S-Monofluoromethyl phosphorothioates represent an important class of organofluorine compounds and are re ported here for the first time.A series of S-monofluoromethyl phosphorothioates are conveniently synthesized fro...S-Monofluoromethyl phosphorothioates represent an important class of organofluorine compounds and are re ported here for the first time.A series of S-monofluoromethyl phosphorothioates are conveniently synthesized from different P^(Ⅴ)-H compounds and PhSO_(2)SCH_(2)F under mild conditions.The method is compatible with common functional groups and provides potential opportunities to synthesize new bioactive molecules for medicinal chemistry.展开更多
Double-stranded RNA-mediated interference (RNAi), antisense oligonucleotides (ASO), and ribozymes have excellent specificity to their target oncogenic mRNA. They also seem to show great promise when it comes to treati...Double-stranded RNA-mediated interference (RNAi), antisense oligonucleotides (ASO), and ribozymes have excellent specificity to their target oncogenic mRNA. They also seem to show great promise when it comes to treating cancer. The problem is that RNAi, ASO, and ribozymes have poor stability and are constantly being degraded by nucleases. Researchers have made some efforts to increase antisense oligonucleotides’ stability by creating phospharimidate and Phosphorothioate. Currently, ribozymes, antisense oligonucleotides, and (RNAi) are the three main methods used to target RNA. These methods are currently undergoing clinical trials for the purpose of focusing on specific RNAs involved in disorders like cancer and neurodegeneration. In fact, ASOs that target amyotrophic lateral sclerosis and spinal muscular atrophy have produced promising results in clinical trials. The formation of chemical alterations that boost affinity and selectivity while reducing noxiousness owing to off-target impacts are two benefits of ASOs. Another benefit is increased affinity. With a focus on RNAi and ASOs, this review illustrated the main therapeutic strategies of RNA therapy now in use.展开更多
The phosphorothioate oligonucleotides are used more and more for cancer treatment.Fast analysis of phosphorothioate oligonucleotide is important for quality control of this drug.In this paper,FT-ICR-MS was used to ana...The phosphorothioate oligonucleotides are used more and more for cancer treatment.Fast analysis of phosphorothioate oligonucleotide is important for quality control of this drug.In this paper,FT-ICR-MS was used to analyze a phosphorothioate oligonucleotide with 6 321 u.Experimental results showed that accurate relative molecular mass of phosphorothioate oligonucleotide determined by FT-ICR-MS can be used for confirmation of its composition.展开更多
Aim: To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor-α (TNF-α-induced apoptosis in prostate cancer cells (PC3). Meth...Aim: To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor-α (TNF-α-induced apoptosis in prostate cancer cells (PC3). Methods: Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) and polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA was measured by reverse transcription PCR (RT-PCR) assay and gel-image system, hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by morphological method and determined by flow cytometry. Results: The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide (S PS-ODN) and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-α treatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-α treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group. Conclusion: hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-α- induced apoptosis of PC3 cells.展开更多
AIM: To explore effects of telomerase RNA-targeting phosphorothioate antisense oligodeoxynucleotides (PS-ASODN) on growth of human gastrointestinal stromal tumors transplanted in mice. METHODS: A SCID mouse model for ...AIM: To explore effects of telomerase RNA-targeting phosphorothioate antisense oligodeoxynucleotides (PS-ASODN) on growth of human gastrointestinal stromal tumors transplanted in mice. METHODS: A SCID mouse model for transplantation of human gastrointestinal stromal tumors (GISTs) was established using tumor cells from a patient who was diagnosed with GIST and consequently had been treated with imatinib. GIST cells cultured for 10 passages were used for inoculation into mice. Transfection of PS-ASODN was carried out with Lipotap Liposomal Transfection Reagent. GISTs that subsequently developed in SCID mice were subjected to intratumoral injection once daily from day 7 to day 28 postinoculation, and mice were divided