Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in th...Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in the genome to generate doublestrand breaks（DSBs）in vivo is a prerequisite for any genome editing efforts.展开更多
Objective: To study whether 2 hepatitis delta virus genomic ribozymes(g. Rz) have trans-cleaving activity. Methods: g. Rz74 and g. Rz55 were transcribed from their templates which were synthesized and cloned into plas...Objective: To study whether 2 hepatitis delta virus genomic ribozymes(g. Rz) have trans-cleaving activity. Methods: g. Rz74 and g. Rz55 were transcribed from their templates which were synthesized and cloned into plasmid pGEM-4Z; Homologous substrate RNA was transcribed for pRz277B and labelled withα-32 P-UTP. The trans-cleaving activity was studied by mixing the ribozymes and substrate in appropriate who under certain conditions. Results: The trans-cleaved products were generated after mixing the ribozyme with the substrate for 30 min, and accumulated more for 90 min. Conclusion: Both g. Rz74 and g. Rz55 possess trans-cleaving activity.展开更多
To reverse multidrug resistance（MDR） of HepG2 by anti-MDR1 hammerhead ribozyme, an anti-MDR1 hammerhead ribozyme was developed and delivered to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retrovira...To reverse multidrug resistance（MDR） of HepG2 by anti-MDR1 hammerhead ribozyme, an anti-MDR1 hammerhead ribozyme was developed and delivered to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase Ⅲ promoter. The expression of mdrl/Pgp and Rz was detected in HepG2, HepG2 muhidrug-resistant cell line and HepG2 Rz-transfected cells by semi-quantitative RT-PCR and Western blot methods. Moreover, MTT assay was employed to detect the sensitivity of these ribozyme-transfected cells, and Rhodamine123 （Rh123） was used to test the function of Pgp. The Rz- transfected HepG2 cells became doxorubicin-sensitive, which was concomitant with the decreased MDR1 expression. The study showed that the retrovirus vector encoding the anti-MDR1 ribozyme may be applicable to the treatment of MDR cells.展开更多
Objective: To investigate the in vitro cleavage ability and effects on apoptosis and cell growth of the bcr-abl fusion gene specific multi-unit ribozymes. Methods: Three fusion point specific ribozymes were designed ...Objective: To investigate the in vitro cleavage ability and effects on apoptosis and cell growth of the bcr-abl fusion gene specific multi-unit ribozymes. Methods: Three fusion point specific ribozymes were designed and the multi-unit ribozymes?in vitro transcription vector and retroviral vector were constructed. The in vitro cleavage ability was tested. The retroviral vector was transfected into K562 cell and the effects on proliferation, apoptosis, cell cycle and cell structure were observed. Results: Multi-unit ribozymes had in vitro cleavage efficiency of 70.8%, which was more efficient than single-unit and double-unit ribozymes. Transfection of the retroviral vector of the ribozyme into K562 cells, induced inhibition of cell growth and apoptosis. The incorporation rate of DNA in ribozymes transfected K562 cells was greatly decreased along with time passed, with an inhibition rate of more than 50% after 96 h of transfection. Under FCM, 18.4% of the cells underwent apoptosis 72 h after transfection and more cells were blocked in G phase, with the ratio in S phase greatly decreased (41.9%). Under electron microscope, compaction of nuclear chromatin and apoptosis bodies were observed. Conclusion: Multi-unit ribozymes specific to bcr-abl fusion gene can be used to treat CML and to purge bone marrow for self-grafting.展开更多
Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hep...Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hepatitis delta virus(HDV) ribozyme was chosen and used to demonstrate the effective cleavage in vitro and in vivo. Methods Three hammerhead ribozymes for potential target sites(S, X and C genes) and co-expression plasmid(pTr-dB, pTdδ-dB, pTrX-dB and pTrC-dB) as well as four HDV-ribozyme chimera constructs with HBV(pTdXX, pTdXC, pTdSX and pTdSC) were severally chosen to validate the inhibition of the replication and expression of HBV. The co-expression plasmids(pTdδ and pTr-Db) in physiological saline were hydrodynamically injected to mice by tail vein. Results Compared with the group injected with pTr-dB in Huh-7 cell, hepatitis B surface antigen(HBsAg) was reduced by 31% in the group injected with pTdδ-dB, by 54%, 26%, 72% and 97% in