Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein.

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found that Marinomonas mediterranea RT-Cas1/Cas2 adds short 3'-DNA (dN) tails to RNA protospacers, enabling their direct integration into CRISPR arrays as 3'-dN-RNAs or 3'-dN-RNA/cDNA duplexes at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers is initiated at 3' proximal sites by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of near full-length cDNAs of diverse RNAs without fixed sequence requirements. The integration of 3'-dN-RNAs or single-stranded DNAs (ssDNAs) is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage defense nucleases. Our findings reveal mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.

Insights into LINE-1 reverse transcription guide therapy development.

Subsets of long interspersed nuclear element 1 (LINE-1) retrotransposons can 'retrotranspose' throughout the human genome at a cost to host cell fitness, as observed in some cancers. Pharmacological inhibition of LINE-1 retrotransposition requires a comprehensive understanding of the LINE-1 ORF2p reverse transcriptase. Two recent publications, by Thawani et al. and Baldwin et al., report structures of LINE-1 ORF2p and address long-standing mechanistic gaps regarding LINE-1 retrotransposition. Both studies will be critical to design new specific inhibitors of the LINE-1 ORF2p reverse transcriptase.

[Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin].

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

Low-Cost Arduino Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) for Sensitive Nucleic Acid Detection.

This work presents a low-cost transcription loop-mediated isothermal amplification (RT-LAMP) instrument for nucleic acid detection, employing an Arduino Nano microcontroller. The cooling system includes customized printed circuit boards (PCBs) that serve as electrical resistors and incorporate fans. An aluminum block is designed to accommodate eight vials. The system also includes two PCB heaters-one for sample heating and the other for vial lid heating to prevent condensation. The color detection system comprises a TCS3200 color 8-sensor array coupled to one side of the aluminum heater body and a white 8-LED array coupled to the other side, controlled by two Multiplexer/Demultiplexer devices. LED light passes through the sample, reaching the color sensor and conveying color information crucial for detection. The top board is maintained at 110 ± 2 °C, while the bottom board is held at 65 ± 0.5 °C throughout the RT-LAMP assay. Validation tests successfully demonstrated the efficacy of the colorimetric RT-LAMP reactions using SARS-CoV-2 RNA amplification as a sample viability test, achieving 100% sensitivity and 97.3% specificity with 66 clinical samples. Our instrument offers a cost-effective (USD 100) solution with automated result interpretation and superior sensitivity compared to visual inspection. While the prototype was tested with SARS-CoV-2 RNA samples, its versatility extends to detecting other pathogens using alternative primers, showcasing its potential for broader applications in biosensing.

Cohort profile: PRESTIGIO, an Italian prospective registry-based cohort of people with HIV-1 resistant to reverse transcriptase, protease and integrase inhibitors.

PURPOSE: The PRESTIGIO Registry was established in 2017 to collect clinical, virological and immunological monitoring data from people living with HIV (PLWH) with documented four-class drug resistance (4DR). Key research purposes include the evaluation of residual susceptibility to specific antiretrovirals and the validation of treatment and monitoring strategies in this population. PARTICIPANTS: The PRESTIGIO Registry collects annual plasma and peripheral blood mononuclear cell samples and demographic, clinical, virological, treatment and laboratory data from PLWH followed at 39 Italian clinical centres and characterised by intermediate-to-high genotypic resistance to ≥1 nucleoside reverse transcriptase inhibitors, ≥1 non-nucleoside reverse transcriptase inhibitors, ≥1 protease inhibitors, plus either intermediate-to-high genotypic resistance to ≥1 integrase strand transfer inhibitors (INSTIs) or history of virological failure to an INSTI-containing regimen. To date, 229 people have been recorded in the cohort. Most of the data are collected from the date of the first evidence of 4DR (baseline), with some prebaseline information obtained retrospectively. Samples are collected from the date of enrollment in the registry. FINDINGS TO DATE: The open-ended cohort has been used to assess (1) prognosis in terms of survival or development of AIDS-related or non-AIDS-related clinical events; (2) long-term efficacy and safety of different antiretroviral regimens and (3) virological and immunological factors predictive of clinical outcome and treatment efficacy, especially through analysis of plasma and cell samples. FUTURE PLANS: The registry can provide new knowledge on how to implement an integrated approach to study PLWH with documented resistance to the four main antiretroviral classes, a population with a limited number of individuals characterised by a high degree of frailty and complexity in therapeutic management. Given the scheduled annual updates of PLWH data, the researchers who collaborate in the registry can send study proposals at any time to the steering committee of the registry, which evaluates every 3 months whether the research studies can be conducted on data and biosamples from the registry and whether they are aimed at a better understanding of a specific health condition, the emergence of comorbidities or the effect of potential treatments or experimental drugs that may have an impact on disease progression and quality of life. Finally, the research studies should aim to be inclusive, innovative and in touch with the communities and society as a whole. TRIAL REGISTRATION NUMBER: NCT04098315.

