Development of Polymer-Based In-Plane Nanopore for DNA Sequencing

Mechanically robust solid-state nanopores have the potential to be the next generation DNA sensing platforms. However, mass production and limited base-calling accuracy are the hurdles for solid-state nanopore based DNA sensing. In order to solve these problems, a polymer dual-nanopore device fabricated via high throughput nanoimprint lithography (NIL) was proposed to sequence DNA by time-of-flight (ToF) measurement. As a proof of concept, this study presents mononucleotides discrimination via ToF measurement using polymer in-plane dual-nanopore device. First, fabrication of polymer in-plane nanopore with controllable dimensions was studied in consideration of experimental conditions and materials selection. Then, surface charge density effect on DNA translocation through in-plane nanopore was studied numerically and experimentally using fabricated nanopore devices on PEGDA, PMMA and COC. λ-DNA sensing was only observed in PEGDA device with a surface charge density lower than the threshold surface charge density predicted by COMSOL simulation. With demonstrated single molecule sensing ability, mononucleotides were introduced to PEGDA dual-nanopore with 500 nm flight tube and discriminated under various conditions. At pH 8.0, mononucleotides were driven by eletrophoretic motion and their ToF was in a decreasing order of dGMP \u3e dAMP \u3e dCMP \u3e dTMP. At pH 10.0, mononucleotides were driven by electroosmotic flow (EOF) due to a higher surface charge density on nanochannel walls and ToF was in the same order as pH 8.0 with an average identification accuracy of 55%. Dual-nanopore device with 1 μm flight tube was then used to improve the average identification accuracy to 75%. Finally, dGMP and dTMP in a mix solution were dicriminated by their ToF difference

Exploring molecular biology in sequence space: the road to next-generation single-molecule biophysics

Next-generation sequencing techniques have led to a new quantitative dimension in the biological sciences. In particular, integrating sequencing techniques with biophysical tools allows sequence-dependent mechanistic studies. Using the millions of DNA clusters that are generated during sequencing to perform high-throughput binding affinity and kinetics measurements enabled the construction of energy landscapes in sequence space, uncovering relationships between sequence, structure, and function. Here, we review the approaches to perform ensemble fluorescence experiments on next-generation sequencing chips for variations of DNA, RNA, and protein sequences. As the next step, we anticipate that these fluorescence experiments will be pushed to the single-molecule level, which can directly uncover kinetics and molecular heterogeneity in an unprecedented high-throughput fashion. Molecular biophysics in sequence space, both at the ensemble and single-molecule level, leads to new mechanistic insights. The wide spectrum of applications in biology and medicine ranges from the fundamental understanding of evolutionary pathways to the development of new therapeutics.Biological and Soft Matter Physic

Evaluation of nanopore-based sequencing technology for gene marker based analysis of complex microbial communities. Method development for accurate 16S rRNA gene amplicon sequencing

Nucleic acid sequencing can provide a detailed overview of microbial communities in comparison with standard plate-culture methods. Expansion of high-throughput sequencing (HTS) technologies and reduction in analysis costs has allowed for detailed exploration of various habitats with use of amplicon, metagenomics, and metatranscriptomics approaches. However, due to a capital cost of HTS platforms and requirements for batch analysis, genomics-based studies are still not being used as a standard method for the comprehensive examination of environmental or clinical samples for microbial characterization. This research project investigated the potential of a novel nanopore-based sequencing platform from Oxford Nanopore Technologies (ONT) for rapid and accurate analysis of various environmentally complex samples. ONT is an emerging company that developed the first-ever portable nanopore-based sequencing platform called MinIONTM. Portability and miniaturised size of the device gives an immense opportunity for de-centralised, in-field, and real-time analysis of environmental and clinical samples. Nonetheless, benchmarking of this new technology against the current gold-standard platform (i.e., Illumina sequencers) is necessary to evaluate nanopore data and understand its benefits and limitations. The focus of this study is on the evaluation of nanopore sequencing data: read quality, sequencing errors, alignment quality but also bacterial community structure. For this reason, mock bacterial community samples were generated, sequenced and analysed with use of multiple bioinformatics approaches. Furthermore, this study developed sophisticated library preparation and data analyses methods to enable high-accuracy analysis of amplicon libraries from complex microbial communities for sequencing on the nanopore platform. Besides, the best performing library preparation and data analyses methods were used for analysis of environmental samples and compared to high-quality Illumina metagenomics data. This work opens a new possibility for accurate, in-field amplicon analysis of complex samples with the use of MinIONTM and for the development of autonomous biosensing technology for culture-free detection of pathogenic and non-pathogenic microorganisms in water, soil, food, drinks or blood

