The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
Dirk Grimm - One of the best experts on this subject based on the ideXlab platform.
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Blocking Sense-Strand activity improves potency, safety and specificity of anti-hepatitis B virus short hairpin RNA.
EMBO molecular medicine, 2016Co-Authors: Thomas Michler, Stefan Mockenhaupt, Stefanie Große, Natalie Röder, Ferdinand Stückler, Bettina Knapp, Mathias Heikenwalder, Ulrike Protzer, Dirk GrimmAbstract:Abstract Hepatitis B virus (HBV) is a promising target for therapies based on RNA interference (RNAi) since it replicates via RNA transcripts that are vulnerable to RNAi silencing. Clinical translation of RNAi technology, however, requires improvements in potency, specificity and safety. To this end, we systematically compared different strategies to express anti‐HBV short hairpin RNA (shRNA) in a pre‐clinical immunocompetent hepatitis B mouse model. Using recombinant Adeno‐associated virus (AAV) 8 vectors for delivery, we either (i) embedded the shRNA in an artificial mi(cro)RNA under a liver‐specific promoter; (ii) co‐expressed Argonaute‐2, a rate‐limiting cellular factor whose saturation with excess RNAi triggers can be toxic; or (iii) co‐delivered a decoy (“TuD”) directed against the shRNA Sense Strand to curb off‐target gene regulation. Remarkably, all three strategies minimised adverse side effects as compared to a conventional shRNA vector that caused weight loss, liver damage and dysregulation of > 100 hepatic genes. Importantly, the novel AAV8 vector co‐expressing anti‐HBV shRNA and TuD outperformed all other strategies regarding efficiency and persistence of HBV knock‐down, thus showing substantial promise for clinical translation.
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Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Stefan Mockenhaupt, Stefanie Große, Daniel Rupp, Ralf Bartenschlager, Dirk GrimmAbstract:Exogenous RNAi triggers such as shRNAs ideally exert their activities exclusively via the antiSense Strand that binds and silences designated target mRNAs. However, in principle, the Sense Strand also possesses silencing capacity that may contribute to adverse RNAi side effects including off-target gene regulation. Here, we address this concern with a novel strategy that reduces Sense Strand activity of vector-encoded shRNAs via codelivery of inhibitory tough decoy (TuD) RNAs. Using various shRNAs for proof of concept, we validate that coexpression of TuDs can sequester and inactivate shRNA Sense Strands in human cells selectively without affecting desired antiSense activities from the same shRNAs. Moreover, we show how coexpressed TuDs can alleviate shRNA-mediated perturbation of global gene expression by specifically de-repressing off-target transcripts carrying seed matches to the shRNA Sense Strand. Our combination of shRNA and TuD in a single bicistronic gene transfer vector derived from Adeno-associated virus (AAV) enables a wide range of applications, including gene therapies. To this end, we engineered our constructs in a modular fashion and identified simple hairpin design rules permitting adaptation to preexisting or new shRNAs. Finally, we demonstrate the power of our vectors for combinatorial RNAi strategies by showing robust suppression of hepatitis C virus (HCV) with an AAV expressing a bifunctional TuD against an anti-HCV shRNA Sense Strand and an HCV-related cellular miRNA. The data and tools reported here represent an important step toward the next generation of RNAi triggers with increased specificity and thus ultimately safety in humans.