Modeling and Analysis of HIV-1 Pol Polyprotein as a Case Study for Predicting Large Polyprotein Structures.

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). HIV protease, reverse transcriptase, and integrase are targets of current drugs to treat the disease. However, anti-viral drug-resistant strains have emerged quickly due to the high mutation rate of the virus, leading to the demand for the development of new drugs. One attractive target is Gag-Pol polyprotein, which plays a key role in the life cycle of HIV. Recently, we found that a combination of M50I and V151I mutations in HIV-1 integrase can suppress virus release and inhibit the initiation of Gag-Pol autoprocessing and maturation without interfering with the dimerization of Gag-Pol. Additional mutations in integrase or RNase H domain in reverse transcriptase can compensate for the defect. However, the molecular mechanism is unknown. There is no tertiary structure of the full-length HIV-1 Pol protein available for further study. Therefore, we developed a workflow to predict the tertiary structure of HIV-1 NL4.3 Pol polyprotein. The modeled structure has comparable quality compared with the recently published partial HIV-1 Pol structure (PDB ID: 7SJX). Our HIV-1 NL4.3 Pol dimer model is the first full-length Pol tertiary structure. It can provide a structural platform for studying the autoprocessing mechanism of HIV-1 Pol and for developing new potent drugs. Moreover, the workflow can be used to predict other large protein structures that cannot be resolved via conventional experimental methods.

Development of a reverse transcriptase digital droplet polymerase chain reaction-based approach for SARS-CoV-2 variant surveillance in wastewater.

An urgent need for effective surveillance strategies arose due to the global emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although vaccines and antivirals are available, concerns persist about the evolution of new variants with potentially increased infectivity, transmissibility, and immune evasion. Therefore, variant monitoring is crucial for public health decision-making. Wastewater-based surveillance has proven to be an effective tool to monitor SARS-CoV-2 variants within populations. Specific SARS-CoV-2 variants are detected and quantified in wastewater in this study using a reverse transcriptase digital droplet polymerase chain reaction (RT-ddPCR) approach. The 11 designed assays were first validated in silico using a substantial dataset of high-quality SARS-CoV-2 genomes to ensure comprehensive variant coverage. The assessment of the sensitivity and specificity with reference material showed the capability of the developed assays to reliably identify target mutations while minimizing false positives and false negatives. The applicability of the assays was evaluated using wastewater samples from a wastewater treatment plant in Ghent, Belgium. The quantification of the specific mutations linked to the variants of concern present in these samples was calculated using these assays based on the detection of single mutations, which confirms their use for real-world variant surveillance. In conclusion, this study provides an adaptable protocol to monitor SARS-CoV-2 variants in wastewater with high sensitivity and specificity. Its potential for broader application in other viral surveillance contexts highlights its added value for rapid response to emerging infectious diseases. PRACTITIONER POINTS: Robust RT-ddPCR methodology for specific SARS-CoV-2 variants of concern detection in wastewater. Rigorous validation that demonstrates high sensitivity and specificity. Demonstration of real-world applicability using wastewater samples. Valuable tool for rapid response to emerging infectious diseases.

Large Deletions, Cleavage of the Telomeric Repeat Sequence, and Reverse Transcriptase-Mediated DNA Damage Response Associated with Long Interspersed Element-1 ORF2p Enzymatic Activities.

