRV P19 fusion Protein binds double-stranded siRNAs with nanomolar affinity as determined by FRET.
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studies of a viral suppressor of rna silencing P19 cfp fusion Protein a fret based probe for sensing double stranded fluorophore tagged small rnas
Biophysical Chemistry, 2009Co-Authors: Roger Koukiekolo, John Paul Pezacki, Jenny Cheng, Selena M. Sagan, Zygmunt J. JakubekAbstract:Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small Protein, P19, that acts as a suppressor of RNA silencing. This Protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity.
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Studies of the interaction of the viral suppressor of RNA silencing Protein P19 with small RNAs using fluorescence polarization.
Biochemistry, 2008Co-Authors: Jenny Cheng, Selena M. Sagan, Zygmunt J. Jakubek, John Paul PezackiAbstract:Tombusviruses use a 19 kDa Protein (P19) as a suppressor of the RNA silencing pathway during infection. The P19 Protein binds to short-interfering RNA (siRNA) as a dimer and shows a high selectivity for short duplex RNAs over other RNA species. Since P19 can bind to synthetic and RNA silencing generated small RNAs with little sequence dependence and with size selectivity, this Protein has utility as a tool for studying RNA silencing pathways in eukaryotes. However, the ability of p 19 to serve as a tool for studying RNA silencing pathways may be complicated by the presence of other endogenous small RNAs such as micro-RNAs (miRNAs). To understand the importance of endogenous small RNA components with respect to pl9's ability to bind to siRNAs, we examined the interactions of P19 with human miR-122, a 23-nucleotide duplex miRNA containing several mismatched base pairs that is highly abundant in the liver. The binding characteristics were compared with those of an siRNA optimized against the human kinase CSK. The binding studies were performed using fluorescence polarization experiments on duplex oligonucleotides containing Cy3 dye labels at the 5'-end of one of the strands of RNA as well as electrophoretic gel mobility shift assays. Both methods indicate that the synthetic siRNA with no mismatches in base pairing bound with >3-fold selectivity over that of miR-122. Our results suggest that pl9 can distinguish between siRNAs and miRNA species, although the difference in binding constants is not so large that interactions with endogenous miRNAs can be totally ignored.
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effects of ph and salt concentration on the sirna binding activity of the rna silencing suppressor Protein P19
FEBS Letters, 2007Co-Authors: Roger Koukiekolo, John Paul Pezacki, Selena M. SaganAbstract:The RNA silencing pathway is an important component of the anti-viral immune response in eukaryotes, particularly in plants. In turn, many viruses have evolved mechanisms to evade or suppress this pathway. Tombusviruses such as the Carnation Italian ringspot virus (CIRV) express a 19kDa Protein (P19) that is a suppressor of RNA silencing in infected plants. This Protein acts as a dimer and binds specifically to short-interfering RNA (siRNA) through electrostatic interactions between charged residues in the binding cleft. Since pH and salt concentrations can vary widely from host to host, we have investigated the influence of these parameters on the siRNA binding activity of CIRV P19. Previously, we established a convenient fluorescence-based method for assaying CIRV P19:siRNA binding using Ni(2+)-NTA coated 96-well plates. Using this method, we observe that the CIRV P19 Protein binds to siRNA with nanomolar affinity and that this binding is sensitive to pH and salt concentration. The pH-dissociation constant profile shows that CIRV P19:siRNA binding is dependent on three different apparent pK(a) values. The values extrapolated from the curve are 7.1, 8.0 and 10.6 that we interpret as the ionization of one or more histidine, cysteine and lysine residues, respectively. We find that the optimal suppression of RNA silencing by CIRV P19 occurs in the pH range from 6.2 to 7.6.
Roger Koukiekolo - One of the best experts on this subject based on the ideXlab platform.
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erratum to studies of a viral suppressor of rna silencing P19 cfp fusion Protein a fret based probe for sensing double stranded fluorophore tagged small rnas biophysical chemistry 143 2009 166 169
Biophysical Chemistry, 2009Co-Authors: Roger Koukiekolo, Selena M. Sagan, Jenny Cheng, Zygmunt J. Jakubek, John Paul PezackiAbstract:It has been brought to the author's attention that the above paper containsmissing text in the Abstract and they would like to correct this. The text was inadvertently deleted during the typesetting and editing of the proofs. On page 166, the Abstract should read as follows: Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small Protein, P19, that acts as a suppressor of RNA silencing. This Protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity. Here we demonstrate a new approach for rapidly determining the quantities of siRNA using fluorescence resonance energy transfer (FRET) between the Carnation Italian ringspot virus (CIRV) P19-CFP fusion Protein and Cy3-labeled siRNA. The CIRV P19 fusion Protein binds double-stranded siRNAs with nanomolar affinity as determined by FRET.
