A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template.

Primases have a fundamental role in DNA replication. They synthesize a primer that is then extended by DNA polymerases. Archaeoeukaryotic primases require for synthesis a catalytic and an accessory domain, the exact contribution of the latter being unresolved. For the pRN1 archaeal primase, this domain is a 115-amino acid helix bundle domain (HBD). Our structural investigations of this small HBD by liquid- and solid-state nuclear magnetic resonance (NMR) revealed that only the HBD binds the DNA template. DNA binding becomes sequence-specific after a major allosteric change in the HBD, triggered by the binding of two nucleotide triphosphates. The spatial proximity of the two nucleotides and the DNA template in the quaternary structure of the HBD strongly suggests that this small domain brings together the substrates to prepare the first catalytic step of primer synthesis. This efficient mechanism is likely general for all archaeoeukaryotic primases.

Pervasive Regulatory Functions of mRNA Structure Revealed by High-Resolution SHAPE Probing.

mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.

Molecular basis for the initiation of DNA primer synthesis.

During the initiation of DNA replication, oligonucleotide primers are synthesized de novo by primases and are subsequently extended by replicative polymerases to complete genome duplication. The primase-polymerase (Prim-Pol) superfamily is a diverse grouping of primases, which includes replicative primases and CRISPR-associated primase-polymerases (CAPPs) involved in adaptive immunity1-3. Although much is known about the activities of these enzymes, the precise mechanism used by primases to initiate primer synthesis has not been elucidated. Here we identify the molecular bases for the initiation of primer synthesis by CAPP and show that this mechanism is also conserved in replicative primases. The crystal structure of a primer initiation complex reveals how the incoming nucleotides are positioned within the active site, adjacent to metal cofactors and paired to the templating single-stranded DNA strand, before synthesis of the first phosphodiester bond. Furthermore, the structure of a Prim-Pol complex with double-stranded DNA shows how the enzyme subsequently extends primers in a processive polymerase mode. The structural and mechanistic studies presented here establish how Prim-Pol proteins instigate primer synthesis, revealing the requisite molecular determinants for primer synthesis within the catalytic domain. This work also establishes that the catalytic domain of Prim-Pol enzymes, including replicative primases, is sufficient to catalyse primer formation.

Tethered Oligonucleotide-Primed Sequencing, TOP-Seq: A High-Resolution Economical Approach for DNA Epigenome Profiling.

Modification of CG dinucleotides in DNA is part of epigenetic regulation of gene function in vertebrates and is associated with complex human disease. Bisulfite sequencing permits high-resolution analysis of cytosine modification in mammalian genomes; however, its utility is often limited due to substantial cost. Here, we describe an alternative epigenome profiling approach, named TOP-seq, which is based on covalent tagging of individual unmodified CG sites followed by non-homologous priming of the DNA polymerase action at these sites to directly produce adjoining regions for their sequencing and precise genomic mapping. Pilot TOP-seq analyses of bacterial and human genomes showed a better agreement of TOP-seq with published bisulfite sequencing maps as compared to widely used MBD-seq and MRE-seq and permitted identification of long-range and gene-level differential methylation among human tissues and neuroblastoma cell types. Altogether, we propose an affordable single CG-resolution technique well suited for large-scale epigenome studies.

PrimerEvalPy: a tool for in-silico evaluation of primers for targeting the microbiome.

BACKGROUND: The selection of primer pairs in sequencing-based research can greatly influence the results, highlighting the need for a tool capable of analysing their performance in-silico prior to the sequencing process. We therefore propose PrimerEvalPy, a Python-based package designed to test the performance of any primer or primer pair against any sequencing database. The package calculates a coverage metric and returns the amplicon sequences found, along with information such as their average start and end positions. It also allows the analysis of coverage for different taxonomic levels. RESULTS: As a case study, PrimerEvalPy was used to test the most commonly used primers in the literature against two oral 16S rRNA gene databases containing bacteria and archaea. The results showed that the most commonly used primer pairs in the oral cavity did not match those with the highest coverage. The best performing primer pairs were found for the detection of oral bacteria and archaea. CONCLUSIONS: This demonstrates the importance of a coverage analysis tool such as PrimerEvalPy to find the best primer pairs for specific niches. The software is available under the MIT licence at https://gitlab.citius.usc.es/lara.vazquez/PrimerEvalPy .

In situ enzymatic template replication on DNA microarrays.