into the following four groups according to treatment: PS-ASODN group (5.00 μmoL/L of oligonucleotide, each mouse received 0.2 mL once daily); imatinib group (0.1 mg/g body weight); liposome negative control group (0.01 mL/g); and saline group (0.01 mL/g). On day 28, the mice were sacrificed, and tumor attributes including weight and longest and shortest diameters were measured. Tumor growth was compared between treatment groups, and telomerase activity was measured by enzyme-linked immunosorbent assay. Apoptosis was examined by flow cytometry. Real-time polymerase chain reaction was used to detect expression of the mRNA encoding the apoptosis inhibition B-cell leukemia/lymphoma 2 (bcl-2 ) gene. RESULTS: In the PS-ASODN group, tumor growth was inhibited by 59.437%, which was markedly higher than in the imatinib group (11.071%) and liposome negative control group (2.759%) [tumor inhibition=(mean tumor weight of control group - mean tumor weight of treatment group)/(mean tumor weight of control group) × 100%]. Telomerase activity was significantly lower (P < 0.01) in the PS-ASODN group (0.689 ± 0.158) compared with the imatinib group (1.838 ± 0.241), liposome negative control group (2.013 ± 0.273), and saline group (2.004 ± 0.163). Flow cytometry revealed that the apoptosis rate of tumor cells treated with PS-ASODN was 20.751% ± 0.789%, which was higher (P < 0.01) than that of the imatinib group (1.163% ± 0.469%), liposome negative control group (1.212% ± 0.310%), and saline group (1.172% ± 0.403%). Expression of bcl-2 mRNA in the transplanted GISTs was markedly downregulated (P < 0.01) in the PS-ASODN group (7.245 ± 0.739) compared with the imatinib group (14.153 ± 1.618) and liposome negative control group (16.396 ± 1.351).CONCLUSION: PS-ASODN can repress GIST growth, mediated perhaps by inhibition of telomerase activity and downregulation of bcl-2 expression.展开更多
Nucleic acid detection plays a key role in diverse diagnosis and disease control.Currently available nucleic acid detection techniques are challenged by trade-offs among speed,simplicity,precision and cost.Here,we des...Nucleic acid detection plays a key role in diverse diagnosis and disease control.Currently available nucleic acid detection techniques are challenged by trade-offs among speed,simplicity,precision and cost.Here,we described a novel method,designated SENSOR(Sulfur DNA mediated nucleic acid sensing platform),for rapid nucleic acid detection.SENSOR was developed from phosphorothioate(PT)-DNA and sulfur binding domain(SBD)which specifically binds double-stranded PT-modified DNA.SENSOR utilizes PT-DNA oligo and SBD as targeting module,which is linked with split luciferase reporter to generate luminescence signal within 10 min.We tested detection on synthesized nucleic acid and COVID-19 pseudovirus,achieving attomolar sensitivity combined with an amplification procedure.Single nucleotide polymorphisms(SNP)could also be discriminated.Indicating SENSOR a new promising nucleic acid detection technique.展开更多
The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study prelimi- nary molecular mechanisms for its effects. I...The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study prelimi- nary molecular mechanisms for its effects. In order to set up a model in vitro, BEL7402-HBx cell line, stably expressing HBx mRNA, was established by stable transfection of pcDNA-HBx, which contains HBx gene, into hepatoma cell line BEL7402. Control cell line BEL7402-cDNA3, stably transfected with pcDNA3, was set up simultaneously as a control. Trypan blue exclusion test, caspase 3 activity detection and TUNEL assay were performed to detect the apoptosis of BEL7402, BEL7402-cDNA3, BEL7402-HBx induced by TRAIL. The expression of TRAIL recep- tors in three groups was analyzed by Flow cytometry. In addition, phosphorothioated antisense oligonucleotide against the translation initial region of HBx gene (PS-asODNs/HBx) was used to block the expression of HBx in HepG2.2.15 cells and to further confirm the effects of HBx on TRAIL-induced apoptosis. Trypan blue exclusion test indicated that TRAIL had a dose-dependent cytotoxicity on BEL7402, BEL7402-cDNA3 and BEL7402-HBx cells. Under treatment of the same concentration of TRAIL, BEL7402-HBx had a higher apoptosis rate and a higher level of Caspase 3 activation than BEL7402 and BEL7402-cDNA3. TUENL assay showed that the apoptosis rate of BEL7402-HBx induced by 10 μg/L TRAIL was 41.4%±7.2%, signifi- cantly higher than that of BEL7402 and BEL7402-cDNA3 cells. Blockade of HBx expression in Hep G2.2.15 cells partly inhibited the apoptosis induced by TRAIL. The introduction or blockade of HBx did not change the expression pattern of TRAIL receptors. The present study firstly con- firms the effects of HBx on TRAIL- induced apoptosis from two different points and it is not re- lated with the expression level of TRAIL receptors. This would be useful to further clarify the roles of imbalanced apoptosis in pathogenesis of Hepatitis B and related hepatocellular carcinoma.展开更多
