Journal of Nucleic Acids最新文献

{"title":"Dual Detection of Hepatitis B and C Viruses Using CRISPR-Cas Systems and Lateral Flow Assay.","authors":"Syeda Najidah Shahni, Sarah Albogami, Iqbal Azmi, Bijay Pattnaik, Rituparna Chaudhuri, Kapil Dev, Jawed Iqbal, Amit Sharma, Tanveer Ahmad","doi":"10.1155/2024/8819834","DOIUrl":"https://doi.org/10.1155/2024/8819834","url":null,"abstract":"<p><p>The development of sensitive and specific diagnostic tools for hepatitis B virus (HBV) and hepatitis C virus (HCV) remains crucial for effective disease management and control. In this study, we utilized CRISPR-Cas12 and CRISPR-Cas13 systems for the detection of HBV (DNA virus) and HCV (RNA virus), respectively. We designed and tested multiple guide RNAs (gRNAs) targeting both viruses, confirming successful cleavage of target sequences through gel electrophoresis and a fluorescent reporter assay. Using optimized gRNAs, we developed a lateral flow assay (LFA) for sensitive detection of HBV and HCV, demonstrating a concentration-dependent signal increase. Importantly, no cross-reactivity was observed with other viral targets. To further enhance sensitivity, we employed a dual-enzyme approach, combining Cas12 and Cas13 in a single reaction, which significantly improved detection limits for both viruses. Finally, we developed a dual antigen detection LFA strip capable of simultaneously detecting both HBV and HCV in a single sample. This approach holds promise for point-of-care (POC) diagnostics where the specific viral infection is unknown. This study addresses the current limitations in CRISPR-Cas based diagnostics, namely, the need for ultrasensitive detection methods and the ability to detect multiple antigens using a single test strip. Our findings demonstrate the feasibility of using CRISPR-Cas systems for highly sensitive and specific detection of HBV and HCV, paving the way for potential POC application.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11470818/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142467924","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genetic Polymorphisms and Forensic Parameters of Thirteen X-Chromosome Markers in the Iraqi Kurdish Population","authors":"Ara K. Mohammad, Bahez Ismael, Khanzad Ahmed Ali, Balnd M. Albarzinji","doi":"10.1155/2024/9125094","DOIUrl":"https://doi.org/10.1155/2024/9125094","url":null,"abstract":"X-chromosome short tandem repeat (X-STR) tools are crucial in forensic genetics and human population fields. This study presents the development and validation of a multiplex STR system consisting of thirteen X-STR loci and amelogenin specific to the human X chromosome. The system was optimized and tested for species specificity, sensitivity, stability, and DNA mixture using 9947A female and 9948 male control genomic DNA. The amplified products of nine loci were sequenced to determine the correct amplicon length. Allele frequencies, forensic parameters, mean exclusion chance (MEC), linkage disequilibrium (LD), and allelic patterns were investigated using DNA samples from 225 (159 male, 66 female) unrelated Kurdish individuals who live in Sulaymaniyah province in the Kurdistan region of Iraq. The most informative locus in the Kurdish population was GATA172D05, while the least informative locus was DXS10164. The results demonstrated that the 13 X-STR system is highly polymorphic and sensitive for forensic DNA identification. Genetic distance-based clustering, metric multidimensional scaling (MDS), and correlation matrix were analyzed for 19 ethnic groups and populations. The phylogenetic tree showed that populations clustered according to their ethnogeographic relationships. The findings revealed genetic links between the Iraqi Kurds, Caucasians, Iraqi Arabs, United States (U.S.) ethnic groups, and Chinese populations.","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140699302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis and Evaluation of MGB Polyamide-Oligonucleotide Conjugates as Gene Expression Control Compounds.","authors":"Kazuo Kamaike,&nbsp;Mutsumi Sano,&nbsp;Daisuke Sakata,&nbsp;Yu Nishihara,&nbsp;Hiroaki Amino,&nbsp;Akihiro Ohtsuki,&nbsp;Yui Okada,&nbsp;Takafumi Miyakawa,&nbsp;Makoto Kogawara,&nbsp;Mai Tsutsumi,&nbsp;Misato Takahashi,&nbsp;Etsuko Kawashima,&nbsp;Koichiro Ota,&nbsp;Hiroaki Miyaoka","doi":"10.1155/2023/2447998","DOIUrl":"https://doi.org/10.1155/2023/2447998","url":null,"abstract":"<p><p>MGB polyamide-oligonucleotide conjugates <b>ON 1</b>-<b>4</b> with linked MGB polyamides at the 2-exocyclic amino group of a guanine base using aminoalkyl linkers were synthesized and evaluated in terms of binding affinity for complementary DNA containing the MGB polyamide binding sequence using <i>T</i> _m and CD analyses. The MGB polyamides comprised pyrrole polyamides (Py₄- and Py₃-), which possess binding