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699. shRNA Sense Strand Neutralization Reduces Off-Targeting, Ameliorates Toxicity and Enhances Efficacy of RNAi-Based HBV Gene Therapy
Molecular Therapy, 2015Co-Authors: Thomas Michler, Stefan Mockenhaupt, Stefanie Große, Ulrike Protzer, Dirk GrimmAbstract:A decade after first reports of successful inhibition of hepatitis B virus (HBV) replication in vivo using shRNAs, only a single RNAi therapeutic is under phase II efficacy investigation in Hepatitis B patients. This slow clinical translation is puzzling, as current standard-of-care requires life-long and daily drug treatment which is often accompanied by side effects and occurrence of resistant HBV mutants. Also, while preventing the generation of infectious HBV virions, reverse-transcriptase inhibitors fail to suppress HBV surface and e-antigen, which enable viral persistence by modulating the host immune system and contribute to HBV-associated hepatocellular carcinoma. These problems may be solved using RNAi as anti-HBV therapeutic since (i) long-term suppression can be achieved by a single dose of an shRNA-encoding vector, and (ii) all four HBV transcripts share a common 3 end, allowing concurrent inhibition of HBV pre-genomic RNA and all viral proteins with a single RNAi molecule, and potentially restoring antiviral immunity and preventing carcinogenesis. Still, clinical translation remains hampered by safety concerns as shRNA over-expression in the mouse liver causes elevated liver transaminases, jaundice and weight loss. One explanation is that ectopic RNAi triggers overload the endogenous miRNA pathway, perturbing miRNA biogenesis and/or activity, and causing cytotoxicity. Evidence for this saturation model is that (i) shRNAs circumvent Drosha processing, a gatekeeper for miRNA biogenesis, (ii) embedding shRNAs in a recombinant miRNA context yields lower and safer levels of mature RNAi triggers, and (iii) overexpression of Argonaute-2 (Ago2) ameliorates toxicity and enhances RNAi efficacy. Moreover, RNAi triggers can perturb cell physiology through unwanted inhibition of off-target genes with partial complementarities to one of the two Strands of the RNAi molecule. To alleviate such unintended gene silencing, we have developed a novel bi-cistronic AAV vector that expresses, in addition to the shRNA, a second RNA hairpin called tough decoy or TuD. In cell culture, the TuD effectively sequestered and inactivated shRNA Sense Strands, and thereby improved RNAi specificity. These remarkable features translated well into an HBV-transgenic mouse model, where Sense Strand off-target activity was likewise prevented, as validated by transcriptome analysis of liver RNA, coinciding with ameliorated toxicity. Enhanced in vivo safety was also noted for two alternative AAV vectors, either co-expressing the shRNA and Ago2, or embedding the shRNA in a miR-122 backbone. Notably, the new shRNA/TuD vector outperformed the other expression strategies regarding efficacy with stable HBV reduction of up to 98% over 3 months. We speculate that its enhanced antiviral efficacy results from increased loading of the desired antiSense Strand into RISC in absence of the Sense Strand, possibly also further amending toxicity by attenuating RISC saturation. The simple TuD design and the versatility of our new AAV vector pave the way for adaptation of our strategy to other applications and should facilitate clinical translation.
Jean-paul Desaulniers - One of the best experts on this subject based on the ideXlab platform.
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Stability and evaluation of siRNAs labeled at the Sense Strand with a 3'-azobenzene unit.
Bioorganic & medicinal chemistry letters, 2018Co-Authors: Matthew L. Hammill, Ayushi Patel, Maria Abd Alla, Jean-paul DesaulniersAbstract:Abstract siRNAs bearing a 3′-azobenzene derivative on the Sense Strand were evaluated for their gene silencing ability in mammalian cell culture and nuclease stability in nuclease-rich media. Azobenzene can be isomerized between cis and trans isomers through the incubation of UV (cis isomer) and visible light (trans isomer). It was demonstrated that subtle differences in nuclease stability and activity were observed. These small changes can be used to photochemically fine-tune the activity of an siRNA for gene-silencing applications.
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Effective gene-silencing of siRNAs that contain functionalized spacer linkages within the central region
RSC Advances, 2017Co-Authors: Jean-paul Desaulniers, Gordon Hagen, Jocelyn Anderson, Chris Mckim, Blake RobertsAbstract:Short-interfering RNAs containing a variety of functional groups at the central region of the Sense Strand were synthesized and evaluated. The gene-silencing data suggest that these siRNAs are biocompatible and are very effective in cell-based assays.
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Evaluation of siRNAs that contain internal variable-length spacer linkages.