Background Telomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells. Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that...Background Telomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells. Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-α (TNF-α)-induced apoptosis. Methods Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry. Results AS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-α while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group. Conclusions AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase activity.展开更多
Here we tell a 20-year long story.It began with an easily overlooked DNA degradation(Dnd)phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by fi...Here we tell a 20-year long story.It began with an easily overlooked DNA degradation(Dnd)phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by five dnd genes.This unusual DNA modification,called phosphorothioation,is the first physiological modification identified on the DNA backbone,in which the nonbridging oxygen is replaced by sulfur in a sequence selective and stereo-specific manner.Homologous dnd gene clusters have been identified in diverse and distantly related bacteria and thus have drawn immediate attention of the entire microbial scientific community.Here,we summarize the progress in chemical,genetic,enzymatic,bioinformatical and analytical aspects of this novel postreplicative DNA modification.We also discuss perspectives on the physiological functions of the DNA phosphorothioate modification in bacteria and their implications.展开更多
With numerous advancements in novel biochemical techniques, our knowledge of the role of RNAs in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless,...With numerous advancements in novel biochemical techniques, our knowledge of the role of RNAs in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless, detailed information regarding RNA processing, trafficking, and localization in living cells has been lacking due to technical limitations in imaging single RNA transcripts in living cells with high spatial and temporal resolution. In this review, we discuss tech- niques that have shown great promise for single RNA imaging, followed by highlights in our recent work in the development of molecular beacons (MBs), a class of nanoscale oligonucleotide-probes, for detecting individual RNA transcripts in living cells. With further refinement of MB design and development of more sophisticated fluorescence microscopy techniques, we envision that MB-based approaches could promote new discoveries of RNA functions and activities.展开更多
PEGylation, the attachment of poly(ethylene glycol) (PEG), has been adopted to improve the pharmacokinetic properties of oligonucleotide therapeutics for nearly 30 years. Prior efforts mainly focused on the invest...PEGylation, the attachment of poly(ethylene glycol) (PEG), has been adopted to improve the pharmacokinetic properties of oligonucleotide therapeutics for nearly 30 years. Prior efforts mainly focused on the investigation of linear or slightly branched PEG having different molecular weights, terminal functional groups, and possible oligonucleotide sites for functionalization. Recent studies on highly branched PEG (including brush, star, and micellar structures) indicate superior properties in several areas including cellular uptake, gene regulation efficac~ reduction of side effects, and biodistribution. This review focuses on comparing the effects of PEG architecture on the physiochemical and biological properties of the PEGylated oligonucleotide.展开更多
基金supported by the Natural Science Foundation of Shanghai(No.17ZR1447100)the Science and Technology Commission of Shanghai Municipality(No.19DZ2271100)。
文摘S-Monofluoromethyl phosphorothioates represent an important class of organofluorine compounds and are re ported here for the first time.A series of S-monofluoromethyl phosphorothioates are conveniently synthesized from different P^(Ⅴ)-H compounds and PhSO_(2)SCH_(2)F under mild conditions.The method is compatible with common functional groups and provides potential opportunities to synthesize new bioactive molecules for medicinal chemistry.