affinity for A-T base pairs, and imidazole (Im₃-) and pyrrole-<i>γ</i>-imidazole (Py₃-<i>γ</i>-Im₃-) polyamide hairpin motifs, which possess binding affinity for C-G base pairs. It was found that the stability of modified dsDNA was greatly influenced by the linker length. Py₄- and Py₃-oligonucleotide conjugates (<b>ON 1</b> (<i>n</i> = 4) and <b>ON 2</b> (<i>n</i> = 4)) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA. Although Im₃-oligonucleotide conjugate <b>ON 3</b> (<i>n</i> = 4) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA, stabilization of dsDNA by the imidazole amide moiety of <b>ON 3</b> (<i>n</i> = 4) was lower compared with the pyrrole amide moiety of <b>ON 2</b> (<i>n</i> = 4). The Py₃-<i>γ</i>-Im₃-oligonucleotide conjugate <b>ON 4</b> (<i>n</i> = 2), which possesses binding affinity for C-G base pairs via a pyrrole/imidazole combination and contains a 2-aminoethyl linker, showed high binding ability for complementary DNA. Furthermore, the DNA sequence recognition of MGB polyamide-oligonucleotide conjugates was investigated using single-base mismatch DNAs, which possess a mismatch base in the MGB polyamide binding sequence. The Py₃-<i>γ</i>-Im₃-oligonucleotide conjugate <b>ON 4</b> (<i>n</i> = 2) showed high sequence recognition ability for complementary DNA.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10030224/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9523227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparing Two Methods for the Isolation of Exosomes.","authors":"Mohammad A Aziz,&nbsp;Benedict Seo,&nbsp;Haizal M Hussaini,&nbsp;Merilyn Hibma,&nbsp;Alison M Rich","doi":"10.1155/2022/8648373","DOIUrl":"https://doi.org/10.1155/2022/8648373","url":null,"abstract":"<p><p>Exosomes are membrane-bound nanovesicles released by cells into their extracellular environment. They carry different types of RNA including mRNA which may be useful in the diagnosis of various diseases. Exosome isolation has been a challenge because of their small size; therefore, two exosome isolation methods were compared in this study. The Exoquick-TC PLUS™ exosome isolation kit (kit) was compared with the classic ultracentrifugation (UC) method for exosome isolation. In samples obtained using both methods, cryo-electron microscopy showed round or slightly elongated vesicles with diameters ranging from 50 to 150 nm and delimited by a bilayered membrane. Dynamic light scattering resulted in multiple peaks for kit exosomes, whereas a single peak was observed for UC exosomes. Significantly, more total RNA was present in UC exosomes in contrast to kit exosomes (<i>P</i> < 0.0001). This was reflected in subsequent mRNA analysis using qPCR, where UC exosomes had lower Ct values compared to kit exosomes. In conclusion, exosome characterization revealed the presence of exosomes in both UC and the kit samples. The kit samples presented additional peaks from DLS which might be due to impurities. Overall, due to a higher total RNA and mRNA content, UC is a better option for subsequent mRNA analysis; nevertheless, the kit can still be used if an ultracentrifuge is not available as four out of the five genes selected were detected and quantified using the kit.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2022-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9626211/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40443240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a Reference Method and Materials for Quantitative Measurement of UV-Induced DNA Damage in Mammalian Cells: Comparison of Comet Assay and Cell Viability.","authors":"Donald H Atha, Alessandro Tona, Vytas Reipa","doi":"10.1155/2022/9188636","DOIUrl":"10.1155/2022/9188636","url":null,"abstract":"<p><p>Application of DNA damage diagnostic tests is rapidly growing, in particular for ovarian, prostate, and skin cancers; environmental monitoring; chronic and degenerative diseases; and male infertility. Such tests suffer from significant variability among different laboratories due the lack of standardization, experimental validation, and differences in data interpretation. Reference methods and materials for quantitative measurement of UVA-induced DNA damage in mammalian cells are frequently needed. In this study, we examined the use of the single-cell gel electrophoresis (comet) assay to assess the UVA-induced DNA damage in surface-attached Chinese hamster ovary (CHO) cells treated with a photosensitizer as a candidate cellular oxidative damage reference material. We found that the comet images became diffused and the viability of the cells decreased substantially (>20%) as the UVA dose and benzo [a] pyrene (BaP) concentration exceeded 6.3 J/cm² and 10^-6 mol/L BaP. Maintaining the conditions of exposure within this range can improve DNA damage measurement fidelity, particularly if used as a quantitative reference method and to produce materials considered as an <i>in vitro</i> standard for the comet assay.