the group injected with recombinant-ribozymes pTdSX, pTdSC, pTdXC and pTdXX, respectively. The inhibiting effects of endogenous ribozymes RzX and RzC on the HBsAg expression were 66% and 57%, respectively. Compared with the positive control, the amount of HBsAg was decreased in mice injected with pTdXX through tail vein by 88% and 96% on the second day and the third day, respectively. HBsAg was undetectable on the 6th day and could not primitively be detected on the 9th day in the sera from all mice. HBV DNA was not detected in the sera of BALB/c mice injected with pTdXX-dB, pTrX-dB or replicating-defective plasmid pHBV, while HBV DNA replication in control group could be detected on the 6th day. While HBcAg could not be detected in liver tissues of mice injected with plasmid pTdXX-dB on the 3rd day. Conclusions Encoding regions of HBV S, C and X gene were the effective cleavage sites for hammerhead ribozyme in vitro and in vivo, which provides basis for further construction of therapeutic recombinant HDV and the development of targeting antiviral gene therapy.展开更多
The discovery that RNA can act as an enzyme led Thomas Cech to win the Nobel Prize in Chemistry and led immediately to the next wave of attempts to find an effective RNA-based therapy.The tantalizing idea that RNA enz...The discovery that RNA can act as an enzyme led Thomas Cech to win the Nobel Prize in Chemistry and led immediately to the next wave of attempts to find an effective RNA-based therapy.The tantalizing idea that RNA enzymes called trans-cleaving ribozymes enables them to act as potential antiviral and powerful tool for functional genomic studies.The efficacy of ribozyme function in a complex intracellular environment is dependent on the intracellular fate of the RNA that is being targeted.Recently,ribozymes have been used successfully to inhibit gene expression in a variety of biological systems in vitro and in vivo.Ribozyme has also been used successfully to combat many cases of viral infection,as clinical trial.Despite it needs to be investigated and explored as far as its structural and functional aspects are concern.In view of the significance of ribozyme in modern medicine,we reviewed the recent literature on general approach to control viral infection.展开更多
Objective: To compare the cleavage activity of ribozymes directed against 2 sites of PDGF receptorP subunit cDNA gene in vitro. Methods: The 608 bp fragment of PDGF receptor β subunit cDNA was clonedinto T-vector und...Objective: To compare the cleavage activity of ribozymes directed against 2 sites of PDGF receptorP subunit cDNA gene in vitro. Methods: The 608 bp fragment of PDGF receptor β subunit cDNA was clonedinto T-vector under the control of T7 promoter, named pPDGFR-β. Two ribozymes were designed to cleavethe CUU sequence at codon 45 and codon 252 of PDGF receptor β subunit mRNA respectively. These 2 ham-merhead ribozyme genes were cloned into vector PI. 5 between 5' -cis ribozyme and 3' -cis ribozyme to gener-ate the plasmids of pRZ1 and pRZ2. The pPDGFR-β, pRZl and pRZ2 were linearized and then transcribedwith T7 promoter in vitro. Results: The RZ1 showed high cleavage activity in vitro, but the RZ2 showed nocleavage activity under the same condition. Conclusion: The cleavage site selection is an important factor in-fluencing the cleavage activities of ribozymes.展开更多
Synthetic RNA-based switches provide distinctive merits in modulating gene expression.Simple and flexible RNA-based switches are crucial for advancing the field of gene regulation,paving the way for innovative tools t...Synthetic RNA-based switches provide distinctive merits in modulating gene expression.Simple and flexible RNA-based switches are crucial for advancing the field of gene regulation,paving the way for innovative tools that can sense and manipulate cellular processes.In this research,we have developed programmable ribozymes that are capable of suppressing gene expression in response to specific,endogenously expressed trigger RNAs.We engineer ribozymes by introducing upstream antisense sequences(anti-ribozymes)to inhibit the self-cleaving activity of the hammerhead ribozyme and open the expression of the target gene.The trigger RNA is designed to recognize and bind to complementary sequences within the anti-ribozymes,thereby inhibiting their ability to direct protein synthesis.The anti-ribozyme performance is optimized by regulating the essential sequence modules that play a crucial role in determining the specificity and efficiency of the anti-ribozyme’s interaction with its trigger RNA.By applying this switch mechanism to various ribozyme designs,we have shown that it is possible to achieve control over gene expression across a wide range of trigger RNAs.By exploiting these pro-grammable anti-ribozymes,we aim to create a powerful tool for controlling gene expression in mammalian cells,which could have important implications for basic research,disease diagnosis,and therapeutic interventions.展开更多
