Primer For Reverse Transcription Research Articles

Exploring the impact of primer length on efficient gene detection via high-throughput sequencing

Reverse transcription (RT) is a crucial step in most RNA analysis methods. Optimizing protocols for this initial stage is critical for effective target detection, particularly when working with limited input RNA. Several factors, such as the input material quality and reaction conditions, influence RT efficiency. However, the effect of RT primer length on gene detection efficiency remains largely unknown. Thus, we investigate its impact by generating RNA-seq libraries with random RT primers of 6, 12, 18, or 24 nucleotides. To our surprise, the 18mer primer shows superior efficiency in overall transcript detection compared to the commonly used 6mer primer, especially in detecting longer RNA transcripts in complex human tissue samples. This study highlights the critical role of primer length in RT efficiency, which has significant potential to benefit various transcriptomic assays, from basic research to clinical diagnostics, given the central role of RT in RNA-related analyses.

Determinants of the interaction between the 5′-leader of HIV-1 genome and human lysyl-tRNA synthetase in reverse transcription primer release process

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) initiates from the 3′ end of human tRNALys3. The primer tRNALys3 is selectively packaged into the virus in the form of a complex with human lysyl-tRNA synthetase (LysRS). To facilitate reverse transcription initiation, part of the 5′ leader (5′L) of HIV-1 genomic RNA (gRNA) evolves a tRNA anticodon-like element (TLE), which binds LysRS and releases tRNALys3 for primer annealing and reverse transcription initiation. Although TLE has been identified as a key element in 5′L responsible for LysRS binding, how the conformations and various hairpin structures of 5′L regulate 5′L-LysRS interaction is not fully understood. Here, these factors have been individually investigated using direct and competitive fluorescence anisotropy binding experiments. Our data showed that the conformation of 5′L significantly influences its binding affinity with LysRS. The 5′L conformation favoring gRNA dimerization and packaging exhibits much weaker binding affinity with LysRS compared to the alternative 5′L conformation that is not selected for packaging. Additionally, dimerization of 5′L impairs LysRS-5′L interaction. Furthermore, among various regions of 5′L, both the primer binding site/TLE domain and the stem-loop 3 are important for LysRS interaction, whereas the dimerization initiation site and the splicing donor plays a minor role. In contrast, the presence of the transacting responsive and the polyadenylation signal hairpins slightly inhibit LysRS binding. These findings reveal that the conformation and various regions of the 5′L of HIV-1 genome regulate its interaction with human LysRS and the reverse transcription primer release process.

Human T-cell leukemia virus type 1 uses a specific tRNAPro isodecoder to prime reverse transcription.

Human T-cell leukemia virus type 1 (HTLV-1) is the only oncogenic human retrovirus discovered to date. All retroviruses are believed to use a host cell tRNA to prime reverse transcription (RT). In HTLV-1, the primer-binding site (PBS) in the genomic RNA is complementary to the 3' 18 nucleotides (nt) of human tRNAPro The human genome encodes 20 cytoplasmic tRNAPro genes representing seven isodecoders, all of which share the same 3' 18 nt sequence but vary elsewhere. Whether all tRNAPro isodecoders are used to prime RT in cells is unknown. A previous study showed that a 3' 18 nt tRNAPro-derived fragment (tRFPro) is packaged into HTLV-1 particles and can serve as an RT primer in vitro. The role of this tRNA fragment in the viral life cycle is unclear. In retroviruses, N1-methylation of the tRNA primer at position A58 (m1A) is essential for successful plus-strand transfer. Using primer-extension assays performed in chronically HTLV-1-infected cells, we found that A58 of tRNAPro is m1A-modified, implying that full-length tRNAPro is capable of facilitating successful plus-strand transfer. Analysis of HTLV-1 RT primer extension products indicated that full-length tRNAPro is likely to be the primer. To determine which tRNAPro isodecoder is used as the RT primer, we sequenced the minus-strand strong-stop RT product containing the intact tRNA primer and established that HTLV-1 primes RT using a specific tRNAPro UGG isodecoder. Further studies are required to understand how this primer is annealed to the highly structured HTLV-1 PBS and to investigate the role of tRFPro in the viral life cycle.

