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1.
Biochem J ; 478(14): 2789-2791, 2021 07 30.
Article in English | MEDLINE | ID: covidwho-1526112

ABSTRACT

Post-translational modifications (PTMs) on histone proteins are known as epigenetic marks that demarcate the status of chromatin. These modifications are 'read' by specific reader proteins, which in turn recruit additional factors to modulate chromatin accessibility and the activity of the underlying DNA. Accumulating evidence suggests that these modifications are not restricted solely to histones, many non-histone proteins may function in a similar way through mimicking the histones. In this commentary, we briefly discuss a systematic study of the discovery of histone H3 N-terminal mimicry proteins (H3TMs), and their implications in chromatin regulation and drug discoveries.


Subject(s)
Chromatin/metabolism , DNA/metabolism , Histones/metabolism , Protein Processing, Post-Translational , Animals , Chromatin/genetics , Chromatin Assembly and Disassembly , DNA/genetics , Humans , Lysine/metabolism , Methylation , Models, Biological
2.
NPJ Biofilms Microbiomes ; 7(1): 81, 2021 11 18.
Article in English | MEDLINE | ID: covidwho-1526078

ABSTRACT

The oral microbiome has been connected with lung health and may be of significance in the progression of SARS-CoV-2 infection. Saliva-based SARS-CoV-2 tests provide the opportunity to leverage stored samples for assessing the oral microbiome. However, these collection kits have not been tested for their accuracy in measuring the oral microbiome. Saliva is highly enriched with human DNA and reducing it prior to shotgun sequencing may increase the depth of bacterial reads. We examined both the effect of saliva collection method and sequence processing on measurement of microbiome depth and diversity by 16S rRNA gene amplicon and shotgun metagenomics. We collected 56 samples from 22 subjects. Each subject provided saliva samples with and without preservative, and a subset provided a second set of samples the following day. 16S rRNA gene (V4) sequencing was performed on all samples, and shotgun metagenomics was performed on a subset of samples collected with preservative with and without human DNA depletion before sequencing. We observed that the beta diversity distances within subjects over time was smaller than between unrelated subjects, and distances within subjects were smaller in samples collected with preservative. Samples collected with preservative had higher alpha diversity measuring both richness and evenness. Human DNA depletion before extraction and shotgun sequencing yielded higher total and relative reads mapping to bacterial sequences. We conclude that collecting saliva with preservative may provide more consistent measures of the oral microbiome and depleting human DNA increases yield of bacterial sequences.


Subject(s)
Microbiota/genetics , Saliva/microbiology , Adult , Bacteria/genetics , COVID-19/genetics , DNA/genetics , DNA, Bacterial/genetics , Female , Humans , Male , Metagenome/genetics , Metagenomics/methods , Middle Aged , RNA, Ribosomal, 16S/genetics , SARS-CoV-2/pathogenicity , Sequence Analysis, DNA/methods
3.
Biosens Bioelectron ; 197: 113739, 2022 Feb 01.
Article in English | MEDLINE | ID: covidwho-1487616

ABSTRACT

The molecular biomarkers are molecules that are closely related to specific physiological states. Numerous molecular biomarkers have been identified as targets for disease diagnosis and biological research. To date, developing highly efficient probes for the precise detection of biomarkers has become an attractive research field which is very important for biological and biochemical studies. During the past decades, not only the small chemical probe molecules but also the biomacromolecules such as enzymes, antibodies, and nucleic acids have been introduced to construct of biosensor platform to achieve the detection of biomarkers in a highly specific and highly efficient way. Nevertheless, improving the performance of the biosensors, especially in clinical applications, is still in urgent demand in this field. A noteworthy example is the Corona Virus Disease 2019 (COVID-19) that breaks out globally in a short time in 2020. The COVID-19 was caused by the virus called SARS-CoV-2. Early diagnosis is very important to block the infection of the virus. Therefore, during these months scientists have developed dozens of methods to achieve rapid and sensitive detection of the virus. Nowadays some of these new methods have been applied for producing the commercial detection kit and help people against the disease worldwide. DNA-based biosensors are useful tools that have been widely applied in the detection of molecular biomarkers. The good stability, high specificity, and excellent biocompatibility make the DNA-based biosensors versatile in application both in vitro and in vivo. In this paper, we will review the major methods that emerged in recent years on the design of DNA-based biosensors and their applications. Moreover, we will also briefly discuss the possible future direction of DNA-based biosensors design. We believe this is helpful for people interested in not only the biosensor field but also in the field of analytical chemistry, DNA nanotechnology, biology, and disease diagnosis.


