Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change,insert,or remove a genomic sequence of interest.These advanced molecular tools include meganucleases...Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change,insert,or remove a genomic sequence of interest.These advanced molecular tools include meganucleases,zinc finger nucleases,transcription activator-like effector nucleases and RNA-guided engineered nucleases(RGENs),which create double-strand breaks at specific target sites in the genome,and repair DNA either by homologous recombination in the presence of donor DNA or via the error-prone non-homologous end-joining mechanism.A recently discovered group of RGENs known as CRISPR/Cas9 gene-editing systems allowed precise genome manipulation revealing a causal association between disease genotype and phenotype,without the need for the reengineering of the specific enzyme when targeting different sequences.CRISPR/Cas9 has been successfully employed as an ex vivo gene-editing tool in embryonic stem cells and patient-derived stem cells to understand pancreatic beta-cell development and function.RNA-guided nucleases also open the way for the generation of novel animal models for diabetes and allow testing the efficiency of various therapeutic approaches in diabetes,as summarized and exemplified in this manuscript.展开更多
Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China.In this study,sandwich hybridization integrated with nuclease protection assay(NPA-SH)was used to qu...Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China.In this study,sandwich hybridization integrated with nuclease protection assay(NPA-SH)was used to qualitatively and quantitatively detect P. globosa.Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields.The linear regression equation for P.globosa was obtained,and the lowest detection number of cells was 1.8×104 c...展开更多
Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects...Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay （NPA-SH） was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, and the regression equation is y=4× 10^- 6x＋ 0.694 9, in which x is the cell number, and y is the optical density, with r2=0.953 5. These results show that the NPA-SH method has good feasibility in the detection of P. donghaiense. Results of NPA-SH and microscopy are excellent for each sample. The NPA-SH method was a simple way in quantitative detection of P. donghaiense, and the whole process could be finished in about six hours, which provided a new approach in high-throughput sampling and long-term monitoring of P. donghaiense.展开更多
Animal studies have shown that amphoteric detergent and nuclease(DNase I and ribonuclease A) is the most reliable decellularization method of the peripheral nerve. However, the optimal combination of chemical reagents...Animal studies have shown that amphoteric detergent and nuclease(DNase I and ribonuclease A) is the most reliable decellularization method of the peripheral nerve. However, the optimal combination of chemical reagents for decellularization of human nerve allograft needs further investigation. To find the optimal protocol to remove the immunogenic cellular components of the nerve tissue and preserve the basal lamina and extracellular matrix and whether the optimal protocol can be applied to larger-diameter human peripheral nerves, in this study, we decellularized the median and sural nerves from the cadavers with two different methods: nonionic and anionic detergents(Triton X-100 and sodium deoxycholate) and amphoteric detergent and nuclease(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS), deoxyribonuclease I, and ribonuclease A). All cellular components were successfully removed from the median and sural nerves by amphoteric detergent and nuclease. Not all cellular components were removed from the median nerve by nonionic and anionic detergent. Both median and sural nerves treated with amphoteric detergent and nuclease maintained a completely intact extracellular matrix. Treatment with nonionic and anionic detergent decreased collagen content in both median and sural nerves, while the amphoteric detergent and nuclease treatment did not reduce collagen content. In addition, a contact cytotoxicity assay revealed that the nerves decellularized by amphoteric detergent and nuclease was biocompatible. Strength failure testing demonstrated that the biomechanical properties of nerves decellularized with amphoteric detergent and nuclease were comparable to those of fresh controls. Decellularization with amphoteric detergent and nuclease better remove cellular components and better preserve extracellular matrix than decellularization with nonionic and anionic detergents, even in large-diameter human peripheral nerves. In Korea, cadaveric studies are not yet legally subject to Institutional Review Board review.展开更多
Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectrosc...Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics research. We first introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside cavities of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at sites inside the protein molecule＇s cavities clearly reveals characteristics of the local environment. These solvation behaviors are directly correlated to enzyme activity.展开更多