A programmable quantum dot nanosensor guided by three-way junction skeleton-mediated cascade signal amplification for sensitive detection of circRNAs in breast cancer

Circular RNAs (circRNAs) are endogenous covalent circular molecules that can regulate various biological processes in eukaryotes, and their dysregulation is associated with tumorigenesis. Herein, we demonstrate the construction of a programmable quantum dot (QD) nanosensor guided by three-way junction (TWJ) skeleton-mediated cascade signal amplification for sensitive detection of circRNA in clinical breast tissues. The TWJ probe contains a TWJ primer and a TWJ template. The presence of circRNA can specifically hybridize with TWJ probe to form a stable TWJ skeleton, initiating cascade signal amplification to produce abundant linker probes. The resultant linker probes can bind with Cy5-modified signal probes and biotinylated capture probes to yield the sandwich duplexes. Eventually, the immobilization of sandwich duplexes on the 605QD surface yields the 605QD-dsDNA-Cy5 nanoassembly, resulting in effective fluorescence resonance energy transfer (FRET). This nanosensor is free from exogenous reverse transcription primers and complicated probes design, and it exhibits high amplification efficiency, single-base mismatch specificity, and near-zero background signal. It can detect circRNA with attomolar sensitivity, measure endogenous circRNA at the single-cell level, and discriminate circRNA level in breast cancer tissues from adjacent normal tissues. Moreover, this nanosensor can be expanded to detect other circRNAs (e.g., circHIPK3) by easily changing the corresponding target recognition sequences of TWJ probes, with potential applications in biomedical research and molecular diagnosis.

Validating post-enrichment steps in environmental RNA analysis for improving its availability from water samples.

Environmental RNA (eRNA) analysis is expected to inclusively provide the physiological information of a population and community without individual sampling, having the potential for the improved monitoring of biodiversity and ecosystem function. Protocol development for maximizing eRNA availability is crucial to interpret its detection and quantification results with high accuracy and reliability, but the methodological validation and improvement of eRNA collection and processing methods are scarce. In this study, the technical steps after eRNA extraction, including genomic DNA (gDNA) removal and reverse transcription, were focused on and their performances were compared by zebrafish (Danio rerio) aquarium experiments. Additionally, this study also focused on the eRNA quantification variabilities between replicates and compared them between the PCR and sample levels. Results showed that (i) there was a trade-off between gDNA removal approaches and eRNA yields and an excess gDNA removal could lead to the false-negative eRNA detection, (ii) the use of the gene-specific primers for reverse transcription could increase the eRNA yields for multiple mitochondrial and nuclear genes compared with the random hexamer primers, and (iii) the coefficient of variation (CV) values of eRNA quantifications between PCR replicates were substantially lower for those between samples. Including the study, further knowledge for the sensitive and precise detection of macro-organismal eRNA should be needed for increasing the reliability and robustness of eRNA-based biomonitoring.

Stem-loop RT-qPCR system for multiplex miRNA profiling and its application in wound healing-specific biomarker identification

MiRNAs are biomarkers widely used in research but their clinical application is still challenging due to their low expression levels. Current methods for miRNA detection involve separate transcription and quantification for each target, which is costly and unsuitable for large sample sizes. This study provides a strategy for designing and screening miRNA-specific stem-loop reverse transcription (RT) primers, which enable the simultaneous transcription of three miRNAs and U6, and the concurrent detection of miRNA and U6 in the same transcript using TaqMan probes labeled with different dyes. The strategy was successfully employed to establish multiplex RT-PCR and dual-quantitative PCR (qPCR) quantification systems for 21 differentially expressed miRNAs during wound healing. The corresponding system can accurately quantify the cell culture samples containing miR-7a-5p mimic, miR-7a-5p inhibitor, or negative control. In summary, our results demonstrate that this strategy could efficiently accomplish the design, screening, and analysis of stem-loop RT primers for multiplex miRNA detection. Compared with the commercially customized miRNA assay kits, our system showed a higher degree of automation, more accurate qPCR assay capabilities, and lower assay costs, which could provide practical value for clinical diagnosis.

FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity

Recombinase polymerase amplification (RPA) running at 37–42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

First report of mume virus A infecting Prunus salicina worldwide and Prunus mume in Korea.

Mume virus A (MuVA) of the genus Capillovirus in the family Betaflexiviridae was first isolated from a Japanese apricot tree (Prunus mume) exhibiting symptoms of diffuse chlorotic spots (Marais et al. 2018). MuVA infection has been reported in Japanese apricot trees in Japan as well as in peach (P. persica) and Japanese apricot trees in China (Marais et al. 2018; Zhang et al. 2021; Zheng et al. 2020). In the present study, the diversity of viruses and viroids infecting Chinese plum trees (P. salicina) was investigated using high-throughput sequencing (HTS). Ten flowers each from 50 trees without obvious symptoms related to virus and/or viroid infection were randomly collected from five orchards in Gimcheon, Korea, in April 2020. The samples from each Chinese plum tree were pooled, and the same amounts of 50 individual samples prepared in advance were pooled for the extraction of total RNA using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Removal of ribosomal RNA and construction of cDNA library from the extracted total RNA were conducted using the TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina, San Diego, CA, USA). Paired-end RNA sequencing using Illumina NovaSeq 6000 System (paired-end reads of 101 bp and a total of 162,845,322 reads) and data analysis were performed at Macrogen (Daejeon, Korea). Adaptor and low-quality sequences of reads were removed using Trimmomatic program. Trimmed reads were assembled into contigs using Trinity program, and several databases including NCBI Nucleotide and Kyoto Encyclopedia of Genes and Genomes were used for functional annotation. HTS identified plum bark necrosis stem pitting-associated virus (PBNSPaV; four contigs ranging from 2081 to 3202 nucleotides) and hop stunt viroid (HSVd; one contig of 618 nucleotides). PBNSPaV and HSVd were also detected by RT-PCR (PBNSPaV det-F and PBNSPaV det-R for PBNSPaV [Al Rwahnih et al. 2007]; VP-19 and VP-20 for HSVd [Astruc et al. 1996]) and confirmed by Sanger sequencing of the amplified products. Interestingly, one contig derived from MuVA, which was not previously reported in Korea, was also detected. The contig was 7,618-nucleotide long (15,205 reads), and NCBI BLASTN search revealed 98.74% homology (100% query coverage) with the MuVA isolate pm14 (GenBank accession number MG783575). To design diagnostic primers for reverse transcription (RT)-polymerase chain reaction (PCR), the contig sequence and MuVA sequences available in NCBI GenBank (GenBank accession numbers MG783575 and MN412555) were aligned using CLC Main Workbench 6.9.1 (QIAGEN, Redwood, CA, USA). The following primer set (expected size of 1,143 bp) was prepared: MuVA-2F (5'-CAGCTTTGTGACTCYAACCC-3') and MuVA-2R (5'-AATGGCTTGAGGRCCTGCAG-3'). The primers target a partial region (nt position 1185 to 2327 on the basis of the reference genome sequence of MuVA, GenBank accession no. NC_040568) of the polyprotein gene (ORF1). Each of the 50 samples was tested for the presence of MuVA using the above-mentioned RT-PCR primers with SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, Korea). MuVA was detected in three samples collected from the same orchard. The three amplicons were inserted into a T&A cloning vector (RBC Bioscience, Taipei, Taiwan) and sequenced at Macrogen. Three consensus sequences obtained by Sanger sequencing were registered in NCBI GenBank under accession numbers MW589492, MW589493, and MW589494. NCBI BLASTN search revealed that the Korean isolates of MuVA shared high homology with isolate pm14 [98.16%, 98.08%, and 98.16% (100% query coverage), respectively]. To confirm additional MuVA infections, leaf samples of Chinese plum trees were collected from orchards in Uiseong (70 trees) and Seongju (50 trees) as well as a Japanese apricot tree in Chuncheon, from April to July 2021. RT-PCR confirmed additional MuVA infections from Uiseong (one tree) and Seongju (one tree) as well as from the Japanese apricot tree in Chuncheon. NCBI BLASTN search of the three additional amplicons (GenBank accession numbers OM210030, OM210031, and OM210032) revealed high homology with isolate pm14 [98.25%, 98.08%, and 97.90% (100% query coverage)]. To the best of our knowledge, this is the first report of MuVA infecting P. mume in Korea and P. salicina worldwide. Further research is needed to investigate MuVA infections on various Prunus spp. including P. persica in Korea.