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studies of a viral suppressor of rna silencing P19 cfp fusion Protein a fret based probe for sensing double stranded fluorophore tagged small rnas
Biophysical Chemistry, 2009Co-Authors: Roger Koukiekolo, John Paul Pezacki, Jenny Cheng, Selena M. Sagan, Zygmunt J. JakubekAbstract:Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small Protein, P19, that acts as a suppressor of RNA silencing. This Protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity.
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effects of ph and salt concentration on the sirna binding activity of the rna silencing suppressor Protein P19
FEBS Letters, 2007Co-Authors: Roger Koukiekolo, John Paul Pezacki, Selena M. SaganAbstract:The RNA silencing pathway is an important component of the anti-viral immune response in eukaryotes, particularly in plants. In turn, many viruses have evolved mechanisms to evade or suppress this pathway. Tombusviruses such as the Carnation Italian ringspot virus (CIRV) express a 19kDa Protein (P19) that is a suppressor of RNA silencing in infected plants. This Protein acts as a dimer and binds specifically to short-interfering RNA (siRNA) through electrostatic interactions between charged residues in the binding cleft. Since pH and salt concentrations can vary widely from host to host, we have investigated the influence of these parameters on the siRNA binding activity of CIRV P19. Previously, we established a convenient fluorescence-based method for assaying CIRV P19:siRNA binding using Ni(2+)-NTA coated 96-well plates. Using this method, we observe that the CIRV P19 Protein binds to siRNA with nanomolar affinity and that this binding is sensitive to pH and salt concentration. The pH-dissociation constant profile shows that CIRV P19:siRNA binding is dependent on three different apparent pK(a) values. The values extrapolated from the curve are 7.1, 8.0 and 10.6 that we interpret as the ionization of one or more histidine, cysteine and lysine residues, respectively. We find that the optimal suppression of RNA silencing by CIRV P19 occurs in the pH range from 6.2 to 7.6.
Zygmunt J. Jakubek - One of the best experts on this subject based on the ideXlab platform.
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erratum to studies of a viral suppressor of rna silencing P19 cfp fusion Protein a fret based probe for sensing double stranded fluorophore tagged small rnas biophysical chemistry 143 2009 166 169
Biophysical Chemistry, 2009Co-Authors: Roger Koukiekolo, Selena M. Sagan, Jenny Cheng, Zygmunt J. Jakubek, John Paul PezackiAbstract:It has been brought to the author's attention that the above paper containsmissing text in the Abstract and they would like to correct this. The text was inadvertently deleted during the typesetting and editing of the proofs. On page 166, the Abstract should read as follows: Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small Protein, P19, that acts as a suppressor of RNA silencing. This Protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity. Here we demonstrate a new approach for rapidly determining the quantities of siRNA using fluorescence resonance energy transfer (FRET) between the Carnation Italian ringspot virus (CIRV) P19-CFP fusion Protein and Cy3-labeled siRNA. The CIRV P19 fusion Protein binds double-stranded siRNAs with nanomolar affinity as determined by FRET.
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studies of a viral suppressor of rna silencing P19 cfp fusion Protein a fret based probe for sensing double stranded fluorophore tagged small rnas
Biophysical Chemistry, 2009Co-Authors: Roger Koukiekolo, John Paul Pezacki, Jenny Cheng, Selena M. Sagan, Zygmunt J. JakubekAbstract:Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small Protein, P19, that acts as a suppressor of RNA silencing. This Protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity.
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Studies of the interaction of the viral suppressor of RNA silencing Protein P19 with small RNAs using fluorescence polarization.
Biochemistry, 2008Co-Authors: Jenny Cheng, Selena M. Sagan, Zygmunt J. Jakubek, John Paul PezackiAbstract:Tombusviruses use a 19 kDa Protein (P19) as a suppressor of the RNA silencing pathway during infection. The P19 Protein binds to short-interfering RNA (siRNA) as a dimer and shows a high selectivity for short duplex RNAs over other RNA species. Since P19 can bind to synthetic and RNA silencing generated small RNAs with little sequence dependence and with size selectivity, this Protein has utility as a tool for studying RNA silencing pathways in eukaryotes. However, the ability of p 19 to serve as a tool for studying RNA silencing pathways may be complicated by the presence of other endogenous small RNAs such as micro-RNAs (miRNAs). To understand the importance of endogenous small RNA components with respect to pl9's ability to bind to siRNAs, we examined the interactions of P19 with human miR-122, a 23-nucleotide duplex miRNA containing several mismatched base pairs that is highly abundant in the liver. The binding characteristics were compared with those of an siRNA optimized against the human kinase CSK. The binding studies were performed using fluorescence polarization experiments on duplex oligonucleotides containing Cy3 dye labels at the 5'-end of one of the strands of RNA as well as electrophoretic gel mobility shift assays. Both methods indicate that the synthetic siRNA with no mismatches in base pairing bound with >3-fold selectivity over that of miR-122. Our results suggest that pl9 can distinguish between siRNAs and miRNA species, although the difference in binding constants is not so large that interactions with endogenous miRNAs can be totally ignored.
