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PLoS Computational Biology Oct 2023Long-read RNA sequencing has arisen as a counterpart to short-read sequencing, with the potential to capture full-length isoforms, albeit at the cost of lower depth. Yet...
Long-read RNA sequencing has arisen as a counterpart to short-read sequencing, with the potential to capture full-length isoforms, albeit at the cost of lower depth. Yet this potential is not fully realized due to inherent limitations of current long-read assembly methods and underdeveloped approaches to integrate short-read data. Here, we critically compare the existing methods and develop a new integrative approach to characterize a particularly challenging pool of low-abundance long noncoding RNA (lncRNA) transcripts from short- and long-read sequencing in two distinct cell lines. Our analysis reveals severe limitations in each of the sequencing platforms. For short-read assemblies, coverage declines at transcript termini resulting in ambiguous ends, and uneven low coverage results in segmentation of a single transcript into multiple transcripts. Conversely, long-read sequencing libraries lack depth and strand-of-origin information in cDNA-based methods, culminating in erroneous assembly and quantitation of transcripts. We also discover a cDNA synthesis artifact in long-read datasets that markedly impacts the identity and quantitation of assembled transcripts. Towards remediating these problems, we develop a computational pipeline to "strand" long-read cDNA libraries that rectifies inaccurate mapping and assembly of long-read transcripts. Leveraging the strengths of each platform and our computational stranding, we also present and benchmark a hybrid assembly approach that drastically increases the sensitivity and accuracy of full-length transcript assembly on the correct strand and improves detection of biological features of the transcriptome. When applied to a challenging set of under-annotated and cell-type variable lncRNA, our method resolves the segmentation problem of short-read sequencing and the depth problem of long-read sequencing, resulting in the assembly of coherent transcripts with precise 5' and 3' ends. Our workflow can be applied to existing datasets for superior demarcation of transcript ends and refined isoform structure, which can enable better differential gene expression analyses and molecular manipulations of transcripts.
Topics: DNA, Complementary; RNA, Long Noncoding; Sequence Analysis, RNA; Transcriptome; Gene Library; Protein Isoforms; High-Throughput Nucleotide Sequencing
PubMed: 37883581
DOI: 10.1371/journal.pcbi.1011576 -
Nanoscale Horizons Jul 2023Due to the high complexity, diversity and heterogeneity of tumor occurrence and development, multi-mode synergistic therapy is more effective than single treatment modes...
Due to the high complexity, diversity and heterogeneity of tumor occurrence and development, multi-mode synergistic therapy is more effective than single treatment modes to improve the antitumor efficacy. Also, multifunctional probes are crucial to realize synergistic therapy. Herein, a multifunctional DNA tetrahedron nanoprobe was ingeniously designed to simultaneously achieve chemodynamic therapy (CDT) and gene silencing for synergistic antitumor. The multifunctional DNA tetrahedron nanoprobe, DNA tetrahedron-silver nanocluster-antagomir-21 (D-sgc8-DTNS-AgNCs-Anta-21), integrated a CDT reagent (DNA-AgNCs) and miRNA-21 inhibitor (Anta-21) with a specific recognition probe (aptamer). After targeted entry in cancer cells, D-sgc8-DTNS-AgNCs-Anta-21 silenced endogenous miRNA-21 by Anta-21 and produced highly toxic ˙OH by reacting with HO, which induced apoptosis in the tumor cells. The targeted recognition of aptamers led to the concentration-dependent death of HeLa cells. On the contrary, the cell survival rate of normal cells was basically unaffected with an increase in the concentration of D-sgc8-DTNS-AgNCs-Anta-21. Therefore, the diverse functions, biocompatibility and programmability of DNA provide a useful and easy way to assemble multifunctional probes for synergistic therapy.
Topics: Humans; HeLa Cells; Hydrogen Peroxide; DNA; MicroRNAs; Gene Silencing
PubMed: 37317707
DOI: 10.1039/d2nh00575a -
Journal of Clinical Laboratory Analysis Oct 2023Targeted next-generation sequencing (NGS) is a powerful and suitable approach to comprehensively identify multiple types of variants in tumors. RNA-based NGS is...
BACKGROUND
Targeted next-generation sequencing (NGS) is a powerful and suitable approach to comprehensively identify multiple types of variants in tumors. RNA-based NGS is increasingly playing an important role in precision oncology. Both parallel and sequential DNA- and RNA-based approaches are expensive, burdensome, and have long turnaround times, which can be impractical in clinical practice. A streamlined, unified DNA- and RNA-based NGS approach is urgently needed in clinical practice.
METHODS
A DNA/RNA co-hybrid capture sequencing (DRCC-Seq) approach was designed to capture pre-capture DNA and RNA libraries in a single tube and convert them into one NGS library. The performance of the DRCC-Seq approach was evaluated by a panel of reference standards and clinical samples.