HIV-1 Gag Binds the Multi-Aminoacyl-tRNA Synthetase Complex via the EPRS Subunit.

Host factor tRNAs facilitate the replication of retroviruses such as human immunodeficiency virus type 1 (HIV-1). HIV-1 uses human tRNALys3 as the primer for reverse transcription, and the assembly of HIV-1 structural protein Gag at the plasma membrane (PM) is regulated by matrix (MA) domain-tRNA interactions. A large, dynamic multi-aminoacyl-tRNA synthetase complex (MSC) exists in the cytosol and consists of eight aminoacyl-tRNA synthetases (ARSs) and three other cellular proteins. Proteomic studies to identify HIV-host interactions have identified the MSC as part of the HIV-1 Gag and MA interactomes. Here, we confirmed that the MA domain of HIV-1 Gag forms a stable complex with the MSC, mapped the primary interaction site to the linker domain of bi-functional human glutamyl-prolyl-tRNA synthetase (EPRS), and showed that the MA-EPRS interaction was RNA dependent. MA mutations that significantly reduced the EPRS interaction reduced viral infectivity and mapped to MA residues that also interact with phosphatidylinositol-(4,5)-bisphosphate. Overexpression of EPRS or EPRS fragments did not affect susceptibility to HIV-1 infection, and knockdown of EPRS reduced both a control reporter gene and HIV-1 protein translation. EPRS knockdown resulted in decreased progeny virion production, but the decrease could not be attributed to selective effects on virus gene expression, and the specific infectivity of the virions remained unchanged. While the precise function of the Gag-EPRS interaction remains uncertain, we discuss possible effects of the interaction on either virus or host activities.

Microparticles as Viral RNA Carriers from Stool for Stable and Sensitive Surveillance.

Since its discovery, polymerase chain reaction (PCR) has emerged as an important technology for the diagnosis and identification of infectious diseases. It is a highly sensitive and reliable nucleic acids (NA) detection tool for various sample types. However, stool, which carries the most abundant micro-organisms and physiological byproducts, remains to be the trickiest clinical specimen for molecular detection of pathogens. Herein, we demonstrate the novel application of hydrogel microparticles as carriers of viral RNA from stool samples without prior RNA purification for real-time polymerase chain reaction (qPCR). In each microparticle of primer-incorporated network (PIN) as a self-sufficient reaction compartment, immobilized reverse transcription (RT) primers capture the viral RNA by hybridization and directly initiate RT of RNA to generate a pool of complementary DNA (PIN-cDNA pool). Through a simple operation with a portable thermostat device, a PIN-cDNA pool for influenza A virus (IAV) was obtained in 20 min. The PIN-cDNA pools can be stored at room temperature, or directly used to deliver cDNA templates for qPCR. The viral cDNA templates were freely released in the subsequent qPCR to allow amplification efficiency of over 91%. The assay displayed good linearity, repeatability, and comparable limit of detection (LoD) with a commercialized viral RNA purification kit. As a proof of concept, this technology carries a huge potential for onsite application to improve human and animal infectious disease surveillance activities using stool samples without the need for a laboratory or centrifuge for sample preparation.