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":1.3,"publicationDate":"2022-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9509282/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40376855","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Update on the Development of Toehold Switch-Based Approach for Molecular Diagnostic Tests of COVID-19","authors":"Almando Geraldi, N. N. T. Puspaningsih, Fatiha Khairunnisa","doi":"10.1155/2022/7130061","DOIUrl":"https://doi.org/10.1155/2022/7130061","url":null,"abstract":"A high volume of diagnostic tests is needed during the coronavirus disease 2019 (COVID-19) pandemic to obtain representative results. These results can help to design and implement effective policies to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis using current gold standard methods, i.e., real-time quantitative PCR (RT-qPCR), is challenging, especially in areas with limited trained personnel and health-related infrastructure. The toehold switch-based diagnostic system is a promising alternative method for detecting SARS-CoV-2 that has advantages such as inexpensive cost per testing, rapid, and highly sensitive and specific analysis. Moreover, the system can be applied to paper-based platforms, simplifying the distribution and utilization in low-resource settings. This review provides insight into the development of toehold switch-based diagnostic devices as the most recent methods for detecting SARS-CoV-2.","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2022-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48508335","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exposure to a Pathological Condition May Be Required for the Cells to Secrete Exosomes Containing mtDNA Aberration","authors":"Manjusha Vaidya, Sandeep Sreerama, Mariana Gaviria, K. Sugaya","doi":"10.1155/2022/7960198","DOIUrl":"https://doi.org/10.1155/2022/7960198","url":null,"abstract":"Exosomes, nanovesicles secreted by all cells, carry out intercellular communication by transmitting biologically active cargo comprising DNA, RNA, and proteins. These biomolecules reflect the status of their parent cells and can be altered by pathological conditions. Therefore, the researchers have been investigating differential sequences and quantities of DNA associated with exosomes as valuable biomarkers of diseases. Exosomes carry different types of DNA molecules, including genomic, cytoplasmic, and mitochondrial (mtDNA). The mtDNA aberrations are reported to be a hallmark of diseases involving oxidative stress, such as cancer and neurodegenerative diseases. Establishing robust in vitro models comprising appropriate cell lineages is the first step towards investigating disease-specific anomalies and testing therapeutics. Induced pluripotent stem (iPS) cells from patients with diseases have been used for this purpose since they can differentiate into various cells. The current study investigated mtDNA aberrations in exosomes secreted by primary cancer cells and neural stem cells (NSCs) differentiated from iPS cells. The primary cancer cells were isolated from surgically removed glioblastoma multiforme (GBM) tissue, and the iPS cells were produced from control and Alzheimer's disease (AD) subjects' B lymphocytes. We detected aberrations in mtDNA associated with exosomes secreted from GBM cells but not from the NSCs. This result indicates that the cells may not secrete exosomes carrying mtDNA aberration without exposure to a pathological condition. Thus, we may need to consider this fact when we use iPS cell-derived cells as an in vitro disease model.","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2022-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48842269","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Perturbing the Normal Level of SIDT1 Suppresses the Naked ASO Effect.","authors":"Masayuki Takahashi,&nbsp;Mineaki Seki,&nbsp;Masayuki Nashimoto,&nbsp;Tomohiro Kabuta","doi":"10.1155/2021/2458470","DOIUrl":"https://doi.org/10.1155/2021/2458470","url":null,"abstract":"<p><p>Although antisense oligonucleotide (ASO) therapeutics can be taken up by living cells without carrier molecules, a large part of incorporated ASOs are trapped in the endosomes and do not exert therapeutic effects. To improve their therapeutic effects, it would be important to elucidate the mechanism of cellular uptake and intracellular trafficking of ASOs. In this study, we investigated how SIDT1 affects cellular uptake and intracellular trafficking of ASOs. Fluorescence microscopic analysis suggested that most of naked ASOs are trafficked to the lysosomes via the endosomes. The data obtained from flow cytometry and fluorescence microscopy together showed that although the SIDT1 level barely affects the total cellular uptake of ASOs, it appears to affect the intracellular trafficking of ASOs. We also showed that SIDT1 exists mainly in the endoplasmic reticulum and that perturbing the normal level of SIDT1 suppresses the antisense effect of the naked ASO targeting miR-16.