Based upon computer-assisted predictions on the secondary structures of tobacco mosaicvirus (TMV) genomic RNA (both polarities), hammerhead type ribozymes were synthesizedin vitro, which all shared a conserved domain ...Based upon computer-assisted predictions on the secondary structures of tobacco mosaicvirus (TMV) genomic RNA (both polarities), hammerhead type ribozymes were synthesizedin vitro, which all shared a conserved domain adapted from satellite tobacco ringspot virus(sTobRV)RNA. Ribozymes RZ1, RZ2 and RZ3 were designed to cleave the phosphodiester bondsimmediate to the 3'--end of GUC between the residues 5384-5385 and 6312--6313 on the plusstrand and 1214-1215 on the minus strand, respectively. The in vitro data indicated that RZ1 wasable to cleave completely its substrates BT1(+ ) and BT2(+ ), representing partial sequencesof the plus strand of the TMV MP region at 50, 37 and 30℃ with a molar ratio of ribozymeto the target as low as 1:1. Its two iso-ribozymes RZ1A and RZ1B which were respectivelymodified to contain a CUUCGG sequence in the conserved region and in an additional 3'-ter-minal stem-loop of UUUUUCUUCGGAAAAA were able to cleave BT1(+) and BT2(+) asefficiently as RZ1. Ribozyme RZ3 cleaved, with less efficiency, its substrate BT2(-), repre-senting the minus strand of the TMV MP gene at 50, 37 or 30℃ even with a molar ratio ofribozyme to the target as high as 4:1. Ribozyme RZ2, however, had not any visible activityto its substrate BT3(+ ), representing the sequence was involved in the 3'--terminal tRNA--likesequence even though it was incubated for 5 min at 65℃ before being exposed to the ribozyme.展开更多
Accurate identification of the correct,biologically relevant RNA structures is critical to understanding various aspects of RNA biology since proper folding represents the key to the functionality of all types of RNA ...Accurate identification of the correct,biologically relevant RNA structures is critical to understanding various aspects of RNA biology since proper folding represents the key to the functionality of all types of RNA molecules and plays pivotal roles in many essential biological processes.Thus,a plethora of approaches have been developed to predict,identify,or solve RNA structures based on various computational,molecular,genetic,chemical,or physicochemical strategies.Purely computational approaches hold distinct advantages over all other strategies in terms of the ease of implementation,time,speed,cost,and throughput,but they strongly underperform in terms of accuracy that significantly limits their broader application.Nonetheless,the advantages of these methods led to a steady development of multiple in silico RNA secondary structure prediction approaches including recent deep learning-based programs.Here,we compared the accuracy of predictions of biologically relevant secondary structures of dozens of self-cleaving ribozyme sequences using seven in silico RNA folding prediction tools with tasks of varying complexity.We found that while many programs performed well in relatively simple tasks,their performance varied significantly in more complex RNA folding problems.However,in general,a modern deep learning method outperformed the other programs in the complex tasks in predicting the RNA secondary structures,at least based on the specific class of sequences tested,suggesting that it may represent the future of RNA structure prediction algorithms.展开更多