Absolute quantification of a plasma tRNA-derived fragment for the diagnosis and prognosis of gastric cancer.

The transition from a healthy gastric mucosa to gastric cancer is a multi-step process. Early screening can significantly improve the survival rate of gastric cancer patients. A reliable liquid biopsy for gastric cancer prediction is urgently needed and since tRNA-derived fragments (tRFs) are abundant in various body fluids, tRFs are possible new biomarkers for gastric cancer. A total of 438 plasma samples from patients with different gastric mucosal lesions as well as healthy individuals were collected. A specific reverse transcription primer, a forward primer, a reverse primer, and a TaqMan probe were designed. A standard curve was constructed and an absolute quantitation method was devised for detection of tRF-33-P4R8YP9LON4VDP in plasma samples of individuals with differing gastric mucosa lesions. Receiver operating characteristic curves were constructed to evaluate the diagnostic values of tRF-33-P4R8YP9LON4VDP for individual with differing gastric mucosa. A Kaplan-Meier curve was established to calculate the prognostic value of tRF-33-P4R8YP9LON4VDP for advanced gastric cancer patients. Finally, a multivariate Cox regression analysis was performed to assess the independent prognostic value of tRF-33-P4R8YP9LON4VDP for advanced gastric cancer patients. A detection method for plasma tRF-33-P4R8YP9LON4VDP was successfully established. Levels of plasma tRF-33-P4R8YP9LON4VDP were shown to reflect a gradient change from healthy individuals to gastritis patients to early and advanced gastric cancer patients. Significant differences were found among individuals with differing gastric mucosa, with reduced levels of tRF-33-P4R8YP9LON4VDP significantly related to a poor prognosis. tRF-33-P4R8YP9LON4VDP was found to be an independent predictor of an unfavorable survival outcome. In this study, we developed a quantitative detection method for plasma tRF-33-P4R8YP9LON4VDP that exhibited hypersensitivity, convenience, and specificity. Detection of tRF-33-P4R8YP9LON4VDP was found to be a valuable means by which to monitor different gastric mucosa and to predict patient prognosis.

Establishment of an Absolute Quantitative Method to Detect a Plasma tRNA-Derived Fragment and Its Application in the Non-Invasive Diagnosis of Gastric Cancer

(1) Transfer RNA (tRNA)-derived fragments (tRFs) are a new category of regulatory non-coding RNAs with distinct biological functions in cancer. They are produced from pre-tRNAs or mature tRNAs and their sequences are relatively short; thus, the amplification of tRFs, especially those in body fluids, is faced with certain technical difficulties. In this study, we established a quantitative method to detect plasma tRF-27-87R8WP9N1E5 (tRF-27) and used it to screen gastric cancer patients. (2) A specific stem-loop-structure reverse transcription primer, a TaqMan probe, and amplification primers for tRF-27 were prepared, and the absolute quantitative method was used to measure plasma tRF-27 levels. To determine the noninvasive diagnostic value of tRF-27 in gastric cancer, plasma tRF-27 levels in patients with benign and malignant lesions (120 healthy individuals, 48 patients with benign lesions, 48 patients with precancerous lesions, and 72 patients with early gastric cancer) were analyzed. Plasma tRF-27 levels were also analyzed in 106 preoperative gastric cancer patients, 106 postoperative gastric cancer patients, and 120 healthy individuals. Survival curves and Cox regression models were established and analyzed. (3) A new absolute quantitative method to determine the plasma tRF-27 copy number was established. Plasma tRF-27 levels were significantly increased in gastric cancer patients compared to healthy individuals, and the area under the receiver operating characteristic curve was 0.7767, when the cutoff value was 724,807 copies/mL, with sensitivity and specificity values of 0.6226 and 0.8917, respectively. The positive predictive and negative predictive values were 83.50% and 72.80%, respectively. Plasma tRF-27 levels in postoperative gastric cancer patients were significantly decreased compared to preoperative gastric cancer patients and tended to the levels of healthy individuals. Moreover, tRF-27 levels were closely related to tumor size and Ki67 expression in gastric cancer patients. Prognostic analysis showed that tRF-27 may be an independent predictor of overall survival. (4) This novel and non-invasive method of measuring plasma tRF-27 levels was valuable in the early diagnosis of gastric cancer.