文摘Double-stranded RNA-mediated interference (RNAi), antisense oligonucleotides (ASO), and ribozymes have excellent specificity to their target oncogenic mRNA. They also seem to show great promise when it comes to treating cancer. The problem is that RNAi, ASO, and ribozymes have poor stability and are constantly being degraded by nucleases. Researchers have made some efforts to increase antisense oligonucleotides’ stability by creating phospharimidate and Phosphorothioate. Currently, ribozymes, antisense oligonucleotides, and (RNAi) are the three main methods used to target RNA. These methods are currently undergoing clinical trials for the purpose of focusing on specific RNAs involved in disorders like cancer and neurodegeneration. In fact, ASOs that target amyotrophic lateral sclerosis and spinal muscular atrophy have produced promising results in clinical trials. The formation of chemical alterations that boost affinity and selectivity while reducing noxiousness owing to off-target impacts are two benefits of ASOs. Another benefit is increased affinity. With a focus on RNAi and ASOs, this review illustrated the main therapeutic strategies of RNA therapy now in use.
文摘The phosphorothioate oligonucleotides are used more and more for cancer treatment.Fast analysis of phosphorothioate oligonucleotide is important for quality control of this drug.In this paper,FT-ICR-MS was used to analyze a phosphorothioate oligonucleotide with 6 321 u.Experimental results showed that accurate relative molecular mass of phosphorothioate oligonucleotide determined by FT-ICR-MS can be used for confirmation of its composition.
文摘Aim: To investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor-α (TNF-α-induced apoptosis in prostate cancer cells (PC3). Methods: Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) and polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA was measured by reverse transcription PCR (RT-PCR) assay and gel-image system, hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by morphological method and determined by flow cytometry. Results: The telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide (S PS-ODN) and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-α treatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-α treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group. Conclusion: hTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-α- induced apoptosis of PC3 cells.
基金Supported by The Natural Science Foundation of Zhejiang Province, No. Y201016273
文摘AIM: To explore effects of telomerase RNA-targeting phosphorothioate antisense oligodeoxynucleotides (PS-ASODN) on growth of human gastrointestinal stromal tumors transplanted in mice. METHODS: A SCID mouse model for transplantation of human gastrointestinal stromal tumors (GISTs) was established using tumor cells from a patient who was diagnosed with GIST and consequently had been treated with imatinib. GIST cells cultured for 10 passages were used for inoculation into mice. Transfection of PS-ASODN was carried out with Lipotap Liposomal Transfection Reagent. GISTs that subsequently developed in SCID mice were subjected to intratumoral injection once daily from day 7 to day 28 postinoculation, and mice were divided into the following four groups according to treatment: PS-ASODN group (5.00 μmoL/L of oligonucleotide, each mouse received 0.2 mL once daily); imatinib group (0.1 mg/g body weight); liposome negative control group (0.01 mL/g); and saline group (0.01 mL/g). On day 28, the mice were sacrificed, and tumor attributes including weight and longest and shortest diameters were measured. Tumor growth was compared between treatment groups, and telomerase activity was measured by enzyme-linked immunosorbent assay. Apoptosis was examined by flow cytometry. Real-time polymerase chain reaction was used to detect expression of the mRNA encoding the apoptosis inhibition B-cell leukemia/lymphoma 2 (bcl-2 ) gene. RESULTS: In the PS-ASODN group, tumor growth was inhibited by 59.437%, which was markedly higher than in the imatinib group (11.071%) and liposome negative control group (2.759%) [tumor inhibition=(mean tumor weight of control group - mean tumor weight of treatment group)/(mean tumor weight of control group) × 100%]. Telomerase activity was significantly lower (P < 0.01) in the PS-ASODN group (0.689 ± 0.158) compared with the imatinib group (1.838 ± 0.241), liposome negative control group (2.013 ± 0.273), and saline group (2.004 ± 0.163). Flow cytometry revealed that the apoptosis rate of tumor cells treated with PS-ASODN was 20.751% ± 0.789%, which was higher (P < 0.01) than that of the imatinib group (1.163% ± 0.469%), liposome negative control group (1.212% ± 0.310%), and saline group (1.172% ± 0.403%). Expression of bcl-2 mRNA in the transplanted GISTs was markedly downregulated (P < 0.01) in the PS-ASODN group (7.245 ± 0.739) compared with the imatinib group (14.153 ± 1.618) and liposome negative control group (16.396 ± 1.351).CONCLUSION: PS-ASODN can repress GIST growth, mediated perhaps by inhibition of telomerase activity and downregulation of bcl-2 expression.