Linear mRNA vaccines are constrained by exonuclease susceptibility and instability,leading to compromised antigen expression.Circular RNA(circRNA) lacking canonical 5' and 3' untranslated regions demonstrates ...Linear mRNA vaccines are constrained by exonuclease susceptibility and instability,leading to compromised antigen expression.Circular RNA(circRNA) lacking canonical 5' and 3' untranslated regions demonstrates intrinsic exonuclease resistance.Current circularization strategies face three principal limitations:chemical methods produce non-native 2',5'-phosphodiester bonds;ribozyme-mediated approaches are restricted to RNA fragments shorter than 500 nucleotides;the Anabaena Group I intron system retains immunogenic exon sequences.In contrast,the self-splicing Group I intron ribozyme from Tetrahymena enables precisely controlled circularization through autonomous structural rearrangement,yielding exonfree constructs.Through optimized purification protocols,historical scalability challenges are systematically addressed.This Perspective establishes the mechanistic rationale and therapeutic superiority of this engineered RNA circularization platform.展开更多
Sugars are one of the major metabolites and are essential for nucleic acid synthesis and energy production.In addition,sugars can act as signaling molecules.To study sugar signaling at the systemic level,there is an u...Sugars are one of the major metabolites and are essential for nucleic acid synthesis and energy production.In addition,sugars can act as signaling molecules.To study sugar signaling at the systemic level,there is an urgent need to systematically identify sugar-sensing proteins and nucleic acids.I propose the terms“swodkoreceptor”and“swodkocrine signaling,”derived from the Polish word“slodki”meaning“sweet,”to comprise all sugar-sensing proteins and signaling events,respectively,regardless of their cellular location and signaling domains.This proposal is intended to facilitate the inclusion of proteins such as the Escherichia coli Lac I repressor as an allolactose receptor,human glucokinase regulatory protein(GCKR)as a fructose receptor,and other sugar-binding based allosterically regulated enzymes and transcription factors as sugar-sensing receptors.In addition,enzyme-interacting proteins whose interaction state is regulated by sugar binding have also been proposed as sugar receptors.The systemic study of protein-and nucleic-acid-based swodkoreceptors may help to identify organelle-specific swodkoreceptors and to also address receptor duality.The study of intra-and inter-organism swodkocrine signaling and its crosstalk with gasocrine signaling may help to understand the etiology of diseases due to dysregulation in sugar homeostasis and signaling.展开更多
<span style="font-family:""><span style="font-family:Verdana;">For just about 30 years, researchers have considered the likelihood to utilize </span><span style="font...<span style="font-family:""><span style="font-family:Verdana;">For just about 30 years, researchers have considered the likelihood to utilize </span><span style="font-family:Verdana;">nucleic acids as antiviral therapeutics. In principle, small single-stranded</span><span style="font-family:Verdana;"> nuc</span><span style="font-family:Verdana;">leotide sequence (oligonucleotide) could hybridize to a particular gene or</span><span style="font-family:Verdana;"> mes</span><span style="font-family:Verdana;">senger RNA and diminish transcription or translation, respectively, in this</span><span style="font-family:Verdana;"> manner decreasing the amount of protein that is synthesized. Until now, an incredible number of antisense oligonucleotides, double-stranded oligonucleotides, aptamers, ribozymes, deoxyribozymes, interfering RNAs, chimeric RNA</span></span><span style="font-family:Verdana;">-</span><span style="font-family:""><span style="font-family:Verdana;">DNA molecules, antibody genes has been created artificially and ap</span><span style="font-family:Verdana;">plied effectively for comprehension and manipulating biological processe</span><span style="font-family:Verdana;">s and in clinical preliminaries to treat a variety of diseases. Their versatility and potency make them similarly fit candidates for fighting viral infections. However, troubles with their efficiency, off-target effects, toxicity, delivery, and stability halted the development of nucleic acid-based therapeutics that can be utilized in the clinic. The potential for nucleic acid therapeutic agents is significant and is quite recently beginning to be realized. In this review, we have summarized some of the recent advancements made in the area of nucleic acid based therapeutics and focused on the methods of their delivery and associated challenges.展开更多
基金supported by a National Transgenic Science and Technology Program (2016ZX08010002)to R.W.a startup fund from the Huazhong Agricultural University
文摘Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in the genome to generate doublestrand breaks（DSBs）in vivo is a prerequisite for any genome editing efforts.