DNA microarrays are very useful tools to study the realm of nucleic acids interactions at high throughput. The conventional approach to microarray synthesis employs phosphoramidite chemistry and yields unmodified DNA generally attached to a surface at the 3' terminus. Having a freely accessible 3'-OH instead of 5'-OH is desirable too, and being able to introduce nucleoside analogs in a combinatorial manner is highly relevant in the context of nucleic acid therapeutics and in aptamer research. Here, we describe an enzymatic approach to the synthesis of high-density DNA microarrays that can also contain chemical modifications. The method uses a standard DNA microarray, to which a DNA primer is covalently bound through photocrosslinking. The extension of the primer with a DNA polymerase yields double-stranded DNA but is also amenable to the incorporation of modified dNTPs. Further processing with T7 exonuclease, which catalyzes the degradation of DNA in a specific (5'→3') direction, results in template strand removal. Overall, the method produces surface-bound natural and non-natural DNA oligonucleotides, is applicable to commercial microarrays and paves the way for the preparation of combinatorial, chemically modified aptamer libraries.

Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli.

Chemical modifications of RNA 5'-ends enable "epitranscriptomic" regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates for use as initiating entities, providing an ab initio mechanism for altering the RNA 5'-end. In Escherichia coli cells, RNAs with a 5'-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, "primer-dependent initiation." Here, we use massively systematic transcript end readout (MASTER) to detect and quantify RNA 5'-ends generated by primer-dependent initiation for ∼410 (∼1,000,000) promoter sequences in E. coli The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2NTSS-1NTSSWTSS+1, where TSS is the transcription start site, NTSS-1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2) through interchain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally encoded Ecoli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.

Long Non-Coding RNA Detection Based on Multi-Probe-Induced Rolling Circle Amplification for Hepatocellular Carcinoma Early Diagnosis.

Long non-coding RNAs (lncRNAs) played vital roles in physiological and pathological conditions. Consistent results from cell experiments, animal experiments, and clinical studies suggested that lncRNA HULC was an oncogenic lncRNA serving as a potential diagnostic and prognostic marker of hepatocellular carcinoma. In this study, we developed a fluorescent biosensor for lncRNA HULC detection based on rolling circle amplification (RCA) induced by multi-primer probes. Multiple primer probes can not only combine with lncRNA to break its secondary structure, which was conducive to lncRNA captured by Y-shaped probes, but also trigger multiple RCA reactions to achieve signal amplification and the goal of sensitive detection of lncRNA. Compared to previous detection methods, in this scheme, we took advantage of the long sequence characteristics of lncRNA to make it a carrier that can bind multiple primers to initiate RCA. This newly designed biosensor provided a linear range from 1 pM to 100 nM with a detection limit of 0.06 pM. This method can provide a new idea for the application of isothermal amplification in detecting lncRNA. Furthermore, the application of the biosensor in liver cancer cell lines and whole blood samples from hepatocellular carcinomatosis patients also confirmed that the method had good selectivity and sensitivity to lncRNA HULC. This method offered a new way for transforming specific lncRNA into clinical application for diagnosis, prognosis, or predicting treatment response.

Kinetics of protein-assisted nucleic acid interconversion monitored by transient time resolved fluorescence in microfluidic droplets.

Interconversions between nucleic acid structures play an important role in transcriptional and translational regulation and also in repair and recombination. These interconversions are frequently promoted by nucleic acid chaperone proteins. To monitor their kinetics, Förster resonance energy transfer (FRET) is widely exploited using ensemble fluorescence intensity measurements in pre-steady-state stopped-flow experiments. Such experiments only provide a weighted average of the emission of all species in solution and consume large quantities of materials. Herein, we lift these limitations by combining time-resolved fluorescence (TRF) with droplet microfluidics (DmF). We validate the innovative TRF-DmF approach by investigating the well characterized annealing of the HIV-1 (+)/(-) Primer Binding Sequences (PBS) promoted by a HIV-1 nucleocapsid peptide. Upon rapid mixing of the FRET-labelled (-)PBS with its complementary (+)PBS sequence inside microdroplets, the TRF-DmF set-up enables resolving the time evolution of sub-populations of reacting species and reveals an early intermediate with a ∼50 ps donor fluorescence lifetime never identified so far. TRF-DmF also favorably compares with single molecule experiments, as it offers an accurate control of concentrations with no upper limit, no need to graft one partner on a surface and no photobleaching issues.

Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Polymerase Domain Insertion That Confers Dominant Exonuclease Activity.