基金supported by the National Natural Science Foundation of China(31900060)National Key Research and Development Program of China(2020YFA0907800,2022YFC3400200,2022YFA0912200)+1 种基金Natural Science Foundation of Shanghai(20ZR1414500)Shanghai Pilot Program for Basic Research-Shanghai Jiao Tong University(21TQ1400204).
文摘Nucleic acid detection plays a key role in diverse diagnosis and disease control.Currently available nucleic acid detection techniques are challenged by trade-offs among speed,simplicity,precision and cost.Here,we described a novel method,designated SENSOR(Sulfur DNA mediated nucleic acid sensing platform),for rapid nucleic acid detection.SENSOR was developed from phosphorothioate(PT)-DNA and sulfur binding domain(SBD)which specifically binds double-stranded PT-modified DNA.SENSOR utilizes PT-DNA oligo and SBD as targeting module,which is linked with split luciferase reporter to generate luminescence signal within 10 min.We tested detection on synthesized nucleic acid and COVID-19 pseudovirus,achieving attomolar sensitivity combined with an amplification procedure.Single nucleotide polymorphisms(SNP)could also be discriminated.Indicating SENSOR a new promising nucleic acid detection technique.
文摘The purpose of this study is to observe the effects of HBx on the apoptosis of hepatoma cells induced by TNF-related apoptosis-inducing ligand (TRAIL) and to study prelimi- nary molecular mechanisms for its effects. In order to set up a model in vitro, BEL7402-HBx cell line, stably expressing HBx mRNA, was established by stable transfection of pcDNA-HBx, which contains HBx gene, into hepatoma cell line BEL7402. Control cell line BEL7402-cDNA3, stably transfected with pcDNA3, was set up simultaneously as a control. Trypan blue exclusion test, caspase 3 activity detection and TUNEL assay were performed to detect the apoptosis of BEL7402, BEL7402-cDNA3, BEL7402-HBx induced by TRAIL. The expression of TRAIL recep- tors in three groups was analyzed by Flow cytometry. In addition, phosphorothioated antisense oligonucleotide against the translation initial region of HBx gene (PS-asODNs/HBx) was used to block the expression of HBx in HepG2.2.15 cells and to further confirm the effects of HBx on TRAIL-induced apoptosis. Trypan blue exclusion test indicated that TRAIL had a dose-dependent cytotoxicity on BEL7402, BEL7402-cDNA3 and BEL7402-HBx cells. Under treatment of the same concentration of TRAIL, BEL7402-HBx had a higher apoptosis rate and a higher level of Caspase 3 activation than BEL7402 and BEL7402-cDNA3. TUENL assay showed that the apoptosis rate of BEL7402-HBx induced by 10 μg/L TRAIL was 41.4%±7.2%, signifi- cantly higher than that of BEL7402 and BEL7402-cDNA3 cells. Blockade of HBx expression in Hep G2.2.15 cells partly inhibited the apoptosis induced by TRAIL. The introduction or blockade of HBx did not change the expression pattern of TRAIL receptors. The present study firstly con- firms the effects of HBx on TRAIL- induced apoptosis from two different points and it is not re- lated with the expression level of TRAIL receptors. This would be useful to further clarify the roles of imbalanced apoptosis in pathogenesis of Hepatitis B and related hepatocellular carcinoma.