Bioorganic & medicinal chemistry letters, 2012Co-Authors: Tim C. Efthymiou, Brandon J. Peel, Vanthi Huynh, Jean-paul DesaulniersAbstract:Abstract The most widely accepted mechanism of RNAi-silencing involves the RNA-induced silencing complex (RISC) liberating the active antiSense Strand from the Sense Strand of an siRNA duplex to form an active RISC-antiSense complex. This involves cleaving the Sense Strand between positions 9 and 10 from the 5′ end of the Strand prior to dissociation. Destabilizing modifications near the center of the duplex in some cases can enhance the efficacy of the resultant construct and may trigger an alternative mechanism through which the Sense Strand is removed. By introducing alkyl spacers of varying lengths near or within the Sense Strand’s cleavage site, this study illustrates that siRNAs, in most cases, retained potent RNAi-silencing activity. Our results highlight that by substituting the scissile phosphodiester linkage on the Sense Strand with non-cleavable alkyl chains provides a novel and alternative method to destabilize the central region of siRNAs.
Zhenjun Yang - One of the best experts on this subject based on the ideXlab platform.
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Isonucleotide incorporation into middle and terminal siRNA duplexes exhibits high gene silencing efficacy and nuclease resistance.
Organic & biomolecular chemistry, 2017Co-Authors: Shuang Liu, Ye Huang, Lihe Zhang, Lijun Zhong, Yusi Wang, Yuanhe Zhao, Zhu Guan, Zhenjun YangAbstract:In this study, we introduced a pair of nucleotide enantiomers, D-/L-isonucleotides (D-/L-isoNA), to examine the interactions between siRNAs and their related proteins. The serum stability and gene-silencing activity of the modified siRNAs were systematically evaluated. Gene-silencing activity had a site-specific effect, and the incorporation of a single D-isoNA at the 8th position (counting from the 5′-terminus) in the antiSense Strand improved the gene-silencing activity by improving RISC loading and affecting the movement of the PIWI domain. D-isoNA incorporated at the terminus of siRNA including the 2nd position in the antiSense Strand and 3′-overhangs in the Sense Strand, especially the latter, enhanced nuclease resistance and prolonged the silencing retention time. In addition, L-isoNA incorporation into the middle of the Sense Strand enhanced activity. These results provide a chemical strategy for the modulation of siRNA gene-silencing activity and nuclease resistance.
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Exploring directional invasion of serum nuclease into siRNA duplexes by asymmetrical terminal modifications.
ChemMedChem, 2014Co-Authors: Ye Huang, Lihe Zhang, Yujia Guo, Lang Zou, Hongwei Jin, Lijun Zhong, Zhenjun YangAbstract:In this study we demonstrated that ribonuclease A (RNase A) can recognize the thermodynamic asymmetry of siRNA duplexes, similar to other proteins involved in siRNA function such as argonaute 2. RNase A preferentially invades the siRNA duplex through the less stable terminus, i.e., the 3' terminus of the Sense Strand. As evidence, only phosphorothioate (PS) modification at the Sense Strand overhang improved serum stability, whereas PS modification at the antiSense Strand overhang did not affect stability. Moreover, the improvement in stability caused by modification at the Sense Strand overhang was found to correlate with the terminal base pair composition of the siRNA. Gel electrophoresis and MALDI-TOF MS analysis indicated that modifications at the Sense Strand overhang improved the serum stability of siRNAs by inhibiting the directional invasion of RNase A. The blocking effect was not brought about by stabilization of the siRNA duplexes because there was no clear difference between the melting temperatures of siRNAs with PS modifications at each 3' overhang.
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loss of silencing activity caused by 5 terminal modification with d l isonucleotide isona or locked nucleic acid lna could not be restored by 5 terminal phosphorylation
Science China-chemistry, 2014Co-Authors: Ye Huang, Zhuo Chen, Zhuo Wang, Yue Chen, Zhenjun Yang, Lihe ZhangAbstract:In this study, a series of 5′-phosphorylated siRNAs with d-/l-isonucleotide (isoNA) or locked nucleic acid (LNA) incorporated at the 5′-terminus were synthesized. It was found that after incorporating either isoNA or LNA at the 5′-terminus of the antiSense Strand or Sense Strand, the silencing activity of modified Strands has been inhibited, which cannot be recovered by phosphorylation at the 5′-terminus; however, the silencing activity of unmodified Strand to its own target was increased. This work indicates that the isoNA and LNA modification at 5′-terminus can interfere with the Strand selection during the RISC assembling process, and the disturbance of the 5′-phosporylation should not be the only viable mechanism.