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2021-11-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8610720/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39660013","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DNAzymes, Novel Therapeutic Agents in Cancer Therapy: A Review of Concepts to Applications.","authors":"I B K Thomas,&nbsp;K A P Gaminda,&nbsp;C D Jayasinghe,&nbsp;D T Abeysinghe,&nbsp;R Senthilnithy","doi":"10.1155/2021/9365081","DOIUrl":"https://doi.org/10.1155/2021/9365081","url":null,"abstract":"<p><p>The past few decades have witnessed a rapid evolution in cancer drug research which is aimed at developing active biological interventions to regulate cancer-specific molecular targets. Nucleic acid-based therapeutics, including ribozymes, antisense oligonucleotides, small interference RNA (siRNA), aptamer, and DNAzymes, have emerged as promising candidates regulating cancer-specific genes at either the transcriptional or posttranscriptional level. Gene-specific catalytic DNA molecules, or DNAzymes, have shown promise as a therapeutic intervention against cancer in various <i>in vitro</i> and <i>in vivo</i> models, expediting towards clinical applications. DNAzymes are single-stranded catalytic DNA that has not been observed in nature, and they are synthesized through <i>in vitro</i> selection processes from a large pool of random DNA libraries. The intrinsic properties of DNAzymes like small molecular weight, higher stability, excellent programmability, diversity, and low cost have brought them to the forefront of the nucleic acid-based therapeutic arsenal available for cancers. In recent years, considerable efforts have been undertaken to assess a variety of DNAzymes against different cancers. However, their therapeutic application is constrained by the low delivery efficiency, cellular uptake, and target detection within the tumour microenvironment. Thus, there is a pursuit to identify efficient delivery methods <i>in vivo</i> before the full potential of DNAzymes in cancer therapy is realized. In this light, a review of the recent advances in the use of DNAzymes against cancers in preclinical and clinical settings is valuable to understand its potential as effective cancer therapy. We have thus sought to firstly provide a brief overview of construction and recent improvements in the design of DNAzymes. Secondly, this review stipulates the efficacy, safety, and tolerability of DNAzymes developed against major hallmarks of cancers tested in preclinical and clinical settings. Lastly, the recent advances in DNAzyme delivery systems along with the challenges and prospects for the clinical application of DNAzymes as cancer therapy are also discussed.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2021-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8575636/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39609707","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genotoxic Effects of Etoposide, Bleomycin, and Ethyl Methanesulfonate on Cultured CHO Cells: Analysis by GC-MS/MS and Comet Assay.","authors":"Donald H Atha, Erdem Coskun, Onur Erdem, Alessandro Tona, Vytas Reipa, Bryant C Nelson","doi":"10.1155/2020/8810105","DOIUrl":"10.1155/2020/8810105","url":null,"abstract":"<p><p>To evaluate methods for analysis of genotoxic effects on mammalian cell lines, we tested the effect of three common genotoxic agents on Chinese hamster ovary (CHO) cells by single-cell gel electrophoresis (comet assay) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Suspension-grown CHO cells were separately incubated with etoposide, bleomycin, and ethyl methanesulfonate and analyzed by an alkaline comet assay and GC-MS/MS. Although DNA strand breaks were detected by the comet assay after treatment with all three agents, GC-MS/MS could only detect DNA nucleobase lesions oxidatively induced by bleomycin. This demonstrates that although GC-MS/MS has limitations in detection of genotoxic effects, it can be used for selected chemical genotoxins that contribute to oxidizing processes. The comet assay, used in combination with GC-MS/MS, can be a more useful approach to screen a wide range of chemical genotoxins as well as to monitor other DNA-damaging factors.</p>","PeriodicalId":16575,"journal":{"name":"Journal of Nucleic Acids","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2020-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414336/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38271527","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}