Subject(s)
Biosensing Techniques , COVID-19 , Biomarkers , DNA/genetics , Humans , SARS-CoV-2
4.
Cells ; 10(10)2021 10 11.
Article in English | MEDLINE | ID: covidwho-1463567

ABSTRACT

As of September 2021, twenty-one anti-COVID-19 vaccines have been approved in the world. Their utilization will expedite an end to the current pandemic. Besides the usual vaccine formats that include inactivated viruses (eight approved vaccines) and protein-based vaccines (four approved vaccines), three new formats have been validated: recombinant adenovirus (six approved vaccines), DNA (one approved vaccine), and messenger RNA (mRNA, two approved vaccines). The latter was the fastest (authorized in 2020 in the EU, the USA, and Switzerland). Most Western countries have reserved or use the protein vaccines, the adenovirus vaccines, and mRNA vaccines. I describe here the different vaccine formats in the context of COVID-19, detail the three formats that are chiefly reserved or used in Europe, Canada, and the USA, and discuss why the mRNA vaccines appear to be the superior format.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , RNA, Messenger , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Adenoviridae/genetics , Animals , Canada , DNA/genetics , Drug Approval , Europe , Humans , Mice , Patient Safety , United States
5.
Nano Lett ; 21(11): 4643-4653, 2021 06 09.
Article in English | MEDLINE | ID: covidwho-1303734

ABSTRACT

DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays. We further leveraged the microdroplet-enabled confinement effect to perform an ultralocalized droplet Cas12a assay, obtaining excellent specificity and single-molecule sensitivity. Moreover, we demonstrated its versatility and quantification capability by direct counting of diverse virus's DNAs (African swine fever virus, Epstein-Barr virus, and Hepatitis B virus) from clinical serum samples with a wide range of viral titers. Given the flexible programmability of crRNA, we envision this amplification-free technique as a versatile and quantitative platform for molecular diagnosis.


Subject(s)
African Swine Fever Virus , Epstein-Barr Virus Infections , African Swine Fever Virus/genetics , Animals , CRISPR-Cas Systems , DNA/genetics , Herpesvirus 4, Human , Swine
6.
Int J Mol Sci ; 22(7)2021 Mar 26.
Article in English | MEDLINE | ID: covidwho-1299435

ABSTRACT

The importance of gene expression regulation in viruses based upon G-quadruplex may point to its potential utilization in therapeutic targeting. Here, we present analyses as to the occurrence of putative G-quadruplex-forming sequences (PQS) in all reference viral dsDNA genomes and evaluate their dependence on PQS occurrence in host organisms using the G4Hunter tool. PQS frequencies differ across host taxa without regard to GC content. The overlay of PQS with annotated regions reveals the localization of PQS in specific regions. While abundance in some, such as repeat regions, is shared by all groups, others are unique. There is abundance within introns of Eukaryota-infecting viruses, but depletion of PQS in introns of bacteria-infecting viruses. We reveal a significant positive correlation between PQS frequencies in dsDNA viruses and corresponding hosts from archaea, bacteria, and eukaryotes. A strong relationship between PQS in a virus and its host indicates their close coevolution and evolutionarily reciprocal mimicking of genome organization.


Subject(s)
Computational Biology/methods , DNA/genetics , G-Quadruplexes , Genome, Viral , Viral Proteins/genetics , Archaea/virology , Bacteria/virology , Gene Expression Regulation , Genome , Humans , Viruses/genetics
7.
Forensic Sci Int Genet ; 54: 102561, 2021 09.
Article in English | MEDLINE | ID: covidwho-1292724

ABSTRACT

Due to advances in DNA profiling sensitivity as well as the implementation of various types of software to analyse these profiles, forensic biologists can provide opinions about results generated from very low levels of template DNA. The ability to obtain DNA profiles from such 'trace' DNA brings into question the mechanisms of transfer which led to it being deposited. This study investigates the level of DNA that is deposited by an individual to their work environment. DNA collection plates were placed at distances from 0.5 to 5 m from individuals' office desks and left for 1 day to 6 weeks before being swabbed and profiled with GlobalFiler. The results from this study indicate that an individual can deposit DNA in areas they were present, even if surfaces and/or objects were not directly contacted and even after only one day. Distance from a person, the length of time and the person themselves all play a role in the quantity of DNA that is deposited to one's surroundings.