L1 elements can cause DNA damage and genomic variation via retrotransposition and the generation of endonuclease-dependent DNA breaks. These processes require L1 ORF2p protein that contains an endonuclease domain, which cuts genomic DNA, and a reverse transcriptase domain, which synthesizes cDNA. The complete impact of L1 enzymatic activities on genome stability and cellular function remains understudied, and the spectrum of L1-induced mutations, other than L1 insertions, is mostly unknown. Using an inducible system, we demonstrate that an ORF2p containing functional reverse transcriptase is sufficient to elicit DNA damage response even in the absence of the functional endonuclease. Using a TK/Neo reporter system that captures misrepaired DNA breaks, we demonstrate that L1 expression results in large genomic deletions that lack any signatures of L1 involvement. Using an in vitro cleavage assay, we demonstrate that L1 endonuclease efficiently cuts telomeric repeat sequences. These findings support that L1 could be an unrecognized source of disease-promoting genomic deletions, telomere dysfunction, and an underappreciated source of chronic RT-mediated DNA damage response in mammalian cells. Our findings expand the spectrum of biological processes that can be triggered by functional and nonfunctional L1s, which have impactful evolutionary- and health-relevant consequences.

Versatility of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) from diagnosis of early pathological infection to mutation detection in organisms.

Loop-mediated isothermal amplification (LAMP) is a rapid, state-of-the-art DNA amplification technology, used primarily for the quick diagnosis and early identification of microbial infection, caused by pathogens such as virus, bacteria and malaria. A target DNA can be amplified within 30 min using the LAMP reaction, taking place at a steady temperature. The LAMP method uses four or six primers to bind eight regions of a target DNA and has a very high specificity. The devices used for conducting LAMP are usually simple since the LAMP method is an isothermal process. When LAMP is coupled with Reverse Transcription (RT), it allows direct detection of RNA in a sample. This greatly enhances the efficiency of diagnosis of RNA viruses in a sample. Recently, the rampant spread of COVID-19 demanded such a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. Loop-mediated isothermal amplification (LAMP) assays are not only used for the detection of microbial pathogens, but there are various other applications such as detection of genetic mutations in food and various organisms. In this review, various implementations of RT-LAMP techniques would be discussed.

Major Drug Resistance Mutations on Reverse Transcriptase Gene in Human Immunodeficiency Virus Type-1 in Indonesia: A Systematic Review.

PURPOSE OF REVIEW: The prevalence of HIV-1 in Indonesia is on a concerning upward trajectory, with a concurrent rise in the development of drug-resistant strains, challenging the efficacy of antiretroviral therapy (ART). Many mutations have been found in the pol gene that makes HIV resistant to ART. We aim to review the major drug resistance mutations (DRMs) of reverse transcriptase (RT) of pol gene in HIV-1 cases in Indonesia. RECENT FINDINGS: A total of eleven articles reporting DRMs in HIV-1 subjects from various regions between 2015-2020 in Indonesia are included. The prevalence of major DRMs on the RT gene in studies included varies from 3.4% to 34%. The CRF01_AE subtype stands out as the predominant variant. Notably, the prevalence of major DRMs in ART-experienced individuals is 22.1%, while ART-naïve individuals show a lower rate of 4.4%. Among the RT gene mutations, M184I/V emerges as the most prevalent (10.5%) within the nucleos(t)ide reverse transcriptase inhibitors (NRTI) group, while K103N leads among the non-NRTI (NNRTI) group, with a frequency of 6.4%. Regionally, North Sulawesi records the highest prevalence of major DRMs in the RT gene at 21.1%, whereas Riau and Central Papua exhibit the lowest rates at 3.4%. Significant variations in drug resistance mutations within the RT gene across Indonesian regions highlight the importance of closely monitoring and evaluating the effectiveness of current antiretroviral therapy (ART) regimens. Considerably, more studies are needed to understand better and overcome the emergence of DRMs on HIV-1 patients in Indonesia.

Development of Moloney Murine Leukemia Virus Reverse Transcriptase Fused with Archaeal DNA-binding Protein Sis7a.