基金Supported by National Natural Science Foundation of China, NO.3960003l
文摘Objective: To study whether 2 hepatitis delta virus genomic ribozymes(g. Rz) have trans-cleaving activity. Methods: g. Rz74 and g. Rz55 were transcribed from their templates which were synthesized and cloned into plasmid pGEM-4Z; Homologous substrate RNA was transcribed for pRz277B and labelled withα-32 P-UTP. The trans-cleaving activity was studied by mixing the ribozymes and substrate in appropriate who under certain conditions. Results: The trans-cleaved products were generated after mixing the ribozyme with the substrate for 30 min, and accumulated more for 90 min. Conclusion: Both g. Rz74 and g. Rz55 possess trans-cleaving activity.
文摘To reverse multidrug resistance（MDR） of HepG2 by anti-MDR1 hammerhead ribozyme, an anti-MDR1 hammerhead ribozyme was developed and delivered to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase Ⅲ promoter. The expression of mdrl/Pgp and Rz was detected in HepG2, HepG2 muhidrug-resistant cell line and HepG2 Rz-transfected cells by semi-quantitative RT-PCR and Western blot methods. Moreover, MTT assay was employed to detect the sensitivity of these ribozyme-transfected cells, and Rhodamine123 （Rh123） was used to test the function of Pgp. The Rz- transfected HepG2 cells became doxorubicin-sensitive, which was concomitant with the decreased MDR1 expression. The study showed that the retrovirus vector encoding the anti-MDR1 ribozyme may be applicable to the treatment of MDR cells.
基金National Natural Science Foundation of China (No. 39670330).
文摘Objective: To investigate the in vitro cleavage ability and effects on apoptosis and cell growth of the bcr-abl fusion gene specific multi-unit ribozymes. Methods: Three fusion point specific ribozymes were designed and the multi-unit ribozymes?in vitro transcription vector and retroviral vector were constructed. The in vitro cleavage ability was tested. The retroviral vector was transfected into K562 cell and the effects on proliferation, apoptosis, cell cycle and cell structure were observed. Results: Multi-unit ribozymes had in vitro cleavage efficiency of 70.8%, which was more efficient than single-unit and double-unit ribozymes. Transfection of the retroviral vector of the ribozyme into K562 cells, induced inhibition of cell growth and apoptosis. The incorporation rate of DNA in ribozymes transfected K562 cells was greatly decreased along with time passed, with an inhibition rate of more than 50% after 96 h of transfection. Under FCM, 18.4% of the cells underwent apoptosis 72 h after transfection and more cells were blocked in G phase, with the ratio in S phase greatly decreased (41.9%). Under electron microscope, compaction of nuclear chromatin and apoptosis bodies were observed. Conclusion: Multi-unit ribozymes specific to bcr-abl fusion gene can be used to treat CML and to purge bone marrow for self-grafting.