Trypanosoma brucei and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might be a replicative, proofreading enzyme, remains enigmatic. De novo modeling of POLIB suggested a structure that is divergent from all other Family A polymerases, in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3'-5' exonucleases. Here we demonstrate recombinant POLIB 3'-5' exonuclease prefers DNA vs RNA substrates and degrades single- and double-stranded DNA nonprocessively. Exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB extends an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborates the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The enzymatic properties of POLIB suggest this paralog is not a replicative kDNA polymerase, and the noncanonical polymerase domain provides another example of exquisite diversity among DNA polymerases for specialized function.

Quick validation of genetic quality for conditional alleles in mice.

Site-specific conditional inactivation technologies using Cre-loxP or Flp-FRT systems are becoming increasingly important for the elucidation of gene function and disease mechanism in vivo. A large number of gene knockout mouse models carrying complex conditional alleles have been generated by global community efforts and made available for biomedical researchers. The structures of conditional alleles in these mice are becoming increasingly complex and sophisticated, and so the validation of the genetic quality of these alleles is likewise becoming a laborious task for individual researchers. To ensure the reproducibility of conditional experiments, the researcher should confirm that loxP or FRT is integrated at the correct positions in the genome prior to start of the experiments. We report the successful design of universal PCR primers specific to loxP and FRT for the quick validation of conditional floxed and Flrted alleles. The primer set consists of forward and reverse primers complimentary to the loxP or FRT sequences with partial modifications at the 5' end containing 6-base restriction endonuclease recognition sites. The universal primer set was tested to detect genomic intervals between a pair of cis-integrated loxP or FRT and was useful for quickly validating various floxed or Flrted alleles in conditional mice.

Telomere DNA G-quadruplex folding within actively extending human telomerase.

Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K+ versus GQ-destabilizing Li+ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase-DNA complex. Addition of the telomerase processivity factor POT1-TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li+ cations. This result suggests GQ folding synergizes with POT1-TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K+ but not Li+, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.

Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method.

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.

Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes.

BACKGROUND: DNA mismatches can affect the efficiency of PCR techniques if the intended target has mismatches in primer or probe regions. The accepted rule is that mismatches are detrimental as they reduce the hybridization temperatures, yet a more quantitative assessment is rarely performed. METHODS: We calculate the hybridization temperatures of primer/probe sets after aligning to SARS-CoV-2, SARS-CoV-1 and non-SARS genomes, considering all possible combinations of single, double and triple consecutive mismatches. We consider the mismatched hybridization temperature within a range of 5 ∘C to the fully matched reference temperature. RESULTS: We obtained the alignments of 19 PCR primers sets that were recently reported for the detection of SARS-CoV-2 and to 21665 SARS-CoV-2 genomes as well as 323 genomes of other viruses of the coronavirus family of which 10 are SARS-CoV-1. We find that many incompletely aligned primers become fully aligned to most of the SARS-CoV-2 when mismatches are considered. However, we also found that many cross-align to SARS-CoV-1 and non-SARS genomes. CONCLUSIONS: Some primer/probe sets only align substantially to most SARS-CoV-2 genomes if mismatches are taken into account. Unfortunately, by the same mechanism, almost 75% of these sets also align to some SARS-CoV-1 and non-SARS viruses. It is therefore recommended to consider mismatch hybridization for the design of primers whenever possible, especially to avoid undesired cross-reactivity.

Reference gene selection for miRNA and mRNA normalization in potato in response to potato virus Y.

This was the first report on evaluating candidate reference genes for quantifying the expression profiles of both coding (e.g., mRNA) and non-coding (e.g., miRNA) genes in potato response to potato virus Y (PVY) inoculation. The reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) method was employed to quantify the expression profiles of eight selected candidate reference genes; their expression stability was analyzed by four statistical algorithms, i.e., geNorm, BestKeeper, NormFinder and RefFinder. The most stable reference genes were sEF1a, sTUBb and seIF5 with a high stability. The least stable ones were sPP2A, sSUI1 and sGAPDH. The same reference gene allows for normalization of both miRNA and mRNA levels from a single RNA sample using cDNAs synthesized in a single RT reaction, in which a stem-loop primer was used for miRNAs and the oligo (dT) for mRNAs.

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.

Development of a recombinase-aided amplification assay for rapid detection of human norovirus GII.4.