INTRODUCTION: Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) is a common enzyme used to convert RNA sequences into cDNA. However, it still has its shortcomings, especially in terms of processivity and thermostability. According to a previous patent, the fusion of polymerase enzyme to an archaeal DNA-binding protein has been proven to enhance its performance. Furthermore, recent studies have also stated that the fusion of a polymerase enzyme to an archaeal DNA-binding protein is predicted to improve its thermostability and processivity. AIM: As an early stage of enzyme development, this study aimed to design, express, and purify enzymatically active MMLV RT fused with archaeal DNA-binding protein. METHODS: RT fusion proteins were designed and evaluated using in silico methods. The RT fusion enzyme was then expressed in Escherichia coli BL21(DE3) and purified. Its reverse transcriptional activity was proved using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RESULTS: This study showed that MMLV RT fusion with Sis7a protein at its C-terminal end using commercial linker (GGVDMI) produced the best in silico evaluation results. The RT fusion was successfully expressed and purified. It was also known that the optimal condition for expression of the RT fusion was using 0.5 mM IPTG with post-induction incubation at room temperature (± 26°C) for 16 hours. In addition, the activity assay proved that the RT fusion has the reverse transcriptional activity. CONCLUSION: This study shows that the designed MMLV RT Sis7a fusion can be expressed and purified, is enzymatically active, and has the potential to be developed as an improved RT enzyme. Further study is still needed to prove its thermostability and processivity, and further characterize, and plan production scale-up of the MMLV RT Sis7a fusion for commercial use.

Keratocystoma: A Distinctive Salivary Gland Neoplasm Characterized by RUNX2 Rearrangements.

Keratocystoma is a rare salivary gland lesion that has been reported primarily in children and young adults. Because of a scarcity of reported cases, very little is known about it, including its molecular underpinnings, biological potential, and histologic spectrum. Purported to be a benign neoplasm, keratocystoma bears a striking histologic resemblance to benign lesions like metaplastic Warthin tumor on one end of the spectrum and squamous cell carcinoma on the other end. This overlap can cause diagnostic confusion, and it raises questions about the boundaries and definition of keratocystoma as an entity. This study seeks to utilize molecular tools to evaluate the pathogenesis of keratocystoma as well as its relationship with its histologic mimics. On the basis of targeted RNA sequencing (RNA-seq) results on a sentinel case, RUNX2 break-apart fluorescence in situ hybridization (FISH) was successfully performed on 4 cases diagnosed as keratocystoma, as well as 13 cases originally diagnosed as tumors that morphologically resemble keratocystoma: 6 primary squamous cell carcinomas, 3 metaplastic/dysplastic Warthin tumors, 2 atypical squamous cysts, 1 proliferating trichilemmal tumor, and 1 cystadenoma. RNA-seq and/or reverse transcriptase-PCR were attempted on all FISH-positive cases. Seven cases were positive for RUNX2 rearrangement, including 3 of 4 tumors originally called keratocystoma, 2 of 2 called atypical squamous cyst, 1 of 1 called proliferating trichilemmal tumor, and 1 of 6 called squamous cell carcinoma. RNA-seq and/or reverse transcriptase-PCR identified IRF2BP2::RUNX2 in 6 of 7 cases; for the remaining case, the partner remains unknown. The cases positive for RUNX2 rearrangement arose in the parotid glands of 4 females and 3 males, ranging from 8 to 63 years old (mean, 25.4 years; median, 15 years). The RUNX2 -rearranged cases had a consistent histologic appearance: variably sized cysts lined by keratinizing squamous epithelium, plus scattered irregular squamous nests, with essentially no cellular atypia or mitotic activity. The background was fibrotic, often with patchy chronic inflammation and/or giant cell reaction. One case originally called squamous cell carcinoma was virtually identical to the other cases, except for a single focus of small nerve invasion. The FISH-negative case that was originally called keratocystoma had focal cuboidal and mucinous epithelium, which was not found in any FISH-positive cases. The tumors with RUNX2 rearrangement were all treated with surgery only, and for the 5 patients with follow-up, there were no recurrences or metastases (1 to 120 months), even for the case with perineural invasion. Our findings solidify that keratocystoma is a cystic neoplastic entity, one which appears to consistently harbor RUNX2 rearrangements, particularly IRF2BP2::RUNX2 . Having a diagnostic genetic marker now allows for a complete understanding of this rare tumor. They arise in the parotid gland and affect a wide age range. Keratocystoma has a consistent morphologic appearance, which includes large squamous-lined cysts that mimic benign processes like metaplastic Warthin tumor and also small, irregular nests that mimic squamous cell carcinoma. Indeed, RUNX2 analysis has considerable promise for resolving these differential diagnoses. Given that one RUNX2 -rearranged tumor had focal perineural invasion, it is unclear whether that finding is within the spectrum of keratocystoma or whether it could represent malignant transformation. Most important, all RUNX2 -rearranged cases behaved in a benign manner.

