Chain elongation via dinucleotide(dimer)block coupling was considered as an improved chemical technique capable of synthesizing high-quality longer oligonucleotide for de novo DNA synthesis in synthetic biology.Howeve...Chain elongation via dinucleotide(dimer)block coupling was considered as an improved chemical technique capable of synthesizing high-quality longer oligonucleotide for de novo DNA synthesis in synthetic biology.However,this dimer block-wise approach was constrained by readily available dimer phosphoramidite with sufficient quality.Herein,through the usage of a one-pot coupling-oxidation-deprotection cascade process for preparing the key precursors 3'-hydroxyl dimers,then condensation with phosphorodiamidite,purification by flash column chromatography and precipation in methyl tert-butyl ether,a rationally designed dimer phosphoramidite bearing an internucleotide allyl phosphate and aβ-cyanoethyl phosphoramidite at the 3’-hydroxyl was synthesized.All sixteen allylic dimer phosphoramidites 2a-p were smoothly prepared with overall yields exceeding 50%and HPLC purities ranging from 97.40%to 99.69%.With these allylic reagents,oligonucleotides were successfully synthesized using a modified solid-phase phosphoramidite method and were completely deprotected under normal ammonialysis condition.Our results indicated that these dimer block-wise synthesized oligonucleotides were of sufficient quality for gene assembly and protein expression,thus,the allylic phosphate linked dimer phosphoramidite can serve as a promising dimer reagent that will enable the applications of long oligonucleotides.展开更多
Coronavirus disease 2019(COVID-19)messenger RNA(mRNA)vaccines have succeeded unprecedentedly due to high protection efficacy and robust immune responses.However,systematic and longitudinal profiling of immune response...Coronavirus disease 2019(COVID-19)messenger RNA(mRNA)vaccines have succeeded unprecedentedly due to high protection efficacy and robust immune responses.However,systematic and longitudinal profiling of immune responses in mRNA vaccine recipients remains limited.Here,a cohort of ten healthy volunteers who received two doses of ARCoV,a non-modified mRNA vaccine,were enrolled.Peripheral blood samples were collected and analyzed using Olink technology,antibody detection,intracellular cytokine staining,single-cell sequencing,and T cell receptor(TCR)sequencing.ARCoV vaccination induced potent humoral and cellular immune responses,as well as elevated cytokines including CX-C motif chemokine ligand 10(CXCL10)and interferon-gamma(IFN-g).Single-cell sequencing revealed that ARCoV immunization induced an increased relative abundance of interferon-activated T cells,proliferative T cells,and naïve T cells.Monocytes and dendritic cells exhibited activation of the innate immune response,downregulation of hypoxia and glycolysis pathways,and a transient decrease in their proportions.Integrative analysis of single-cell RNA and TCR sequencing identified clonal expansion of effector T cells and killer cell immunoglobulin-like receptor(KIR)-expressing natural killer-like cells after the second dose.These findings deepen our understanding of the immune dynamics following mRNA vaccination and offer valuable insights for designing next-generation vaccines.This study was registered at the Chinese Clinical Trial Registry(ChiCTR2100049104).展开更多
Vaccines are used to protect human beings from various diseases.mRNA vaccines simplify the development process and reduce the production cost of conventional vaccines,making it possible to respond rapidly to acute and...Vaccines are used to protect human beings from various diseases.mRNA vaccines simplify the development process and reduce the production cost of conventional vaccines,making it possible to respond rapidly to acute and severe diseases,such as coronavirus disease 2019.In this study,a universal integrated platform for the streamlined and ondemand preparation of mRNA products directly from DNA templates was established.Target DNA templates were amplified in vitro by a polymerase chain reaction module and transcribed into mRNA sequences,which were magnetically purified and encapsulated in lipid nanoparticles.As an initial example,enhanced green fluorescent protein(eGFP)was used to test the platform.The expression capacity and efficiency of the products were evaluated by transfecting them into HEK-293T cells.The batch production rate was estimated to be 200–300μg of eGFP mRNA in 8 h.Furthermore,an mRNA vaccine encoding the receptor-binding domain(RBD)of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein was produced by this platform.The proposed integrated platform shows advantages for the universal and on-demand preparation of mRNA products,offering the potential to facilitate broad access to mRNA technology and enable the development of mRNA products,including the rapid supply of new mRNA-based vaccines in pandemic situations and personalized mRNA-based therapies for oncology and chronic infectious diseases,such as viral hepatitis and acquired immune deficiency syndrome.展开更多