基金This study was supported by a grant from the Natural Science Foundation of Gansu Province(No.135570380).
文摘Background Telomerase activity is found in 85%-90% of all human cancers but not in their adjacent normal cells. Human telomerase reverse transcriptase (hTERT) is an essential component in the telomerase complex that plays an important role in telomerase activity. This study investigated the effect of the telomerase inhibition with an hTERT antisense oligodeoxynucleotide (ODN) in bladder cancer cells (T24) on tumor necrosis factor-α (TNF-α)-induced apoptosis. Methods Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA expression was measured by reverse transcription polymerase chain reaction (RT-PCR) assay and a gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by a morphological method and determined by flow cytometry. Results AS PS-ODN significantly inhibited telomerase activity and decreased the levels of hTERT mRNA which preceded the decline in the telomerase activity. AS PS-ODN significantly reduced the percentage of positive cells expressing hTERT protein following the decline of hTERT mRNA levels. There was no difference seen in the telomerase activity, hTERT mRNA expression or the protein levels between the sense phosphorothioate oligodeoxynucleotide (SPS-ODN) and the control group. AS PS-ODN treatment significantly decreased the cell viability and enhanced the apoptotic rate of T24 cells in response to TNF-α while there was no difference in cell viability and apoptotic rate between the S PS-ODN and the control group. Conclusions AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression. Treatment with AS PS-ODN may be a potential and most promising strategy for bladder cancer with telomerase activity.
文摘Here we tell a 20-year long story.It began with an easily overlooked DNA degradation(Dnd)phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by five dnd genes.This unusual DNA modification,called phosphorothioation,is the first physiological modification identified on the DNA backbone,in which the nonbridging oxygen is replaced by sulfur in a sequence selective and stereo-specific manner.Homologous dnd gene clusters have been identified in diverse and distantly related bacteria and thus have drawn immediate attention of the entire microbial scientific community.Here,we summarize the progress in chemical,genetic,enzymatic,bioinformatical and analytical aspects of this novel postreplicative DNA modification.We also discuss perspectives on the physiological functions of the DNA phosphorothioate modification in bacteria and their implications.
基金supported by grants from the National Key R&D Program of China (Grant Nos. 2016YFA0501603 and 2016YFA0100702)the National Natural Science Foundation of China (Grant Nos. 31771583 and 81371613)+1 种基金the Beijing Natural Science Foundation (Grant No. 7162114)the 1000 Young Talent Program of China
文摘With numerous advancements in novel biochemical techniques, our knowledge of the role of RNAs in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless, detailed information regarding RNA processing, trafficking, and localization in living cells has been lacking due to technical limitations in imaging single RNA transcripts in living cells with high spatial and temporal resolution. In this review, we discuss tech- niques that have shown great promise for single RNA imaging, followed by highlights in our recent work in the development of molecular beacons (MBs), a class of nanoscale oligonucleotide-probes, for detecting individual RNA transcripts in living cells. With further refinement of MB design and development of more sophisticated fluorescence microscopy techniques, we envision that MB-based approaches could promote new discoveries of RNA functions and activities.
基金Financial support by the National Institute of General Medical Sciences Award Number 1R01GM121612-01, the National Science Foundation (CAREER Award Number 1453255), and the American Chemical Society Petroleum Research Fund (No. PRF 54905-DNI5), is acknowledged.
文摘PEGylation, the attachment of poly(ethylene glycol) (PEG), has been adopted to improve the pharmacokinetic properties of oligonucleotide therapeutics for nearly 30 years. Prior efforts mainly focused on the investigation of linear or slightly branched PEG having different molecular weights, terminal functional groups, and possible oligonucleotide sites for functionalization. Recent studies on highly branched PEG (including brush, star, and micellar structures) indicate superior properties in several areas including cellular uptake, gene regulation efficac~ reduction of side effects, and biodistribution. This review focuses on comparing the effects of PEG architecture on the physiochemical and biological properties of the PEGylated oligonucleotide.