BACKGROUND: Human noroviruses are one of the main causes of foodborne illnesses and represent a serious public health concern. Rapid and sensitive assays for human norovirus detection are undoubtedly necessary for clinical diagnosis, especially in regions without more sophisticated equipment. METHOD: The rapid reverse transcription recombinase-aided amplification (RT-RAA) is a fast, robust and isothermal nucleic acid detection method based on enzyme reaction. This method can complete the sample detection at 39 °C in 30 min. In this study, we successfully established a rapid reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of human norovirus GII.4 and applied this assay to clinical samples, as well as comparison with commercial reverse transcription real-time fluorescence quantitative PCR (RT-qPCR). RESULTS: At 95% probability, the detection sensitivity of RT-RAA was 3.425 log10 genomic copies (LGC)/reaction. Moreover, no cross-reaction was observed with other norovirus genogroups and other common foodborne viruses. Stool samples were examined by RT-RAA and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Compared of RT-qPCR, kappa values for human norovirus detection with RT-RAA were 0.894 (p < 0.001), indicating that both assays were in agreement. CONCLUSION: This RT-RAA assay provides a rapid, specific, and sensitive assay for human norovirus detection and is suitable for clinical testing.

Deciphering the genetic diversity and population structure of Turkish bread wheat germplasm using iPBS-retrotransposons markers.

BACKGROUND: Research activities aiming to investigate the genetic diversity are very crucial because they provide information for the breeding and germplasm conservation activities. Wheat is one of the most important cereal crops globally by feeding more than a third of the human population around the world. METHODS AND RESULTS: During present investigation, a total of 74 Turkish bread wheat accessions (54 landraces and 20 cultivars) were used as plant material and iPBS-retrotransposons marker system was used for the molecular characterization. 13 polymorphic primers used for molecular characterization resulted a total of 152 bands. Range of calculated diversity indices like polymorphism information content (0.11-0.702), effective numbers of alleles (1.026-1.526), Shannon's information index (0.101-0.247) and gene diversity (0.098-0.443) confirmed higher genetic variations in studied germplasm. Bread wheat landraces reflected higher genetic variations compared to commercial cultivars. Analysis of molecular variance resulted that higher (98%) genetic variations are present within populations. The model-based structure algorithm separated 74 bread wheat accessions in to two populations. Diversity indices based on structure evaluated population's revealed population B as a more diverse population. The principal coordinate analysis and neighbor-joining analysis separated 74 bread wheat accessions according to their collection points. Genetic distance for 74 Turkish bread wheat accessions explored Bingol and Asure accessions as genetically diverse that can be used as parents for breeding activities. CONCLUSIONS: The extensive diversity of bread wheat in Turkish germplasm might be used as genetic resource for the exhaustive wheat breeding program. For instance, accessions Bingol and Asure were found genetically diverse and can be used as parents for future breeding activities.

PPL2 translesion polymerase is essential for the completion of chromosomal DNA replication in the African trypanosome.

Faithful copying of the genome is essential for life. In eukaryotes, a single archaeo-eukaryotic primase (AEP), DNA primase, is required for the initiation and progression of DNA replication. Here we have identified additional eukaryotic AEP-like proteins with DNA-dependent primase and/or polymerase activity. Uniquely, the genomes of trypanosomatids, a group of kinetoplastid protozoa of significant medical importance, encode two PrimPol-like (PPL) proteins. In the African trypanosome, PPL2 is a nuclear enzyme present in G2 phase cells. Following PPL2 knockdown, a cell-cycle arrest occurs after the bulk of DNA synthesis, the DNA damage response is activated, and cells fail to recover. Consistent with this phenotype, PPL2 replicates damaged DNA templates in vitro, including templates containing the UV-induced pyrimidine-pyrimidone (6-4) photoproduct. Furthermore, PPL2 accumulates at sites of nuclear DNA damage. Taken together, our results indicate an essential role for PPL2 in postreplication tolerance of endogenous DNA damage, thus allowing completion of genome duplication.

Selection of self-priming molecular replicators.

Self-priming amplification of oligonucleotides is possible based on foldback of 3' ends, self-priming, and concatemerization, especially in the presence of phosphorothioate linkages. Such a simple replicative mechanism may have led to the accumulation of specific replicators at or near the origin of life. To determine how early replicators may have competed with one another, we have carried out selections with phosphorothiolated hairpins appended to a short random sequence library (N10). Upon the addition of deoxynucleoside triphosphates and a polymerase, concatemers quickly formed, and those random sequences that templated the insertion of purines, especially during initiation, quickly predominated. Over several serial transfers, particular sequences accumulated, and in isolation these were shown to outcompete less efficient replicators.