GENOME SEQUENCE ANALYSIS, RT-PCR DIAGNOSTICS AND CONSTRUCTION OF VIRAL PROTEIN-EXPRESSION CASSETES FOR KAZAKH ISOLATE OF ORDINARY AND ANDEAN STRAINS OF POTATO VIRUS S

Potato virus S (PVS) belongs to the genus Carlavirus of family Betaflexiviridae with a positive-sense single-stranded RNA genome of 8.5 Kb. PVS is one of the most prevalent viruses of cultivated potatoes in Kazakhstan. Here we report phylogenetic analysis of complete genome sequences of Kazakh isolates of PVS from potato cultivars Fortuna and Ushkonyr, design of PVS strain-specific diagnostic PCR and construction of expression cassettes for conserved viral proteins from both isolates. The Fortuna and Ushkonyr isolates were found to share 79.9% nucleotide identity with each other and belong respectively to previously-defined ordinary and Andean strains of PVS. Based on analysis of conserved and variable regions of available PVS isolates, we designed primers for reverse transcription (RT)-duplex PCR to detect both strains in single and mixed infections and established their prevalence in Almaty region of Kazakhstan. Coding sequences of triple gene block proteins (25K, 12K, 7K), coat protein (34K) and cysteine-rich protein (11K) as well as methyltransferase, peptidase, helicase and RNA-dependent RNA polymerase domains of viral replicase were subcloned from both Fortuna and Ushkonyr genomic RNAs into the binary vector pBIN19 under the control of CaMV 35S promoter and nopaline synthetase terminator. These expression cassettes will be used to further investigate the biological properties and strain characteristics of viral proteins by their transient expression in plant cells and tissues or their stable expression in transgenic plants.

Phosphomimetic S207D Lysyl-tRNA Synthetase Binds HIV-1 5'UTR in an Open Conformation and Increases RNA Dynamics.

Interactions between lysyl–tRNA synthetase (LysRS) and HIV-1 Gag facilitate selective packaging of the HIV-1 reverse transcription primer, tRNALys3. During HIV-1 infection, LysRS is phosphorylated at S207, released from a multi-aminoacyl–tRNA synthetase complex and packaged into progeny virions. LysRS is critical for proper targeting of tRNALys3 to the primer-binding site (PBS) by specifically binding a PBS-adjacent tRNA-like element (TLE), which promotes release of the tRNA proximal to the PBS. However, whether LysRS phosphorylation plays a role in this process remains unknown. Here, we used a combination of binding assays, RNA chemical probing, and small-angle X-ray scattering to show that both wild-type (WT) and a phosphomimetic S207D LysRS mutant bind similarly to the HIV-1 genomic RNA (gRNA) 5′UTR via direct interactions with the TLE and stem loop 1 (SL1) and have a modest preference for binding dimeric gRNA. Unlike WT, S207D LysRS bound in an open conformation and increased the dynamics of both the PBS region and SL1. A new working model is proposed wherein a dimeric phosphorylated LysRS/tRNA complex binds to a gRNA dimer to facilitate tRNA primer release and placement onto the PBS. Future anti-viral strategies that prevent this host factor-gRNA interaction are envisioned.

Internal oligo(dT) priming introduces systematic bias in bulk and single-cell RNA sequencing count data.