Influenza A virus(IAV)poses a significant threat to human health.The outcome of IAV results from the viral-host interaction,with the underlying molecular mechanisms largely unknown.By integrating the plasma proteomics...Influenza A virus(IAV)poses a significant threat to human health.The outcome of IAV results from the viral-host interaction,with the underlying molecular mechanisms largely unknown.By integrating the plasma proteomics data of the IAV-infected patients into the viral-inflammation protein-protein interaction(VI-PPI)network created in this study,purine nucleoside phosphorylase(PNP),the critical enzyme in purine salvage,was identified as a potential hub gene that connected the different stages of IAV infection.Extended survival rates and reduced pulmonary inflammatory lesions were observed in alveolar epithelial cell(AEC)-specific PNP conditional knockout mice upon H1N1 infection.Mechanistically,PB1-F2 of IAV was revealed as a novel viral transcriptional factor to bind to the TATA box of PNP promoter,leading to enhanced purine salvage in H1N1-challenged AECs.The activation of PNP-mediated purine salvage was verified in IAV-infected patients and A549 cells.PNP knockdown elicited a purine metabolic shift from augmented salvage pathway to de novo synthesis,constraining both viral infection and pro-inflammatory signaling through APRT-AICAR-AMPK activation.Moreover,durdihydroartemisinin(DHA),predicted by VI-PPI as a novel PNP inhibitor,exerted beneficial effects on the survival and weight gain of H1N1-challenged mice via its direct binding to PNP.To reveal for the first time,we found that PNP,activated by IAV,plays a hub role within H1N1-host interaction,simultaneously modulating viral replication and hyperinflammation through purine salvage.Our study sheds new light on a“two-for-one”strategy by targeting purine salvage in combating IAV-related pathology,suggesting PNP as a potential novel anti-influenza host target.展开更多
Immunotherapy has been emerging as a potent strategy for cancer treatment.However,undesirable therapeutic efficacy remains a challenge,including low drug loading,imprecise targeting,and non-specific releasing.The drug...Immunotherapy has been emerging as a potent strategy for cancer treatment.However,undesirable therapeutic efficacy remains a challenge,including low drug loading,imprecise targeting,and non-specific releasing.The drug delivery systems of immunotherapy play a key role in improving therapeutic efficacy and reducing side effects.To address these concerns,functional DNA nanostructures-based materials have been explored to achieve high loading capability,precise targeting,and controllable releasing.This review focuses on the crucial issues of delivery system for cancer immunotherapy and the strategies to improve the delivery efficacy.Specifically,recent advances in DNA nanostructures-based materials that promote the therapeutic efficacy of cancer immunotherapy through rational DNA sequence design to regulate the spatial distribution of immunotherapeutics loading are reviewed.The strategies to enhance precise targeting ability basing on nucleic acid aptamers and further enable immune checkpoint inhibitions are presented.The recent progress on the controllable release of immunotherapeutics triggered by specific stimulus is discussed.In the end,we provide insights for the subsequent realization of applications of DNA nanostructures-based materials for cancer immunotherapy in the future.展开更多
Monkeypox has been declared a public health emergency by the World Health Organization.There is an urgent need for efficient and safe vaccines against the monkeypox virus(MPXV)in response to the rapidly spreading monk...Monkeypox has been declared a public health emergency by the World Health Organization.There is an urgent need for efficient and safe vaccines against the monkeypox virus(MPXV)in response to the rapidly spreading monkeypox epidemic.In the age of COVID-19,mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale preparation.Here,we develop two MPXV quadrivalent mRNA vaccines,named mRNA-A-LNP and mRNA-B-LNP,based on two intracellular mature virus specific proteins(A29L and M1R)and two extracellular enveloped virus specific proteins(A35R and B6R).By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice,mice induce MPXV specific IgG antibodies and potent vaccinia virus(VACV)specific neutralizing antibodies.Further,it elicits efficient MPXV specific Th-1 biased cellular immunity,as well as durable effector memory T and germinal center B cell responses in mice.In addition,two doses of mRNA-A-LNP and mRNA-B-LNP are protective against the VACV challenge in mice.And,the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protects nude mice against the VACV challenge.Overall,our results demonstrate that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics,as well as against outbreaks caused by other orthopoxviruses,including the smallpox virus.展开更多