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Synthesis and biological evaluation of peptide-siRNA conjugates with phosphodiester unit as linker
Science China Chemistry, 2013Co-Authors: Xiao-feng Wang, Ye Huang, Yue Chen, Zhenjun Yang, Hongwei Jin, Yang Liu, Yi Zheng, Lihe ZhangAbstract:In this paper, a series of peptide-siRNA conjugates with phosphodiester unit as the linker targeting to Cdc2 gene were synthesized by solid phase stepwise strategy. The conjugation of peptides at either 3′-terminus of siCdc2 bring no change to the classical A-form of RNA duplex, but slightly compromise the thermodynamic stability. Peptide conjugation at the 3′-terminus of Sense Strand could improve the serum stability obviously, however, the opposite peptide conjugation at the 3′-terminus of antiSense Strand shows no such influence. According to the results of artificial silencing activity assay system, peptide conjugation at 3′-terminus of antiSense Strand slightly weakens the silencing activity of siCdc2. But Sense Strand peptide conjugation exhibits similar silencing activity as native siCdc2, meanwhile, it could mitigate the unwanted off-target effect of Sense Strand targeting to its own mRNA.
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Effects of conformational alteration induced by D-/L-isonucleoside incorporation in siRNA on their stability in serum and silencing activity.
Bioconjugate chemistry, 2013Co-Authors: Ye Huang, Zhuo Chen, Yue Chen, Zhenjun Yang, Hao Zhang, Yichao Zhang, Yi-lei Zhao, Lihe ZhangAbstract:We report here that all of the d- or l-isonucleoside (isoNA) modified siRNAs investigated showed the characteristic A-form conformation in the circular dichroism (CD) spectra compared to native siRNA. The d-isoNA modification had less influence on the thermal stability of siRNAs, but all l-isoNA modification displayed a significant tendency to decrease the thermal stability of siRNA. It was also found that the stabilities of d-/l-isoNA modified siMek1 in serum were different and d-isoNA modification was more potent, i.e., increase of serum stability of siRNA, than l-isoNA modification. When d-isoNA incorporated at position 4 and position 5 at antiSense Strand of siMek1 showed obvious improvement on serum stability, however, l-isoNA incorporated at positions 11 and 12 at antiSense Strand and position 9 at Sense Strand made the siMek1 duplex formed very unstable in serum. The silencing activities of modified siMek1s with d-/l-isoNA at position 1 of antiSense Strand also dropped dramatically; however, the modification at 3'-terminal of the Sense Strand with d- or l-isoNA significantly enhanced the silencing activity targeting the antiSense Strand as reporter and minimized the passenger Strand-specific off-target effect. IsoNA modified in the seed area of siMek1, siMek1 A04D and siMek1 A05L, showed similar activity to the native one and better target selectivity. In the case of modification at the position near the cleavage area, it was found that d- or l-isoNA modified Sense Strand at position 8, 9, or 15 of siMek1 could retain the silencing activities targeting the antiSense Strand as reporter. Especially, both siMek1 S15D and siMek1 S15L showed good silencing activity and high target selectivity compared to native siMek1. The effects of conformational alteration of such isoNA modification of siRNA on their stability in serum and silencing activity are discussed based on computer simulation. Systematic investigation of the relationship between modified siRNA conformation and their physical and biological properties should provide a useful guideline for chemical modification and optimization of siRNA for further clinical application.
Stefan Mockenhaupt - One of the best experts on this subject based on the ideXlab platform.
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Blocking Sense-Strand activity improves potency, safety and specificity of anti-hepatitis B virus short hairpin RNA.