文摘Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hepatitis delta virus(HDV) ribozyme was chosen and used to demonstrate the effective cleavage in vitro and in vivo. Methods Three hammerhead ribozymes for potential target sites(S, X and C genes) and co-expression plasmid(pTr-dB, pTdδ-dB, pTrX-dB and pTrC-dB) as well as four HDV-ribozyme chimera constructs with HBV(pTdXX, pTdXC, pTdSX and pTdSC) were severally chosen to validate the inhibition of the replication and expression of HBV. The co-expression plasmids(pTdδ and pTr-Db) in physiological saline were hydrodynamically injected to mice by tail vein. Results Compared with the group injected with pTr-dB in Huh-7 cell, hepatitis B surface antigen(HBsAg) was reduced by 31% in the group injected with pTdδ-dB, by 54%, 26%, 72% and 97% in the group injected with recombinant-ribozymes pTdSX, pTdSC, pTdXC and pTdXX, respectively. The inhibiting effects of endogenous ribozymes RzX and RzC on the HBsAg expression were 66% and 57%, respectively. Compared with the positive control, the amount of HBsAg was decreased in mice injected with pTdXX through tail vein by 88% and 96% on the second day and the third day, respectively. HBsAg was undetectable on the 6th day and could not primitively be detected on the 9th day in the sera from all mice. HBV DNA was not detected in the sera of BALB/c mice injected with pTdXX-dB, pTrX-dB or replicating-defective plasmid pHBV, while HBV DNA replication in control group could be detected on the 6th day. While HBcAg could not be detected in liver tissues of mice injected with plasmid pTdXX-dB on the 3rd day. Conclusions Encoding regions of HBV S, C and X gene were the effective cleavage sites for hammerhead ribozyme in vitro and in vivo, which provides basis for further construction of therapeutic recombinant HDV and the development of targeting antiviral gene therapy.
文摘The discovery that RNA can act as an enzyme led Thomas Cech to win the Nobel Prize in Chemistry and led immediately to the next wave of attempts to find an effective RNA-based therapy.The tantalizing idea that RNA enzymes called trans-cleaving ribozymes enables them to act as potential antiviral and powerful tool for functional genomic studies.The efficacy of ribozyme function in a complex intracellular environment is dependent on the intracellular fate of the RNA that is being targeted.Recently,ribozymes have been used successfully to inhibit gene expression in a variety of biological systems in vitro and in vivo.Ribozyme has also been used successfully to combat many cases of viral infection,as clinical trial.Despite it needs to be investigated and explored as far as its structural and functional aspects are concern.In view of the significance of ribozyme in modern medicine,we reviewed the recent literature on general approach to control viral infection.
基金Supported by the National Natural Science Foundation of China (No. 39870303)
文摘Objective: To compare the cleavage activity of ribozymes directed against 2 sites of PDGF receptorP subunit cDNA gene in vitro. Methods: The 608 bp fragment of PDGF receptor β subunit cDNA was clonedinto T-vector under the control of T7 promoter, named pPDGFR-β. Two ribozymes were designed to cleavethe CUU sequence at codon 45 and codon 252 of PDGF receptor β subunit mRNA respectively. These 2 ham-merhead ribozyme genes were cloned into vector PI. 5 between 5' -cis ribozyme and 3' -cis ribozyme to gener-ate the plasmids of pRZ1 and pRZ2. The pPDGFR-β, pRZl and pRZ2 were linearized and then transcribedwith T7 promoter in vitro. Results: The RZ1 showed high cleavage activity in vitro, but the RZ2 showed nocleavage activity under the same condition. Conclusion: The cleavage site selection is an important factor in-fluencing the cleavage activities of ribozymes.
基金supported by a grant from the Ministry of Science and Technology of the People’s Republic of China(Grant No.2021YFC2101700 and Grant No.2018YFA0900100)National Key Research and Development Program of China(Grant No.2021YFA0910700)+1 种基金the Chinese Academy of Sciences[No.XDB0480100 of the Strategic Priority Research Program]and CAS Youth Interdisciplinary TeamThis work was also supported by Vanke Special Fund for Public Health and Health Discipline Development,Tsinghua University(2022Z82WKJ006).
文摘Synthetic RNA-based switches provide distinctive merits in modulating gene expression.Simple and flexible RNA-based switches are crucial for advancing the field of gene regulation,paving the way for innovative tools that can sense and manipulate cellular processes.In this research,we have developed programmable ribozymes that are capable of suppressing gene expression in response to specific,endogenously expressed trigger RNAs.We engineer ribozymes by introducing upstream antisense sequences(anti-ribozymes)to inhibit the self-cleaving activity of the hammerhead ribozyme and open the expression of the target gene.The trigger RNA is designed to recognize and bind to complementary sequences within the anti-ribozymes,thereby inhibiting their ability to direct protein synthesis.The anti-ribozyme performance is optimized by regulating the essential sequence modules that play a crucial role in determining the specificity and efficiency of the anti-ribozyme’s interaction with its trigger RNA.By applying this switch mechanism to various ribozyme designs,we have shown that it is possible to achieve control over gene expression across a wide range of trigger RNAs.By exploiting these pro-grammable anti-ribozymes,we aim to create a powerful tool for controlling gene expression in mammalian cells,which could have important implications for basic research,disease diagnosis,and therapeutic interventions.