Subject(s)
DNA Fingerprinting , Touch , DNA/genetics , Humans , Software
8.
Analyst ; 146(13): 4212-4218, 2021 Jun 28.
Article in English | MEDLINE | ID: covidwho-1253999

ABSTRACT

Molecular detection of pathogenic nucleic acids from patient samples requires incubating biochemical reactions at specific temperatures to amplify DNA. This incubation is typically carried out with an electrical heater and a temperature controller. To reduce test cost, to eliminate the need for manufacturing incubators, which may require significant time, and to enable electricity-free operation, we use energetic compounds such as an Mg(Fe) alloy mixed with a phase-change material (PCM) that undergoes phase transformation at the desired incubation temperature. We dubbed this composite Energetic Phase Change Material (EPCM). When the EPCM is brought into contact with water, the magnesium alloy interacts with the water to produce heat. The EPCM heats up to its phase transition temperature. Any excess heat is absorbed as latent heat and the system is maintained at its desired incubation temperature, independent of ambient temperatures, long enough to facilitate enzymatic amplification. The EPCM together with colorimetric amplicon detection facilitates an inexpensive, disposable, point-of-need diagnostic test that does not require any electric power. We demonstrate the feasibility of our approach by detecting SARS-Cov-2 in saliva samples either without any instrumentation or with a palm-size CCD camera that enables us to follow the amplification process in real time.


Subject(s)
COVID-19 , DNA/genetics , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , SARS-CoV-2 , Saliva
10.
Talanta ; 233: 122505, 2021 Oct 01.
Article in English | MEDLINE | ID: covidwho-1230793

ABSTRACT

Colorimetric sensors are recognized as a promising means for target molecule detection as they provide rapid, cost-effective, and facile sensing visible to the naked eye. Challenges remain though in terms of their detection sensitivity and specificity for short-length target genes. Herein, we demonstrate the successful combination of the catalytic hairpin DNA assembly (CHA) approach with enzyme-linked immunosorbent assay (ELISA)-mimicking techniques for a simple, sensitive, and sequence-specific colorimetric assay to detect short SARS-CoV-2 target cDNA. In the developed CHA-based chemiluminescent assay, a low concentration of target cDNA is continuously recycled to amplify dimeric DNA probes from two biotinylated hairpin DNA until the hairpin DNA is completely consumed. The dimeric DNA probes are effectively immobilized in a neutravidin-coated microplate well and then capture neutravidin-conjugated horseradish peroxidase via biotin-neutravidin interactions, resulting in a sensitive and selective colorless-to-blue color change. The developed sensing system exhibits a high sensitivity with a detection limit of ~1 nM for target cDNA as well as the ability to precisely distinguish a single-base mismatched mutant gene within 2 h. As the proposed system does not require complex protocols or expensive equipment to amplify target cDNA, it has the potential to be utilized as a powerful tool to improve the detection sensitivity of target genes for clinical diagnostics with colorimetric detection.


Subject(s)
Biosensing Techniques , COVID-19 , DNA, Catalytic , Colorimetry , DNA/genetics , DNA, Complementary , Humans , Limit of Detection , Luminescent Measurements , SARS-CoV-2
11.
Anal Chim Acta ; 1149: 238130, 2021 Mar 08.
Article in English | MEDLINE | ID: covidwho-985057

ABSTRACT

One challenge in point-of-care (POC) diagnostics is the lack of room-temperature methods for RNA detection based on enzymatic amplification and visualization steps. Here we perform reverse transcription lesion-induced DNA amplification (RT-LIDA), an isothermal amplification method that only requires T4 DNA ligase. RT-LIDA involves the RNA-templated ligation of DNA primers to form complementary DNA (cDNA) followed by toehold-mediated strand displacement of the cDNA and its exponential amplification via our isothermal ligase chain reaction LIDA. Each step is tuned to proceed at 28 °C, which falls within the range of global room temperatures. Using RT-LIDA, we can detect as little as ∼100 amol target RNA and can distinguish RNA target from total cellular RNA. Finally, we demonstrate that the resulting DNA amplicons can be detected colorimetrically, also at room temperature, by rapid, target-triggered disassembly of DNA-modified gold nanoparticles. This integrated amplification/detection platform requires no heating or visualization instrumentation, which is an important step towards realizing instrument-free POC testing.