Template and target-site recognition by human LINE-1 in retrotransposition.

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.

Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Improved protocol for Bst polymerase and reverse transcriptase production and application to a point-of-care diagnostics system.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has raised awareness in the scientific community about the importance of being prepared for sanitary emergencies. Many measures implemented during the COVID pandemic are now being expanded to other applications. In the field of molecular and immunological diagnostics, the need to massively test the population worldwide resulted in the application of a variety of methods to detect viral infection. Besides gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR), the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) arose as an alternative and sensitive method to amplify and detect viral genetic material. We have used openly available protocols and have improved the protein production of RT-LAMP enzymes Bst polymerase and HIV-reverse transcriptase. To optimize enzyme production, we tested different protein tags, and we shortened the protein purification protocol, resulting in reduced processing time and handling of the enzymes and, thus, preserved the protein activity with high purity. The enzymes showed significant stability at 4 °C and 25 °C, over 60 days, and were highly reliable when used as a one-step RT-LAMP reaction in a portable point-of-care device with clinical samples. The enzymes and the reaction setup can be further expanded to detect other infectious diseases agents.

Utility of reverse transcriptase - Multiplex ligation-dependant probe amplification (RT-MLPA) in the molecular classification of Diffuse Large B cell lymphoma (DLBCL) by cell-of-origin (COO).

Classifying diffuse large B cell lymphomas, not otherwise specified (DLBCL, NOS), is based on their cell-of-origin (COO) which is included in the WHO classification (2016), is essential to characterize them better in context of prognostication. While gene expression profiling (GEP) considered the gold standard and more recently, the Nanostring-based approach, classify these tumors accurately, many laboratories with limited resources and instrumentation need an alternate approach that is reliable, inexpensive, and with a reasonable turnaround. The Reverse Transcriptase Multiplex Ligation Dependant Probe Amplification (RT-MLPA) to subtype DLBCL, NOS cases, as designed by CALYM group appears to provide a good alternative but needs to be validated in other centres. Therefore, this study evaluated DLBCL, NOS and compared the results of RT-MLPA to that obtained by immunohistochemistry using the Hans algorithm. Materials and Methods: Sixty-five DLBCL, NOS cases were included and the RT-MLPA was set up and standardized using probes that were designed by the CALYM study group. Briefly, RNA was extracted converted to cDNA and the 21-gene expression classifier that also included probes to detect MYD88 mutations and EBER mRNA was performed by MLPA. The results were analyzed by the open home grown software designed by the same group and compared to the results obtained by IHC. Results: Forty-four of the sixty-five cases provided concordant results (k = 0.35) and if the MYD88 results were to be used as a classifier the concordance would have improved from 67.7% to 82%. The 21 discordant cases were divided into five categories to provide a possible explanation for the discordance. Further 26% and 31% of the samples tested were positive for MYD88 mutations and EBER mRNA, respectively. The test had a turnaround of three days. Conclusion: The test provided moderate (67.7%) concordance when compared with IHC and perhaps would have provided higher concordance if compared with GEP. The test also has the advantage of providing information on the MYD88 and EBV infection status. It was found to be reliable, easy to perform and standardize, requiring only routine instruments available in most molecular laboratories. The RT-MLPA assay therefore provides an alternative for laboratories that would require subtyping of DLBCL, NOS cases in the absence of an access to GEP or other instrument intensive methods.

To Be Mobile or Not: The Variety of Reverse Transcriptases and Their Recruitment by Host Genomes.

Reverse transcriptases (RT), or RNA-dependent DNA polymerases, are unorthodox enzymes that originally added a new angle to the conventional view of the unidirectional flow of genetic information in the cell from DNA to RNA to protein. First discovered in vertebrate retroviruses, RTs were since re-discovered in most eukaryotes, bacteria, and archaea, spanning essentially all domains of life. For retroviruses, RTs provide the ability to copy the RNA genome into DNA for subsequent incorporation into the host genome, which is essential for their replication and survival. In cellular organisms, most RT sequences originate from retrotransposons, the type of self-replicating genetic elements that rely on reverse transcription to copy and paste their sequences into new genomic locations. Some retroelements, however, can undergo domestication, eventually becoming a valuable addition to the overall repertoire of cellular enzymes. They can be beneficial yet accessory, like the diversity-generating elements, or even essential, like the telomerase reverse transcriptases. Nowadays, ever-increasing numbers of domesticated RT-carrying genetic elements are being discovered. It may be argued that domesticated RTs and reverse transcription in general is more widespread in cellular organisms than previously thought, and that many important cellular functions, such as chromosome end maintenance, may evolve from an originally selfish process of converting RNA into DNA.