基金Research suppordet by the Foundation of the President of the Chinese Academy of Sciences, the Foundation of Institute Directors of the Chinese Academy of Sciences and the Shanghai National Laboratory of Plant Genetics of the Chinese Academy of Sciences.
文摘Based upon computer-assisted predictions on the secondary structures of tobacco mosaicvirus (TMV) genomic RNA (both polarities), hammerhead type ribozymes were synthesizedin vitro, which all shared a conserved domain adapted from satellite tobacco ringspot virus(sTobRV)RNA. Ribozymes RZ1, RZ2 and RZ3 were designed to cleave the phosphodiester bondsimmediate to the 3'--end of GUC between the residues 5384-5385 and 6312--6313 on the plusstrand and 1214-1215 on the minus strand, respectively. The in vitro data indicated that RZ1 wasable to cleave completely its substrates BT1(+ ) and BT2(+ ), representing partial sequencesof the plus strand of the TMV MP region at 50, 37 and 30℃ with a molar ratio of ribozymeto the target as low as 1:1. Its two iso-ribozymes RZ1A and RZ1B which were respectivelymodified to contain a CUUCGG sequence in the conserved region and in an additional 3'-ter-minal stem-loop of UUUUUCUUCGGAAAAA were able to cleave BT1(+) and BT2(+) asefficiently as RZ1. Ribozyme RZ3 cleaved, with less efficiency, its substrate BT2(-), repre-senting the minus strand of the TMV MP gene at 50, 37 or 30℃ even with a molar ratio ofribozyme to the target as high as 4:1. Ribozyme RZ2, however, had not any visible activityto its substrate BT3(+ ), representing the sequence was involved in the 3'--terminal tRNA--likesequence even though it was incubated for 5 min at 65℃ before being exposed to the ribozyme.
基金supported by the National Natural Science Foundation of China(Grant No.32000462 to Fei Qi,Grant No.32170619 to Philipp Kapranovand Grant No.32201055 to Yue Chen)+2 种基金the Research Fund for International Senior Scientists from the National Natural Science Foundation of China(Grant No.32150710525 to Philipp Kapranov)the Natural Science Foundation of Fujian Province,China(Grant No.2020J02006 to Philipp Kapranov)the Scientific Research Funds of Huaqiao University,China(Grant No.22BS114 to Fei Qi,Grant No.21BS127 to Yue Chen,and Grant No.15BS101 to Philipp Kapranov).
文摘Accurate identification of the correct,biologically relevant RNA structures is critical to understanding various aspects of RNA biology since proper folding represents the key to the functionality of all types of RNA molecules and plays pivotal roles in many essential biological processes.Thus,a plethora of approaches have been developed to predict,identify,or solve RNA structures based on various computational,molecular,genetic,chemical,or physicochemical strategies.Purely computational approaches hold distinct advantages over all other strategies in terms of the ease of implementation,time,speed,cost,and throughput,but they strongly underperform in terms of accuracy that significantly limits their broader application.Nonetheless,the advantages of these methods led to a steady development of multiple in silico RNA secondary structure prediction approaches including recent deep learning-based programs.Here,we compared the accuracy of predictions of biologically relevant secondary structures of dozens of self-cleaving ribozyme sequences using seven in silico RNA folding prediction tools with tasks of varying complexity.We found that while many programs performed well in relatively simple tasks,their performance varied significantly in more complex RNA folding problems.However,in general,a modern deep learning method outperformed the other programs in the complex tasks in predicting the RNA secondary structures,at least based on the specific class of sequences tested,suggesting that it may represent the future of RNA structure prediction algorithms.