Subject(s)
Metal Nanoparticles , Reverse Transcription , DNA/genetics , Gold , Nucleic Acid Amplification Techniques , RNA/genetics , Sensitivity and Specificity
12.
Anal Chem ; 93(8): 4126-4133, 2021 03 02.
Article in English | MEDLINE | ID: covidwho-1078274

ABSTRACT

The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV-vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.


Subject(s)
CRISPR-Cas Systems , Colorimetry/methods , DNA/chemistry , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Bacterial Proteins , Base Sequence , COVID-19/diagnosis , CRISPR-Associated Proteins , Coronavirus Nucleocapsid Proteins/genetics , DNA/genetics , Endodeoxyribonucleases , Gold/chemistry , Humans , Metal Nanoparticles/chemistry , Phosphoproteins/genetics , Polyproteins/genetics , RNA, Viral/genetics , Reverse Transcription , SARS-CoV-2/chemistry , Surface Plasmon Resonance , Viral Proteins/genetics
13.
Anal Chem ; 92(24): 15872-15879, 2020 12 15.
Article in English | MEDLINE | ID: covidwho-943827

ABSTRACT

Specificity of DNA polymerization plays a critical role in DNA replication and storage of genetic information. Likewise, biotechnological applications, such as nucleic acid detection, DNA amplification, and gene cloning, require high specificity in DNA synthesis catalyzed by DNA polymerases. However, errors in DNA polymerization (such as mis-incorporation and mis-priming) can significantly jeopardize the specificity. Herein, we report our discovery that the specificity of DNA enzymatic synthesis can be substantially enhanced (up to 100-fold higher) by attenuating DNA polymerase kinetics via the phosphorothioate dNTPs. This specificity enhancement allows convenient and sensitive nucleic acid detection, polymerization, PCR, and gene cloning with complex systems (such as human cDNA and genomic DNA). Further, we found that the specificity enhancement offered higher sensitivity (up to 50-fold better) for detecting nucleic acids, such as COVID-19 viral RNAs. Our findings have revealed a simple and convenient strategy for facilitating specificity and sensitivity of nucleic acid detection, amplification, and gene cloning.


Subject(s)
DNA/analysis , RNA, Viral/analysis , DNA/biosynthesis , DNA/genetics , DNA Nucleotidyltransferases/metabolism , Humans , Polymerase Chain Reaction , Polymerization , RNA, Viral/biosynthesis , RNA, Viral/genetics , SARS-CoV-2/genetics
14.
Nat Biomed Eng ; 4(12): 1168-1179, 2020 12.
Article in English | MEDLINE | ID: covidwho-780008

ABSTRACT

The control of viral outbreaks requires nucleic acid diagnostic tests that are sensitive, simple and fast. Here, we report a highly sensitive and specific one-pot assay for the fluorescence-based detection of RNA from pathogens. The assay, which can be performed within 30-50 min of incubation time and can reach a limit of detection of 0.1-attomolar RNA concentration, relies on a sustained isothermal reaction cascade producing an RNA aptamer that binds to a fluorogenic dye. The RNA aptamer is transcribed by the T7 RNA polymerase from the ligation product of a promoter DNA probe and a reporter DNA probe that hybridize with the target single-stranded RNA sequence via the SplintR ligase (a Chlorella virus DNA ligase). In 40 nasopharyngeal SARS-CoV-2 samples, the assay reached positive and negative predictive values of 95 and 100%, respectively. We also show that the assay can rapidly detect a range of viral and bacterial RNAs.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Transcription, Genetic/genetics , COVID-19/virology , Chlorella/metabolism , DNA/genetics , DNA Ligases/metabolism , DNA-Directed RNA Polymerases/metabolism , Diagnostic Tests, Routine/methods , Fluorescence , Humans , Nucleic Acid Amplification Techniques , Pandemics/prevention & control , Sensitivity and Specificity , Viral Proteins/metabolism
15.
Prim Care Diabetes ; 15(1): 4-9, 2021 02.
Article in English | MEDLINE | ID: covidwho-735354