基金supported by the National Key Research and Development Program of China(No.2018YFA0902300)。
文摘Chain elongation via dinucleotide(dimer)block coupling was considered as an improved chemical technique capable of synthesizing high-quality longer oligonucleotide for de novo DNA synthesis in synthetic biology.However,this dimer block-wise approach was constrained by readily available dimer phosphoramidite with sufficient quality.Herein,through the usage of a one-pot coupling-oxidation-deprotection cascade process for preparing the key precursors 3'-hydroxyl dimers,then condensation with phosphorodiamidite,purification by flash column chromatography and precipation in methyl tert-butyl ether,a rationally designed dimer phosphoramidite bearing an internucleotide allyl phosphate and aβ-cyanoethyl phosphoramidite at the 3’-hydroxyl was synthesized.All sixteen allylic dimer phosphoramidites 2a-p were smoothly prepared with overall yields exceeding 50%and HPLC purities ranging from 97.40%to 99.69%.With these allylic reagents,oligonucleotides were successfully synthesized using a modified solid-phase phosphoramidite method and were completely deprotected under normal ammonialysis condition.Our results indicated that these dimer block-wise synthesized oligonucleotides were of sufficient quality for gene assembly and protein expression,thus,the allylic phosphate linked dimer phosphoramidite can serve as a promising dimer reagent that will enable the applications of long oligonucleotides.
基金supported by the National Key Research and Development Project of China(grant number 2021YFC2302400 to C.F.Q.)the National Natural Science Foundation of China(grant numbers 32130005 to C.F.Q.,92169120 to H.Z.,and 82172244 to X.W.).
文摘Coronavirus disease 2019(COVID-19)messenger RNA(mRNA)vaccines have succeeded unprecedentedly due to high protection efficacy and robust immune responses.However,systematic and longitudinal profiling of immune responses in mRNA vaccine recipients remains limited.Here,a cohort of ten healthy volunteers who received two doses of ARCoV,a non-modified mRNA vaccine,were enrolled.Peripheral blood samples were collected and analyzed using Olink technology,antibody detection,intracellular cytokine staining,single-cell sequencing,and T cell receptor(TCR)sequencing.ARCoV vaccination induced potent humoral and cellular immune responses,as well as elevated cytokines including CX-C motif chemokine ligand 10(CXCL10)and interferon-gamma(IFN-g).Single-cell sequencing revealed that ARCoV immunization induced an increased relative abundance of interferon-activated T cells,proliferative T cells,and naïve T cells.Monocytes and dendritic cells exhibited activation of the innate immune response,downregulation of hypoxia and glycolysis pathways,and a transient decrease in their proportions.Integrative analysis of single-cell RNA and TCR sequencing identified clonal expansion of effector T cells and killer cell immunoglobulin-like receptor(KIR)-expressing natural killer-like cells after the second dose.These findings deepen our understanding of the immune dynamics following mRNA vaccination and offer valuable insights for designing next-generation vaccines.This study was registered at the Chinese Clinical Trial Registry(ChiCTR2100049104).
基金This study was supported by the National Key Research and Development Program of China(Grant no.2021YFC2302405).
文摘Vaccines are used to protect human beings from various diseases.mRNA vaccines simplify the development process and reduce the production cost of conventional vaccines,making it possible to respond rapidly to acute and severe diseases,such as coronavirus disease 2019.In this study,a universal integrated platform for the streamlined and ondemand preparation of mRNA products directly from DNA templates was established.Target DNA templates were amplified in vitro by a polymerase chain reaction module and transcribed into mRNA sequences,which were magnetically purified and encapsulated in lipid nanoparticles.As an initial example,enhanced green fluorescent protein(eGFP)was used to test the platform.The expression capacity and efficiency of the products were evaluated by transfecting them into HEK-293T cells.The batch production rate was estimated to be 200–300μg of eGFP mRNA in 8 h.Furthermore,an mRNA vaccine encoding the receptor-binding domain(RBD)of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein was produced by this platform.The proposed integrated platform shows advantages for the universal and on-demand preparation of mRNA products,offering the potential to facilitate broad access to mRNA technology and enable the development of mRNA products,including the rapid supply of new mRNA-based vaccines in pandemic situations and personalized mRNA-based therapies for oncology and chronic infectious diseases,such as viral hepatitis and acquired immune deficiency syndrome.