基金supported by the National Key Research&Development Program of China(Nos.2021YFC2302400,2021YFA1201000,2023YFC2606004)the Fundamental Research Funds for the Central Universities(No.2022CX01013)。
文摘Linear mRNA vaccines are constrained by exonuclease susceptibility and instability,leading to compromised antigen expression.Circular RNA(circRNA) lacking canonical 5' and 3' untranslated regions demonstrates intrinsic exonuclease resistance.Current circularization strategies face three principal limitations:chemical methods produce non-native 2',5'-phosphodiester bonds;ribozyme-mediated approaches are restricted to RNA fragments shorter than 500 nucleotides;the Anabaena Group I intron system retains immunogenic exon sequences.In contrast,the self-splicing Group I intron ribozyme from Tetrahymena enables precisely controlled circularization through autonomous structural rearrangement,yielding exonfree constructs.Through optimized purification protocols,historical scalability challenges are systematically addressed.This Perspective establishes the mechanistic rationale and therapeutic superiority of this engineered RNA circularization platform.
基金supported by the National Science Centre grants,Grant/Award Number:SONATA-BIS 2020/38/E/NZ3/00090 and SONATA 2021/43/D/NZ3/01798。
文摘Sugars are one of the major metabolites and are essential for nucleic acid synthesis and energy production.In addition,sugars can act as signaling molecules.To study sugar signaling at the systemic level,there is an urgent need to systematically identify sugar-sensing proteins and nucleic acids.I propose the terms“swodkoreceptor”and“swodkocrine signaling,”derived from the Polish word“slodki”meaning“sweet,”to comprise all sugar-sensing proteins and signaling events,respectively,regardless of their cellular location and signaling domains.This proposal is intended to facilitate the inclusion of proteins such as the Escherichia coli Lac I repressor as an allolactose receptor,human glucokinase regulatory protein(GCKR)as a fructose receptor,and other sugar-binding based allosterically regulated enzymes and transcription factors as sugar-sensing receptors.In addition,enzyme-interacting proteins whose interaction state is regulated by sugar binding have also been proposed as sugar receptors.The systemic study of protein-and nucleic-acid-based swodkoreceptors may help to identify organelle-specific swodkoreceptors and to also address receptor duality.The study of intra-and inter-organism swodkocrine signaling and its crosstalk with gasocrine signaling may help to understand the etiology of diseases due to dysregulation in sugar homeostasis and signaling.
文摘<span style="font-family:""><span style="font-family:Verdana;">For just about 30 years, researchers have considered the likelihood to utilize </span><span style="font-family:Verdana;">nucleic acids as antiviral therapeutics. In principle, small single-stranded</span><span style="font-family:Verdana;"> nuc</span><span style="font-family:Verdana;">leotide sequence (oligonucleotide) could hybridize to a particular gene or</span><span style="font-family:Verdana;"> mes</span><span style="font-family:Verdana;">senger RNA and diminish transcription or translation, respectively, in this</span><span style="font-family:Verdana;"> manner decreasing the amount of protein that is synthesized. Until now, an incredible number of antisense oligonucleotides, double-stranded oligonucleotides, aptamers, ribozymes, deoxyribozymes, interfering RNAs, chimeric RNA</span></span><span style="font-family:Verdana;">-</span><span style="font-family:""><span style="font-family:Verdana;">DNA molecules, antibody genes has been created artificially and ap</span><span style="font-family:Verdana;">plied effectively for comprehension and manipulating biological processe</span><span style="font-family:Verdana;">s and in clinical preliminaries to treat a variety of diseases. Their versatility and potency make them similarly fit candidates for fighting viral infections. However, troubles with their efficiency, off-target effects, toxicity, delivery, and stability halted the development of nucleic acid-based therapeutics that can be utilized in the clinic. The potential for nucleic acid therapeutic agents is significant and is quite recently beginning to be realized. In this review, we have summarized some of the recent advancements made in the area of nucleic acid based therapeutics and focused on the methods of their delivery and associated challenges.