EMBO molecular medicine, 2016Co-Authors: Thomas Michler, Stefan Mockenhaupt, Stefanie Große, Natalie Röder, Ferdinand Stückler, Bettina Knapp, Mathias Heikenwalder, Ulrike Protzer, Dirk GrimmAbstract:Abstract Hepatitis B virus (HBV) is a promising target for therapies based on RNA interference (RNAi) since it replicates via RNA transcripts that are vulnerable to RNAi silencing. Clinical translation of RNAi technology, however, requires improvements in potency, specificity and safety. To this end, we systematically compared different strategies to express anti‐HBV short hairpin RNA (shRNA) in a pre‐clinical immunocompetent hepatitis B mouse model. Using recombinant Adeno‐associated virus (AAV) 8 vectors for delivery, we either (i) embedded the shRNA in an artificial mi(cro)RNA under a liver‐specific promoter; (ii) co‐expressed Argonaute‐2, a rate‐limiting cellular factor whose saturation with excess RNAi triggers can be toxic; or (iii) co‐delivered a decoy (“TuD”) directed against the shRNA Sense Strand to curb off‐target gene regulation. Remarkably, all three strategies minimised adverse side effects as compared to a conventional shRNA vector that caused weight loss, liver damage and dysregulation of > 100 hepatic genes. Importantly, the novel AAV8 vector co‐expressing anti‐HBV shRNA and TuD outperformed all other strategies regarding efficiency and persistence of HBV knock‐down, thus showing substantial promise for clinical translation.
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Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Stefan Mockenhaupt, Stefanie Große, Daniel Rupp, Ralf Bartenschlager, Dirk GrimmAbstract:Exogenous RNAi triggers such as shRNAs ideally exert their activities exclusively via the antiSense Strand that binds and silences designated target mRNAs. However, in principle, the Sense Strand also possesses silencing capacity that may contribute to adverse RNAi side effects including off-target gene regulation. Here, we address this concern with a novel strategy that reduces Sense Strand activity of vector-encoded shRNAs via codelivery of inhibitory tough decoy (TuD) RNAs. Using various shRNAs for proof of concept, we validate that coexpression of TuDs can sequester and inactivate shRNA Sense Strands in human cells selectively without affecting desired antiSense activities from the same shRNAs. Moreover, we show how coexpressed TuDs can alleviate shRNA-mediated perturbation of global gene expression by specifically de-repressing off-target transcripts carrying seed matches to the shRNA Sense Strand. Our combination of shRNA and TuD in a single bicistronic gene transfer vector derived from Adeno-associated virus (AAV) enables a wide range of applications, including gene therapies. To this end, we engineered our constructs in a modular fashion and identified simple hairpin design rules permitting adaptation to preexisting or new shRNAs. Finally, we demonstrate the power of our vectors for combinatorial RNAi strategies by showing robust suppression of hepatitis C virus (HCV) with an AAV expressing a bifunctional TuD against an anti-HCV shRNA Sense Strand and an HCV-related cellular miRNA. The data and tools reported here represent an important step toward the next generation of RNAi triggers with increased specificity and thus ultimately safety in humans.