RESULTS
The average depth, DNA data ratio, capture ratio, and target coverage 250 (×) of the DNA panel data had a negative correlation with an increase in the proportion of RNA probes. The SNVs, indels, fusions, and MSI status were not affected by the proportion of RNA probes, but the copy numbers of the target genes were higher than expected in the standard materials, and many unexpected gene amplifications were found using D:R (1:2) and D:R (1:4) probe panels. The optimal ratio of DNA and RNA probes in the combined probe panel was 1:1 using the DRCC-Seq approach. The DRCC-Seq approach was feasible and reliable for detecting multiple types of variants in reference standards and real-world clinical samples.
CONCLUSIONS
The DRCC-Seq approach is more cost-effective, with a shorter turnaround time and lower labor requirements than either parallel or sequential targeted DNA NGS and RNA NGS. It is feasible to identify multiple genetic variations at the DNA and RNA levels simultaneously in clinical practice.
Topics: Humans; Neoplasms; RNA; Nucleic Acids; RNA Probes; Precision Medicine; DNA; High-Throughput Nucleotide Sequencing
PubMed: 37877443
DOI: 10.1002/jcla.24977 -
Analytica Chimica Acta Oct 2023Tumor-related mRNA detection is significant and interesting. The current mRNA detection method has the challenge of quantifying long mRNA sequences. Herein, a Y-shaped...
Tumor-related mRNA detection is significant and interesting. The current mRNA detection method has the challenge of quantifying long mRNA sequences. Herein, a Y-shaped DNA probe with three target-binding segments was developed to detect tumor-related mRNA. This Y-shaped DNA probe (Y-probe) was assembled by six single DNA strands. Among these DNA strands, two DNA strands contained the split G-quadruplex sequence, and two DNA strands were modified with a pair of fluorophore and quencher, which were used to produce the detectable signal. In the presence of a long target mRNA sequence, target mRNA was hybridized with the three target-binding segments of the Y-probe, resulting in the increased fluorescence of G-quadruplex specific dye Thioflavin T and the decreased fluorescence of fluorophore, which could achieve the ratio detection of target mRNA. The Y-probe exhibited a low detection limit of 17.53 nM. Moreover, this probe showed high accuracy due to the benefits of three target-binding segments.
Topics: DNA Probes; Fluorescence; Fluorescent Dyes; G-Quadruplexes; Ionophores; RNA, Messenger
PubMed: 37604619
DOI: 10.1016/j.aca.2023.341633 -
Talanta Jun 2024Clustered regularly interspaced short palindromic repeat (CRISPR) system has been explored as an efficient tool for nucleic acid diagnostics. However, it normally needs...
Clustered regularly interspaced short palindromic repeat (CRISPR) system has been explored as an efficient tool for nucleic acid diagnostics. However, it normally needs instrumentation or produces turn-off signals. Herein, a bulged Y-shape DNA (Y-DNA) nanoassembly was designed and synthesized as a novel turn-on probe. A CRISPR/Cas12a and Y-DNA probe mediated colorimetric assay (named as CYMCOA) strategy was developed for visual detection of pathogen DNA. Upon activating Cas12a with pathogen DNA, the Y-DNA bulge is catalytically trans-cleaved, releasing the G-quadruplex sequence embedded in the Y-DNA nanoassembly as a peroxidase-like DNAzyme. Visible signals with chromogen substrates are thus produced. The CYMCOA strategy was combined with recombinase polymerase amplification (RPA), an isothermal amplification technique, in detecting Helicobacter pylori (Hp) bacteria and SARS-CoV-2 N plasmids as two model pathogens. The bioassay has very excellent detection sensitivity and specificity, owing to the triple cascade amplification reactions and the very low mismatch tolerance. The lower limit of detection values were 0.16 cfu⋅mL, 1.5 copies⋅μL, and 0.17 copies⋅μL for Hp bacteria, Hp plasmids, and SARS-CoV-2 N plasmids respectively. The detection is fast and accurate. The colorimetric bioassay strategy provides to be a simple, accurate, fast and instrumentation-free platform for nucleic acids detections in various settings, including crude and emergent situations.
PubMed: 38852348
DOI: 10.1016/j.talanta.2024.126348 -
ArXiv Mar 2024DNA regulation and repair processes require direct interactions between proteins and DNA at specific sites. Local fluctuations of the sugar-phosphate backbones and bases...
DNA regulation and repair processes require direct interactions between proteins and DNA at specific sites. Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA 'breathing') play a central role in such processes. Here we review the development and application of novel spectroscopic methods and analyses - both at the ensemble and single-molecule levels - to study structural and dynamic properties of exciton-coupled cyanine and fluorescent nucleobase analogue dimer-labeled DNA constructs at key positions involved in protein-DNA complex assembly and function. The exciton-coupled dimer probes act as 'sensors' of the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probes. These methods can be used to study the mechanisms of protein binding and function at these sites.
PubMed: 38584614
DOI: No ID Found -
Fa Yi Xue Za Zhi Oct 2023To explore the feasibility of genetic marker detection of semen-specific coding region single nucleotide polymorphism (cSNP) based on SNaPshot technology in semen stains...