基金supported by the National Natural Science Foundation of China(81830101 and 82301991)。
文摘Influenza A virus(IAV)poses a significant threat to human health.The outcome of IAV results from the viral-host interaction,with the underlying molecular mechanisms largely unknown.By integrating the plasma proteomics data of the IAV-infected patients into the viral-inflammation protein-protein interaction(VI-PPI)network created in this study,purine nucleoside phosphorylase(PNP),the critical enzyme in purine salvage,was identified as a potential hub gene that connected the different stages of IAV infection.Extended survival rates and reduced pulmonary inflammatory lesions were observed in alveolar epithelial cell(AEC)-specific PNP conditional knockout mice upon H1N1 infection.Mechanistically,PB1-F2 of IAV was revealed as a novel viral transcriptional factor to bind to the TATA box of PNP promoter,leading to enhanced purine salvage in H1N1-challenged AECs.The activation of PNP-mediated purine salvage was verified in IAV-infected patients and A549 cells.PNP knockdown elicited a purine metabolic shift from augmented salvage pathway to de novo synthesis,constraining both viral infection and pro-inflammatory signaling through APRT-AICAR-AMPK activation.Moreover,durdihydroartemisinin(DHA),predicted by VI-PPI as a novel PNP inhibitor,exerted beneficial effects on the survival and weight gain of H1N1-challenged mice via its direct binding to PNP.To reveal for the first time,we found that PNP,activated by IAV,plays a hub role within H1N1-host interaction,simultaneously modulating viral replication and hyperinflammation through purine salvage.Our study sheds new light on a“two-for-one”strategy by targeting purine salvage in combating IAV-related pathology,suggesting PNP as a potential novel anti-influenza host target.
基金supported by National Natural Science Foundation of China(No.22225505).
文摘Immunotherapy has been emerging as a potent strategy for cancer treatment.However,undesirable therapeutic efficacy remains a challenge,including low drug loading,imprecise targeting,and non-specific releasing.The drug delivery systems of immunotherapy play a key role in improving therapeutic efficacy and reducing side effects.To address these concerns,functional DNA nanostructures-based materials have been explored to achieve high loading capability,precise targeting,and controllable releasing.This review focuses on the crucial issues of delivery system for cancer immunotherapy and the strategies to improve the delivery efficacy.Specifically,recent advances in DNA nanostructures-based materials that promote the therapeutic efficacy of cancer immunotherapy through rational DNA sequence design to regulate the spatial distribution of immunotherapeutics loading are reviewed.The strategies to enhance precise targeting ability basing on nucleic acid aptamers and further enable immune checkpoint inhibitions are presented.The recent progress on the controllable release of immunotherapeutics triggered by specific stimulus is discussed.In the end,we provide insights for the subsequent realization of applications of DNA nanostructures-based materials for cancer immunotherapy in the future.
基金supported by the National Key R&D Program of China (2021YFC2302405)the National Natural Science Foundation of China (Grant No.81830101).
文摘Monkeypox has been declared a public health emergency by the World Health Organization.There is an urgent need for efficient and safe vaccines against the monkeypox virus(MPXV)in response to the rapidly spreading monkeypox epidemic.In the age of COVID-19,mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale preparation.Here,we develop two MPXV quadrivalent mRNA vaccines,named mRNA-A-LNP and mRNA-B-LNP,based on two intracellular mature virus specific proteins(A29L and M1R)and two extracellular enveloped virus specific proteins(A35R and B6R).By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice,mice induce MPXV specific IgG antibodies and potent vaccinia virus(VACV)specific neutralizing antibodies.Further,it elicits efficient MPXV specific Th-1 biased cellular immunity,as well as durable effector memory T and germinal center B cell responses in mice.In addition,two doses of mRNA-A-LNP and mRNA-B-LNP are protective against the VACV challenge in mice.And,the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protects nude mice against the VACV challenge.Overall,our results demonstrate that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics,as well as against outbreaks caused by other orthopoxviruses,including the smallpox virus.