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699. shRNA Sense Strand Neutralization Reduces Off-Targeting, Ameliorates Toxicity and Enhances Efficacy of RNAi-Based HBV Gene Therapy
Molecular Therapy, 2015Co-Authors: Thomas Michler, Stefan Mockenhaupt, Stefanie Große, Ulrike Protzer, Dirk GrimmAbstract:A decade after first reports of successful inhibition of hepatitis B virus (HBV) replication in vivo using shRNAs, only a single RNAi therapeutic is under phase II efficacy investigation in Hepatitis B patients. This slow clinical translation is puzzling, as current standard-of-care requires life-long and daily drug treatment which is often accompanied by side effects and occurrence of resistant HBV mutants. Also, while preventing the generation of infectious HBV virions, reverse-transcriptase inhibitors fail to suppress HBV surface and e-antigen, which enable viral persistence by modulating the host immune system and contribute to HBV-associated hepatocellular carcinoma. These problems may be solved using RNAi as anti-HBV therapeutic since (i) long-term suppression can be achieved by a single dose of an shRNA-encoding vector, and (ii) all four HBV transcripts share a common 3 end, allowing concurrent inhibition of HBV pre-genomic RNA and all viral proteins with a single RNAi molecule, and potentially restoring antiviral immunity and preventing carcinogenesis. Still, clinical translation remains hampered by safety concerns as shRNA over-expression in the mouse liver causes elevated liver transaminases, jaundice and weight loss. One explanation is that ectopic RNAi triggers overload the endogenous miRNA pathway, perturbing miRNA biogenesis and/or activity, and causing cytotoxicity. Evidence for this saturation model is that (i) shRNAs circumvent Drosha processing, a gatekeeper for miRNA biogenesis, (ii) embedding shRNAs in a recombinant miRNA context yields lower and safer levels of mature RNAi triggers, and (iii) overexpression of Argonaute-2 (Ago2) ameliorates toxicity and enhances RNAi efficacy. Moreover, RNAi triggers can perturb cell physiology through unwanted inhibition of off-target genes with partial complementarities to one of the two Strands of the RNAi molecule. To alleviate such unintended gene silencing, we have developed a novel bi-cistronic AAV vector that expresses, in addition to the shRNA, a second RNA hairpin called tough decoy or TuD. In cell culture, the TuD effectively sequestered and inactivated shRNA Sense Strands, and thereby improved RNAi specificity. These remarkable features translated well into an HBV-transgenic mouse model, where Sense Strand off-target activity was likewise prevented, as validated by transcriptome analysis of liver RNA, coinciding with ameliorated toxicity. Enhanced in vivo safety was also noted for two alternative AAV vectors, either co-expressing the shRNA and Ago2, or embedding the shRNA in a miR-122 backbone. Notably, the new shRNA/TuD vector outperformed the other expression strategies regarding efficacy with stable HBV reduction of up to 98% over 3 months. We speculate that its enhanced antiviral efficacy results from increased loading of the desired antiSense Strand into RISC in absence of the Sense Strand, possibly also further amending toxicity by attenuating RISC saturation. The simple TuD design and the versatility of our new AAV vector pave the way for adaptation of our strategy to other applications and should facilitate clinical translation.
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Alleviation of shRNA off-target effects via rAAV vector-encoded Sense Strand decoys
2012Co-Authors: Stefan MockenhauptAbstract:The use of exogenous triggers of RNAi such as short hairpin RNAs (shRNAs) in combination with viral vector-mediated gene delivery holds great potential for applications in gene therapy, in biotechnological processes as well as in basic research. Ideally, shRNAs only exert their activity via the antiSense Strand that binds and silences the designated target mRNA. However, the undesired Sense Strand can also be activated and hence holds a certain silencing potential. In principle, this may contribute to erratic off-targeting which typically occurs via imperfect binding and subsequent inhibition of untargeted cellular mRNAs. The aim of this study was to assess whether shRNA Sense Strand activity plays an important role in producing off-target effects and –if so- to find novel means of counteracting this activity. Functional characterization of relative Strand activities of different shRNAs indeed revealed high levels of undesired Sense Strands activity for most RNAi triggers. This effect was found both upon plasmid transfection and rAAV2-mediated transduction which represents a therapeutically relevant system for gene delivery. We therefore devised a novel strategy for Sense Strand counteraction in which an shRNA is co-expressed with an inhibitor RNA transcript designed to stably sequester and inactivate the Sense but not the antiSense Strand. Proof-of-concept for our approach was obtained in transfected human cells with an shRNA against hepatitis C virus (HCV). By using RNA-polymerase III-transcribed inhibitors known as tough decoys (TuDs) we could efficiently and specifically counteract the Sense Strand of the HCV-shRNA in luciferase and eGFP-based reporter assays. We then tested the shRNA-TuD combination in the context of a single self-complementary rAAV2 vector. TuD co-expression led to impairment of Sense Strand activity upon transduction of HEK293T and Huh7 cells. Inhibition of HCV replication in Huh7 cells was not altered indicating that the desired antiSense Strand activity was unaffected. Our strategy is hence compatible with rAAV-mediated gene delivery. Furthermore, expression profiling in Huh7 cells revealed that TuD co-expression specifically de-repressed endogenous off-target transcripts that carried seed matches to the shRNA Sense Strand leading to lower levels of perturbation of global gene expression. As expected, repression of transcripts carrying antiSense Strand seed matches remained unaffected. These results show that shRNA Sense Strands can indeed contribute to off-targeting and that TuD-mediated inhibition can be used to counteract this effect. Besides our functional data, we also defined rules for TuD and shRNA design as well as promoter choice which allows implementation of the system for other shRNAs. In this study, we provide new insights into the functionalities and relative activities of both Strands of shRNAs. We furthermore present TuD-mediated selective counteraction of shRNA Sense Strands as a novel method to improve the functional Strand bias and thus increase shRNA specificity. We are optimistic that our strategy will facilitate and further foster the clinical implementation of vector-based RNAi.