The distribution and specificity of extracellular nucleases produced by marine fungi belonging to eleven genera, namely: Alternaria, Aspergillus, Aureobasidium, Chaetomium, Fusarium, Gliomastix, Humicola, Penicillium,...The distribution and specificity of extracellular nucleases produced by marine fungi belonging to eleven genera, namely: Alternaria, Aspergillus, Aureobasidium, Chaetomium, Fusarium, Gliomastix, Humicola, Penicillium, Scopulariopsis, Wardomyces, Periconia, have implied its important function in the organic phosphorus and nitrogen circle in the Ocean. The fungal nucleases of 64 isolates tested were more or less specific for single-stranded DNA with a high preferential specificity towards poly-U substrate with forming of 5’-phosphate mononucleotides. A couple of the nucleases were capable of RNA digesting. The highest level of extracellular nucleolytic ability was observed in Penicillium spp. isolates. The tight correlation found between extracellular nuclease activity and the rate of thymidine uptake by actively growing and sporulating marine fungus Penicillium melinii suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.展开更多
Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies.Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis.DNA fragments can be efficie...Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies.Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis.DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time.The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions.Deoxyadenosine monophosphate(dAMP)was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis.The linear range in DNA quantitation by this method is 1.2-5000 ng/mL.In the validation,the inter-day and intra-day accuracies were within 90%-110%,and the inter-day and intra-day precision were acceptable(RSD<10%).The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model.Compared to the quantitation methods using UV absorbance,the reported method provides an enhanced sensitivity,and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.展开更多
Microalgae are photosynthetic microorganisms that function as primary producers in aquatic ecosystems. Some species of microalgae undergo rapid growth and cause harmful blooms in marine ecosystems. Heterocapsa triquet...Microalgae are photosynthetic microorganisms that function as primary producers in aquatic ecosystems. Some species of microalgae undergo rapid growth and cause harmful blooms in marine ecosystems. Heterocapsa triquetra is one of the most common bloom-forming species in estuarine and coastal waters worldwide. Although this species does not produce toxins, unlike some other Heterocapsa species, the high density of its blooms can cause significant ecological damage. We developed a H. triquetra species-specific nuclease protection assay sandwich hybridization（NPA-SH） probe that targets the large subunit of ribosomal RNA（LSU r RNA）. We tested probe specificity and sensitivity with five other dinoflagellates that also cause red tides. Our assay detected H.triquetra at a concentration of 1.5×10^4 cells/m L, more sensitive than required for a red-tide guidance warning by the Korea Ministry of Oceans and Fisheries in 2015（3.0×10^4 cells/m L）. We also used the NPA-SH assay to monitor H. triquetra in the Tongyeong region of the southern sea area of Korea during 2014. This method could detect H.triquetra cells within 3 h. Our assay is useful for monitoring H. triquetra under field conditions.展开更多
Objective: To establish a novel approach for quick and highly efficient verification of human gene imprinting. Methods: A pair of dye-labelled probes, 5' nuclease assay was combined with RT-PCR to determine the ge...Objective: To establish a novel approach for quick and highly efficient verification of human gene imprinting. Methods: A pair of dye-labelled probes, 5' nuclease assay was combined with RT-PCR to determine the genotype of a transcribed single nucleotide polymorphism (SNP) rs705(C>T) of a known imprinted gene, small nuclear ribonucleotide protein N (SNRPN), on both genomic DNA and cDNA of human lym-phoblast cell lines. Results: Allele discrimination showed a clear monoallelic expression pattern of SNRPN, which was confirmed by RT-PCR based restriction fragment length polymorphism (RFLPs). Pedigree analysis verified the paternal origin of expressed allele, which was in consistency with previous report. Conclusion: Transcribed SNP is an ideal marker for detecting gene imprinting by 5' nuclease assay. This approach also may be used to discover differential allele expression of non-imprinted genes, finding out gene cis-acting functional polymorphism.展开更多
Tryptophan （Trp） is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the int...Tryptophan （Trp） is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the integrity of the overall structure. To evaluate this effect, we design thirteen double point mutants of staphylococcal nuclease, each of which has a single Trp residue planted at an internal site. The studies on Trp fluorescence, ANS-binding fluorescence, far- and near-UV CD spectra, and enzymatic activity are carried out. It is found that the mutation at the hydrophobic core of protein generates molten globular state conformation, which is a loose structure compared to their original compactness in wild type （WT）. Its enzyme activity and surface hydrophobicity are also affected. The studies show that by proper site designing and external binding, Trp mutagenesis is a suitable method for carrying out the study on site specified dynamics of proteins.展开更多
基金the Akdeniz University Scientific Research Commission and the Scientific and Technological Research Council of Turkey,No.TUBITAK-215S820.