RT-based Sanger sequencing of RNAs containing complex RNA repetitive elements.

Although next-generation sequencing (NGS) technologies have revolutionized our ability to sequence DNA with high-throughput, the chain termination-based Sanger sequencing method remains a widely used approach for DNA sequence analysis due to its simplicity, low cost and high accuracy. In particular, high accuracy makes Sanger sequencing the "gold standard" for sequence validation in basic research and clinical applications. During the early days of Sanger sequencing development, reverse transcriptase (RT)-based RNA sequencing was also explored and showed great promise, but the approach did not acquire popularity over time due to the limited processivity and low template unwinding capability of Avian Myeloblastosis Virus (AMV) RT, and other RT enzymes available at the time. RNA molecules have complex features, often containing repetitive sequences and stable secondary or tertiary structures. While these features are required for RNA biological function, they represent strong obstacles for retroviral RTs. Repetitive sequences and stable structures cause reverse transcription errors and premature primer extension stops, making chain termination-based methods unfeasible. MarathonRT is an ultra-processive RT encoded group II intron that can copy RNA molecules of any sequence and structure in a single cycle, making it an ideal RT enzyme for Sanger RNA sequencing. In this chapter, we upgrade the Sanger RNA sequencing method by replacing AMV RT with MarathonRT, providing a simple, robust method for direct RNA sequence analysis. The guidance for troubleshooting and further optimization are also provided.

End-to-end RT-PCR of long RNA and highly structured RNA.

RNA molecules play important roles in numerous normal cellular processes and disease states, from protein coding to gene regulation. RT-PCR, applying the power of polymerase chain reaction (PCR) to RNA by coupling reverse transcription with PCR, is one of the most important techniques to characterize RNA transcripts and monitor gene expression. The ability to analyze full-length RNA transcripts and detect their expression is critical to decipher their biological functions. However, due to the low processivity of retroviral reverse transcriptases (RTs), we can only monitor a small fraction of long RNA transcripts, especially those containing stable secondary and tertiary structures. The full-length sequences can only be deduced by computational analysis, which is often misleading. Group II intron-encoded RTs are a new type of RT enzymes. They have evolved specialized structural elements that unwind template structures and maintain close contact with the RNA template. Therefore, group II intron-encoded RTs are more processive than the retroviral RTs. The discovery, optimization and deployment of processive group II intron RTs provide us the opportunity to analyze RNA transcripts with single molecule resolution. MarathonRT, the most processive group II intron RT, has been extensively optimized for processive reverse transcription. In this chapter, we use MarathonRT to deliver a general protocol for long amplicon generation by RT-PCR, and also provide guidance for troubleshooting and further optimization.

RNA G-quadruplex (rG4) structure detection using RTS and SHALiPE assays.

RNA G-quadruplexes (rG4s) are non-canonical RNA secondary structures that were first reported several decades ago. Latest studies have suggested that they are widespread in the transcriptomes of diverse species, and they have been demonstrated to have key roles in various fundamental cellular processes. Among the RNA secondary structure probing assays developed recently, Reverse transcriptase stalling (RTS) and selective 2'-hydroxyl acylation analyzed by lithium ion-based primer extension (SHALiPE) enabled the identification and characterization of distinct structural features of an rG4 structure of interest. Herein, we present an experimental protocol describing in detail the procedures involved in the preparation of in vitro transcribed RNAs, buffers, and reagents for RTS and SHALiPE assays, as well as performing RTS and SHALiPE assays, to examine the formation of rG4 and reveal the rG4 structural conformation at nucleotide resolution in vitro. RTS and SHALiPE assays can be performed by an experienced molecular biologist or chemical biologist with a basic understanding of nucleic acids. The duration for the preparation of in vitro transcription and RNA preparation is around 2 days, and the duration for RTS and SHALiPE assays is approximately 5 h.