OBJECTIVES
To explore the feasibility of genetic marker detection of semen-specific coding region single nucleotide polymorphism (cSNP) based on SNaPshot technology in semen stains and mixed body fluid identification.
METHODS
Genomic DNA (gDNA) and total RNA were extracted from 16 semen stains and 11 mixtures composed of semen and venous blood, and the total RNA was reverse transcribed into complementary DNA (cDNA). The cSNP genetic markers were screened on the validated semen-specific mRNA coding genes. The cSNP multiplex detection system based on SNaPshot technology was established, and samples were genotyped by capillary electrophoresis (CE).
RESULTS
A multiplex detection system containing 5 semen-specific cSNPs was successfully established. In 16 semen samples, except the cSNP located in the gene showed allele loss in cDNA detection results, the gDNA and cDNA typing results of other cSNPs were highly consistent. When detecting semen-venous blood mixtures, the results of cSNP typing detected were consistent with the genotype of semen donor and were not interfered by the genotype of venous blood donor.
CONCLUSIONS
The method of semen-specific cSNPs detection by SNaPshot technology method can be applied to the genotyping of semen (stains) and provide information for determining the origin of semen in mixed body fluids (stains).
Topics: Genetic Markers; Semen; Polymorphism, Single Nucleotide; DNA, Complementary; Body Fluids; RNA, Messenger; DNA; Saliva; Forensic Genetics
PubMed: 38006266
DOI: 10.12116/j.issn.1004-5619.2021.510804 -
Heliyon Jun 2024Cervical cancer is caused by changes in the cervix that lead to precancerous cells and eventually progress to cancer. Human papillomavirus (HPV) infections are the...
Cervical cancer is caused by changes in the cervix that lead to precancerous cells and eventually progress to cancer. Human papillomavirus (HPV) infections are the primary cause of cervical cancer. Early detection of HPV is crucial in preventing cervical cancer, and regular screening for HPV infection can identify cell changes before they develop into cancer. While Pap smear tests are reliable for cervical cancer screening, they are critical, expensive, and labor-intensive. Therefore, researchers are focusing on identifying blood-based biomarkers using biosensors for cervical cancer screening. HPV strains 16, 45, and 18 are common culprits in cervical cancer. This study aimed to develop an HPV-16 DNA biosensor on a zeolite-iron oxide (zeolite-IO) modified interdigitated electrode (IDE) sensor. The DNA probe was immobilized on the IDE through amine-modified zeolite-IO, enhancing the hybridization of the target and DNA probe. The detection limit of the DNA-DNA duplex was found to be 7.5 pM with an R value of 0.9868. Additionally, control experiments with single and triple mismatched sequences showed no increase in current responses, and the identification of target DNA in a serum-spiked sample indicated specific and selective target identification.
PubMed: 38845893
DOI: 10.1016/j.heliyon.2024.e31851 -
American Journal of Cancer Research 2023To develop a novel nano DNA fluorescent probe for in situ detection of CSTF2 in liver cancer (LC) and study its correlation with the development of LC, we developed...
To develop a novel nano DNA fluorescent probe for in situ detection of CSTF2 in liver cancer (LC) and study its correlation with the development of LC, we developed nano-TiO-DNA fluorescent probe which can bind with CSTF2 in LC samples with high efficiency. The detection process of CSTF2 did not involve the use PCR technology, and the concentration of CSTF2 can be directly observed by fluorescence intensity. This probe exhibited excellent physicochemical properties in ethyl alcohol at -20°C and could directly and selectively permeate into Hep-3B cells. By using CSTF2 Nano-TiO-DNA probe, we found that the CSTF2 level increased greatly in LC tissue and cells, and high CSTF2 level was closely associated with high levels of tumor markers and poor prognosis in LC patients. After transfection, CSTF2 was overexpressed or silenced in Hep-3B cells, and we find that high CSTF2 level effectively increased the activity and invasion of Hep-3B cells and reduced their apoptosis. Furthermore, high CSTF2 level significantly increased the tumor volume and weight in mice models by activating PI3K/AKT/mTOR signal pathway. Therefore, CSTF2 can serve as an early biomarker of LC and a novel potential target for its treatment.
PubMed: 38058835
DOI: No ID Found -
Methods in Molecular Biology (Clifton,... 2024Filoviruses are causative agents of severe hemorrhagic fevers with high case fatality rates in humans. For studies of virus biology and the subsequent development of...
Filoviruses are causative agents of severe hemorrhagic fevers with high case fatality rates in humans. For studies of virus biology and the subsequent development of countermeasures, reverse genetic systems, and especially those facilitating the generation of recombinant filoviruses, are indispensable. Here, we describe the generation of recombinant filoviruses from cDNA.
Topics: Humans; Filoviridae; Reverse Genetics; DNA, Complementary; Hemorrhagic Fever, Ebola; Ebolavirus
PubMed: 38064023
DOI: 10.1007/978-1-0716-3533-9_1