文摘Targeted genome editing is a continually evolving technology employing programmable nucleases to specifically change,insert,or remove a genomic sequence of interest.These advanced molecular tools include meganucleases,zinc finger nucleases,transcription activator-like effector nucleases and RNA-guided engineered nucleases(RGENs),which create double-strand breaks at specific target sites in the genome,and repair DNA either by homologous recombination in the presence of donor DNA or via the error-prone non-homologous end-joining mechanism.A recently discovered group of RGENs known as CRISPR/Cas9 gene-editing systems allowed precise genome manipulation revealing a causal association between disease genotype and phenotype,without the need for the reengineering of the specific enzyme when targeting different sequences.CRISPR/Cas9 has been successfully employed as an ex vivo gene-editing tool in embryonic stem cells and patient-derived stem cells to understand pancreatic beta-cell development and function.RNA-guided nucleases also open the way for the generation of novel animal models for diabetes and allow testing the efficiency of various therapeutic approaches in diabetes,as summarized and exemplified in this manuscript.
基金supported by the National Hi-Tech Research and Development Program(863)of China(No.2006AA09Z178,2001AA635090)the National Nat-ural Science Foundation of China(No.40706044)
文摘Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China.In this study,sandwich hybridization integrated with nuclease protection assay(NPA-SH)was used to qualitatively and quantitatively detect P. globosa.Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields.The linear regression equation for P.globosa was obtained,and the lowest detection number of cells was 1.8×104 c...
基金The National High Technology Research and Development Program ("863" Program) of China under contract No 2006AA09Z178 the National Natural Science Foundation of China under contract No 40706044
文摘Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay （NPA-SH） was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, and the regression equation is y=4× 10^- 6x＋ 0.694 9, in which x is the cell number, and y is the optical density, with r2=0.953 5. These results show that the NPA-SH method has good feasibility in the detection of P. donghaiense. Results of NPA-SH and microscopy are excellent for each sample. The NPA-SH method was a simple way in quantitative detection of P. donghaiense, and the whole process could be finished in about six hours, which provided a new approach in high-throughput sampling and long-term monitoring of P. donghaiense.
基金supported by the Korea Health Industry Development Institute,No.HI17C1221(to JKK)National Research Foundation of Korea,Nos.NRF-2017R1A2B4003692,NRF-2020R1A2C1006656(both to JKK)the Asan Institute for Life Sciences,Asan Medical Center,Nos.2018-766,2019IP0766(both to JKK)。
文摘Animal studies have shown that amphoteric detergent and nuclease(DNase I and ribonuclease A) is the most reliable decellularization method of the peripheral nerve. However, the optimal combination of chemical reagents for decellularization of human nerve allograft needs further investigation. To find the optimal protocol to remove the immunogenic cellular components of the nerve tissue and preserve the basal lamina and extracellular matrix and whether the optimal protocol can be applied to larger-diameter human peripheral nerves, in this study, we decellularized the median and sural nerves from the cadavers with two different methods: nonionic and anionic detergents(Triton X-100 and sodium deoxycholate) and amphoteric detergent and nuclease(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS), deoxyribonuclease I, and ribonuclease A). All cellular components were successfully removed from the median and sural nerves by amphoteric detergent and nuclease. Not all cellular components were removed from the median nerve by nonionic and anionic detergent. Both median and sural nerves treated with amphoteric detergent and nuclease maintained a completely intact extracellular matrix. Treatment with nonionic and anionic detergent decreased collagen content in both median and sural nerves, while the amphoteric detergent and nuclease treatment did not reduce collagen content. In addition, a contact cytotoxicity assay revealed that the nerves decellularized by amphoteric detergent and nuclease was biocompatible. Strength failure testing demonstrated that the biomechanical properties of nerves decellularized with amphoteric detergent and nuclease were comparable to those of fresh controls. Decellularization with amphoteric detergent and nuclease better remove cellular components and better preserve extracellular matrix than decellularization with nonionic and anionic detergents, even in large-diameter human peripheral nerves. In Korea, cadaveric studies are not yet legally subject to Institutional Review Board review.
基金Project supported by the National Basic Research Program of China(Grant Nos.2013CB921904,2009CB930504,and 2013CB328700)the National Natural Science Foundation of China(Grant Nos.11074016,11121091,10934001,61177020,11134001,and 10828407)
文摘Internal solvation of protein was studied by site-directed mutagenesis, with which an intrinsically fluorescent probe,tryptophan, is inserted into the desired position inside a protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics research. We first introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside cavities of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at sites inside the protein molecule＇s cavities clearly reveals characteristics of the local environment. These solvation behaviors are directly correlated to enzyme activity.