Lihe Zhang - One of the best experts on this subject based on the ideXlab platform.
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Isonucleotide incorporation into middle and terminal siRNA duplexes exhibits high gene silencing efficacy and nuclease resistance.
Organic & biomolecular chemistry, 2017Co-Authors: Shuang Liu, Ye Huang, Lihe Zhang, Lijun Zhong, Yusi Wang, Yuanhe Zhao, Zhu Guan, Zhenjun YangAbstract:In this study, we introduced a pair of nucleotide enantiomers, D-/L-isonucleotides (D-/L-isoNA), to examine the interactions between siRNAs and their related proteins. The serum stability and gene-silencing activity of the modified siRNAs were systematically evaluated. Gene-silencing activity had a site-specific effect, and the incorporation of a single D-isoNA at the 8th position (counting from the 5′-terminus) in the antiSense Strand improved the gene-silencing activity by improving RISC loading and affecting the movement of the PIWI domain. D-isoNA incorporated at the terminus of siRNA including the 2nd position in the antiSense Strand and 3′-overhangs in the Sense Strand, especially the latter, enhanced nuclease resistance and prolonged the silencing retention time. In addition, L-isoNA incorporation into the middle of the Sense Strand enhanced activity. These results provide a chemical strategy for the modulation of siRNA gene-silencing activity and nuclease resistance.
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Exploring directional invasion of serum nuclease into siRNA duplexes by asymmetrical terminal modifications.
ChemMedChem, 2014Co-Authors: Ye Huang, Lihe Zhang, Yujia Guo, Lang Zou, Hongwei Jin, Lijun Zhong, Zhenjun YangAbstract:In this study we demonstrated that ribonuclease A (RNase A) can recognize the thermodynamic asymmetry of siRNA duplexes, similar to other proteins involved in siRNA function such as argonaute 2. RNase A preferentially invades the siRNA duplex through the less stable terminus, i.e., the 3' terminus of the Sense Strand. As evidence, only phosphorothioate (PS) modification at the Sense Strand overhang improved serum stability, whereas PS modification at the antiSense Strand overhang did not affect stability. Moreover, the improvement in stability caused by modification at the Sense Strand overhang was found to correlate with the terminal base pair composition of the siRNA. Gel electrophoresis and MALDI-TOF MS analysis indicated that modifications at the Sense Strand overhang improved the serum stability of siRNAs by inhibiting the directional invasion of RNase A. The blocking effect was not brought about by stabilization of the siRNA duplexes because there was no clear difference between the melting temperatures of siRNAs with PS modifications at each 3' overhang.