ABSTRACT

BACKGROUND: Diabetes has been found to be one of the leading comorbidities associated with fatality in COVID-19 patients. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is facilitated by interaction with Angiotensin Converting Enzyme-2 (ACE2) and possible polymorphisms in ACE2 can be a determining factor in host-viral protein interaction. A significant shift of healthcare towards 'Telemedicine' is also on the rise. In this review, the possible effects of ACE2 polymorphisms on SARS-CoV-2 entry along with the escalation of 'telemedicine' is discussed. METHOD: An expansive literature search using keywords: "COVID-19", "SARS-CoV-2", "diabetes", "type 2 diabetes'', "type 1 diabetes", "ACE2", "polymorphism", "DPP4" and "telemedicine" was conducted on Pubmed and EMBASE till 7th August 2020. RESULT: Possible polymorphisms in ACE2 gene can play a role in influencing the virus entry in host body. Telemedicine can bring a new revolution for medical sector. CONCLUSION: COVID-19 severity is more heinous among diabetic population. So far, the in-silico studies involving human ACE2-viral Spike (S) interaction showed inconsistent predictions regarding some SNPs. But without actual in-vivo studies, a holistic understanding can't be established.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/epidemiology , DNA/genetics , Diabetes Mellitus, Type 2/genetics , Pandemics , Polymorphism, Genetic , Telemedicine/methods , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/genetics , COVID-19/metabolism , Comorbidity , Diabetes Mellitus, Type 2/epidemiology , Diabetes Mellitus, Type 2/metabolism , Humans , SARS-CoV-2
17.
Biosens Bioelectron ; 167: 112479, 2020 Nov 01.
Article in English | MEDLINE | ID: covidwho-684409

ABSTRACT

COVID-19 pandemic outbreak is the most astounding scene ever experienced in the 21st century. It has been determined to be caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With the global pandemic, the lack of efficient rapid and accurate molecular diagnostic testing tools has hindered the public opportunely response to the emerging viral threat. Herein, a DNA nanoscaffold hybrid chain reaction (DNHCR)-based nucleic acid assay strategy is reported for rapid detection of SARS-CoV-2 RNA. In this method, the DNA nanoscaffolds have been first constructed by the self-assembly of long DNA strands and self-quenching probes (H1). Then, the SARS-CoV-2 RNA will initiate the hybridization of H1 and free H2 DNA probes along the nanoscaffold, and an illuminated DNA nanostring is instantly obtained. By taking advantages of the localization design of the H1 probes and the temperature tolerance of the isothermal amplification, the proposed DNHCR method can detect target at short responding time (within 10 min) and mild condition (15 °C-35 °C). Moreover, the reliability of DNHCR method in serum and saliva samples have also been validated. Therefore, DNHCR-based method is expected to provide a simple and faster alternative to the traditional SARS-CoV-2 qRT-PCR assay.


Subject(s)
Betacoronavirus , Biosensing Techniques/methods , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/statistics & numerical data , Coronavirus Infections/epidemiology , DNA/chemical synthesis , DNA/chemistry , DNA/genetics , Feasibility Studies , Humans , Molecular Diagnostic Techniques/methods , Molecular Diagnostic Techniques/statistics & numerical data , Nanostructures/chemistry , Nanotechnology , Nucleic Acid Amplification Techniques/methods , Pandemics , Pneumonia, Viral/epidemiology , RNA, Viral/analysis , RNA, Viral/genetics , Reproducibility of Results , SARS-CoV-2 , Sensitivity and Specificity
18.
Forensic Sci Int Genet ; 48: 102346, 2020 09.
Article in English | MEDLINE | ID: covidwho-642775

ABSTRACT

The aggressive nature of the new SARS-2 corona virus now referred to as SARS-CoV-2 ; the seriousness and length of the period of infection; the fast and far-reaching transmissibility via liquid droplets that become air-borne when someone coughs, sneezes or speaks with increasing evidence to support actual airborne transmission; the presence of viral particles especially in body fluids and tissues, of viral positive individuals; and the persistence of the virus on different types of surfaces pose serious concerns for forensic practitioners, including forensic DNA analysts. Many forensic laboratories and law enforcement agencies need to address the inevitable changes that must be made in forensic DNA testing. In this article, we explore the effects of the COVID-19 pandemic on the collection, handling, storage and transport of biological samples for downstream DNA testing. This paper aims to open discussions on the urgency of balancing the need to conduct investigations in order to maintain public order with the requirements of effective biosafety protocols specifically formulated to protect human resources within the forensic science community.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , DNA/analysis , Forensic Genetics , Pandemics , Pneumonia, Viral/epidemiology , COVID-19 , Coronavirus Infections/virology , DNA/genetics , Databases, Genetic , Humans , Pneumonia, Viral/virology , SARS-CoV-2
19.
Science ; 367(6483): 1178, 2020 03 13.
Article in English | MEDLINE | ID: covidwho-124609
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