文摘The distribution and specificity of extracellular nucleases produced by marine fungi belonging to eleven genera, namely: Alternaria, Aspergillus, Aureobasidium, Chaetomium, Fusarium, Gliomastix, Humicola, Penicillium, Scopulariopsis, Wardomyces, Periconia, have implied its important function in the organic phosphorus and nitrogen circle in the Ocean. The fungal nucleases of 64 isolates tested were more or less specific for single-stranded DNA with a high preferential specificity towards poly-U substrate with forming of 5’-phosphate mononucleotides. A couple of the nucleases were capable of RNA digesting. The highest level of extracellular nucleolytic ability was observed in Penicillium spp. isolates. The tight correlation found between extracellular nuclease activity and the rate of thymidine uptake by actively growing and sporulating marine fungus Penicillium melinii suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.
文摘Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies.Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis.DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time.The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions.Deoxyadenosine monophosphate(dAMP)was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis.The linear range in DNA quantitation by this method is 1.2-5000 ng/mL.In the validation,the inter-day and intra-day accuracies were within 90%-110%,and the inter-day and intra-day precision were acceptable(RSD<10%).The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model.Compared to the quantitation methods using UV absorbance,the reported method provides an enhanced sensitivity,and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.
基金The Public Welfare&Safety Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Science,ICT&Future Planning under contract No.NRF-2013M3A2A1067529
文摘Microalgae are photosynthetic microorganisms that function as primary producers in aquatic ecosystems. Some species of microalgae undergo rapid growth and cause harmful blooms in marine ecosystems. Heterocapsa triquetra is one of the most common bloom-forming species in estuarine and coastal waters worldwide. Although this species does not produce toxins, unlike some other Heterocapsa species, the high density of its blooms can cause significant ecological damage. We developed a H. triquetra species-specific nuclease protection assay sandwich hybridization（NPA-SH） probe that targets the large subunit of ribosomal RNA（LSU r RNA）. We tested probe specificity and sensitivity with five other dinoflagellates that also cause red tides. Our assay detected H.triquetra at a concentration of 1.5×10^4 cells/m L, more sensitive than required for a red-tide guidance warning by the Korea Ministry of Oceans and Fisheries in 2015（3.0×10^4 cells/m L）. We also used the NPA-SH assay to monitor H. triquetra in the Tongyeong region of the southern sea area of Korea during 2014. This method could detect H.triquetra cells within 3 h. Our assay is useful for monitoring H. triquetra under field conditions.
文摘Objective: To establish a novel approach for quick and highly efficient verification of human gene imprinting. Methods: A pair of dye-labelled probes, 5' nuclease assay was combined with RT-PCR to determine the genotype of a transcribed single nucleotide polymorphism (SNP) rs705(C>T) of a known imprinted gene, small nuclear ribonucleotide protein N (SNRPN), on both genomic DNA and cDNA of human lym-phoblast cell lines. Results: Allele discrimination showed a clear monoallelic expression pattern of SNRPN, which was confirmed by RT-PCR based restriction fragment length polymorphism (RFLPs). Pedigree analysis verified the paternal origin of expressed allele, which was in consistency with previous report. Conclusion: Transcribed SNP is an ideal marker for detecting gene imprinting by 5' nuclease assay. This approach also may be used to discover differential allele expression of non-imprinted genes, finding out gene cis-acting functional polymorphism.
基金Supported by the National Key Basic Research Program of China under Grant Nos 2013CB921904,2013CB328701-2013CB328706the National Natural Science Foundation of China under Grant Nos 11074016,11121091,61177020,11134001and 10828407
文摘Tryptophan （Trp） is an intrinsic fluorescent probe for detecting the site-specified dynamics inside/outside protein. It is found that the Trp can easily be inserted in desired sites of protein, which affects the integrity of the overall structure. To evaluate this effect, we design thirteen double point mutants of staphylococcal nuclease, each of which has a single Trp residue planted at an internal site. The studies on Trp fluorescence, ANS-binding fluorescence, far- and near-UV CD spectra, and enzymatic activity are carried out. It is found that the mutation at the hydrophobic core of protein generates molten globular state conformation, which is a loose structure compared to their original compactness in wild type （WT）. Its enzyme activity and surface hydrophobicity are also affected. The studies show that by proper site designing and external binding, Trp mutagenesis is a suitable method for carrying out the study on site specified dynamics of proteins.