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loss of silencing activity caused by 5 terminal modification with d l isonucleotide isona or locked nucleic acid lna could not be restored by 5 terminal phosphorylation
Science China-chemistry, 2014Co-Authors: Ye Huang, Zhuo Chen, Zhuo Wang, Yue Chen, Zhenjun Yang, Lihe ZhangAbstract:In this study, a series of 5′-phosphorylated siRNAs with d-/l-isonucleotide (isoNA) or locked nucleic acid (LNA) incorporated at the 5′-terminus were synthesized. It was found that after incorporating either isoNA or LNA at the 5′-terminus of the antiSense Strand or Sense Strand, the silencing activity of modified Strands has been inhibited, which cannot be recovered by phosphorylation at the 5′-terminus; however, the silencing activity of unmodified Strand to its own target was increased. This work indicates that the isoNA and LNA modification at 5′-terminus can interfere with the Strand selection during the RISC assembling process, and the disturbance of the 5′-phosporylation should not be the only viable mechanism.
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Synthesis and biological evaluation of peptide-siRNA conjugates with phosphodiester unit as linker
Science China Chemistry, 2013Co-Authors: Xiao-feng Wang, Ye Huang, Yue Chen, Zhenjun Yang, Hongwei Jin, Yang Liu, Yi Zheng, Lihe ZhangAbstract:In this paper, a series of peptide-siRNA conjugates with phosphodiester unit as the linker targeting to Cdc2 gene were synthesized by solid phase stepwise strategy. The conjugation of peptides at either 3′-terminus of siCdc2 bring no change to the classical A-form of RNA duplex, but slightly compromise the thermodynamic stability. Peptide conjugation at the 3′-terminus of Sense Strand could improve the serum stability obviously, however, the opposite peptide conjugation at the 3′-terminus of antiSense Strand shows no such influence. According to the results of artificial silencing activity assay system, peptide conjugation at 3′-terminus of antiSense Strand slightly weakens the silencing activity of siCdc2. But Sense Strand peptide conjugation exhibits similar silencing activity as native siCdc2, meanwhile, it could mitigate the unwanted off-target effect of Sense Strand targeting to its own mRNA.
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Effects of conformational alteration induced by D-/L-isonucleoside incorporation in siRNA on their stability in serum and silencing activity.
Bioconjugate chemistry, 2013Co-Authors: Ye Huang, Zhuo Chen, Yue Chen, Zhenjun Yang, Hao Zhang, Yichao Zhang, Yi-lei Zhao, Lihe ZhangAbstract:We report here that all of the d- or l-isonucleoside (isoNA) modified siRNAs investigated showed the characteristic A-form conformation in the circular dichroism (CD) spectra compared to native siRNA. The d-isoNA modification had less influence on the thermal stability of siRNAs, but all l-isoNA modification displayed a significant tendency to decrease the thermal stability of siRNA. It was also found that the stabilities of d-/l-isoNA modified siMek1 in serum were different and d-isoNA modification was more potent, i.e., increase of serum stability of siRNA, than l-isoNA modification. When d-isoNA incorporated at position 4 and position 5 at antiSense Strand of siMek1 showed obvious improvement on serum stability, however, l-isoNA incorporated at positions 11 and 12 at antiSense Strand and position 9 at Sense Strand made the siMek1 duplex formed very unstable in serum. The silencing activities of modified siMek1s with d-/l-isoNA at position 1 of antiSense Strand also dropped dramatically; however, the modification at 3'-terminal of the Sense Strand with d- or l-isoNA significantly enhanced the silencing activity targeting the antiSense Strand as reporter and minimized the passenger Strand-specific off-target effect. IsoNA modified in the seed area of siMek1, siMek1 A04D and siMek1 A05L, showed similar activity to the native one and better target selectivity. In the case of modification at the position near the cleavage area, it was found that d- or l-isoNA modified Sense Strand at position 8, 9, or 15 of siMek1 could retain the silencing activities targeting the antiSense Strand as reporter. Especially, both siMek1 S15D and siMek1 S15L showed good silencing activity and high target selectivity compared to native siMek1. The effects of conformational alteration of such isoNA modification of siRNA on their stability in serum and silencing activity are discussed based on computer simulation. Systematic investigation of the relationship between modified siRNA conformation and their physical and biological properties should provide a useful guideline for chemical modification and optimization of siRNA for further clinical application.