Significant advances in RNA sequencing have been recently made possible by using oligo(dT) primers for simultaneous mRNA enrichment and reverse transcription priming. The associated increase in efficiency has enabled more economical bulk RNA sequencing methods and the advent of high-throughput single-cell RNA sequencing, already one of the most widely adopted methods in transcriptomics. However, the effects of off-target oligo(dT) priming on gene expression quantification have not been appreciated. In the present study, we describe the extent, the possible causes, and the consequences of internal oligo(dT) priming across multiple public datasets obtained from various bulk and single-cell RNA sequencing platforms. To explore and address this issue, we developed a computational algorithm for RNA counting methods, which identifies the sequencing read alignments that likely resulted from internal oligo(dT) priming and removes them from the data. Directly comparing filtered datasets to those obtained by an alternative method reveals significant improvements in gene expression measurement. Finally, we infer a list of human genes whose expression quantification is most likely to be affected by internal oligo(dT) priming and predict that when measured using these methods, the expression of most genes may be inflated by at least 10% whereby some genes are affected more than others.

Baiting out a full length sequence from unmapped RNA-seq data

BackgroundAs a powerful tool, RNA-Seq has been widely used in various studies. Usually, unmapped RNA-seq reads have been considered as useless and been trashed or ignored.ResultsWe develop a strategy to mining the full length sequence by unmapped reads combining with specific reverse transcription primers design and high throughput sequencing. In this study, we salvage 36 unmapped reads from standard RNA-Seq data and randomly select one 149 bp read as a model. Specific reverse transcription primers are designed to amplify its both ends, followed by next generation sequencing. Then we design a statistical model based on power law distribution to estimate its integrality and significance. Further, we validate it by Sanger sequencing. The result shows that the full length is 1556 bp, with insertion mutations in microsatellite structure.ConclusionWe believe this method would be a useful strategy to extract the sequences information from the unmapped RNA-seq data. Further, it is an alternative way to get the full length sequence of unknown cDNA.

Structural dissection of sequence recognition and catalytic mechanism of human LINE-1 endonuclease.

Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1’s ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5′-TTTTT/AA-3′. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN’s sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes ‘compression’ near the cleavage site. This provides multiple advantages for L1-EN’s role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.

Interplay between Host tRNAs and HIV-1: A Structural Perspective.

The cellular metabolism of host tRNAs and life cycle of HIV-1 cross paths at several key virus–host interfaces. Emerging data suggest a multi-faceted interplay between host tRNAs and HIV-1 that plays essential roles, both structural and regulatory, in viral genome replication, genome packaging, and virion biogenesis. HIV-1 not only hijacks host tRNAs and transforms them into obligatory reverse transcription primers but further commandeers tRNAs to regulate the localization of its major structural protein, Gag, via a specific interface. This review highlights recent advances in understanding tRNA–HIV-1 interactions, primarily from a structural perspective, which start to elucidate their underlying molecular mechanisms, intrinsic specificities, and biological significances. Such understanding may provide new avenues toward developing HIV/AIDS treatments and therapeutics including small molecules and RNA biologics that target these host–virus interfaces.

High allele discrimination in the typing of single nucleotide polymorphisms of miRNA

MicroRNAs (miRNAs) belonging to the same family have similar sequences and are difficult to identify. Herein, we report the reverse transcription-hairpin-probe-polymerase chain reaction (RT-Hpro-PCR) technique, which utilises a reverse transcription (RT) primer containing a 5′-end deoxyribonucleic acid (DNA) tag, to detect miRNAs with similar sequences. This strategy follows a two-step RT-PCR method using 6–7-mer RT-primers with a ~ 10-mer tag sequence at the 5′-end and a probe with a hairpin structure (Hpro), including two C-bulges, attached. The findings demonstrate that the specificity of RT could be increased by shortening the complementary part of the RT primer containing a different base, wherein the PCR could successfully progress with the use of 5′-end DNA tag because of an increase in the length of the hybridised tagged primer. This study shows the potential of RT-Hpro-PCR to precisely detect miRNAs with similar sequences, which could help explore the roles of miRNAs in several biological processes.