Analysis of Transcriptome and Epitranscriptome in Plants Using PacBio Iso-Seq and Nanopore-Based Direct RNA Sequencing

Nanopore sequencing from Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) are revolutionizing the way transcriptomes are analyzed. These methods offer many advantages over most widely used high-throughput short-read RNA sequencing (RNA-Seq) approaches and allow a comprehensive analysis of transcriptomes in identifying full-length splice isoforms and several other post-transcriptional events. In addition, direct RNA-Seq provides valuable information about RNA modifications, which are lost during the PCR amplification step in other methods. Here, we present a comprehensive summary of important applications of these technologies in plants, including identification of complex alternative splicing (AS), full-length splice variants, fusion transcripts, and alternative polyadenylation (APA) events. Furthermore, we discuss the impact of the newly developed nanopore direct RNA-Seq in advancing epitranscriptome research in plants. Additionally, we summarize computational tools for identifying and quantifying full-length isoforms and other co/post-transcriptional events and discussed some of the limitations with these methods. Sequencing of transcriptomes using these new single-molecule long-read methods will unravel many aspects of transcriptome complexity in unprecedented ways as compared to previous short-read sequencing approaches. Analysis of plant transcriptomes with these new powerful methods that require minimum sample processing is likely to become the norm and is expected to uncover novel co/post-transcriptional gene regulatory mechanisms that control biological outcomes during plant development and in response to various stresses

Poly(A) Tail Regulation in the Nucleus

Der Ribonukleinsäure (RNS) Stoffwechsel umfasst verschiedene Schritte, beginnend mit der Transkription der RNS über die Translation bis zum RNA Abbau. Poly(A) Schwänze befinden sich am Ende der meisten der Boten-RNS, schützen die RNA vor Abbau und stimulieren Translation. Die Deadenylierung von Poly(A) Schwänzen limitiert den Abbau von RNS. Bisher wurde RNS Abbau meist im Kontext von cytoplasmatischen Prozessen untersucht, ob und wie RNS Deadenylierung und Abbau in Nukleus erfolgen ist bisher unklar. Es wurde daher eine neue Methode zur genomweiten Bestimmung von Poly(A) Schwanzlänge entwickelt, welche FLAM-Seq genannt wurde. FLAM-Seq wurde verwendet um Zelllinien, Organoide und C. elegans RNS zu analysieren und es wurde eine signifikante Korrelation zwischen 3’-UTR und Poly(A) Länge gefunden, sowie für viele Gene ein Zusammenhang von alternativen 3‘-UTR Isoformen und Poly(A) Länge. Die Untersuchung von Poly(A) Schwänzen von nicht-gespleißten RNS Molekülen zeige, dass deren Poly(A) Schwänze eine Länge von mehr als 200 nt hatten. Die Analyse wurde durch eine Inhibition des Spleiß-Prozesses validiert. Die Verwendung von Methoden zur Markierung von RNS, welche die zeitliche Auflösung der RNS Prozessierung ermöglicht, deutete auf eine Deadenylierung der Poly(A) Schwänze schon wenige Minuten nach deren Synthesis hin. Die Analyse von subzellulären Fraktionen zeigte, dass diese initiale Deadenylierung ein Prozess im Nukleus ist. Dieser Prozess ist gen-spezifisch und Poly(A) Schwänze von bestimmten Typen von Transkripten, wie nuklearen langen nicht-kodierende RNS Molekülen waren nicht deadenyliert. Um Enzyme zu identifizieren, welche die Deadenylierung im Zellkern katalysieren, wurden verschiedene Methoden wie RNS-abbauende Cas Systeme, siRNAs oder shRNA Zelllinien verwendet. Trotz einer effizienten Reduktion der RNS Expression entsprechender Enzymkomplexe konnten keine molekularen Phänotypen identifiziert werden welche die Poly(A) Länge im Zellkern beeinflussen.The RNA metabolism involves different steps from transcription to translation and decay of messenger RNAs (mRNAs). Most mRNAs have a poly(A) tail attached to their 3’-end, which protects them from degradation and stimulates translation. Removal of the poly(A) tail is the rate-limiting step in RNA decay controlling stability and translation. It is yet unclear if and to what extent RNA deadenylation occurs in the mammalian nucleus. A novel method for genome-wide determination of poly(A) tail length, termed FLAM-Seq, was developed to overcome current challenges in sequencing mRNAs, enabling genome-wide analysis of complete RNAs, including their poly(A) tail sequence. FLAM-Seq analysis of different model systems uncovered a strong correlation between poly(A) tail and 3’-UTR length or alternative polyadenylation. Cytosine nucleotides were further significantly enriched in poly(A) tails. Analyzing poly(A) tails of unspliced RNAs from FLAM-Seq data revealed the genome-wide synthesis of poly(A) tails with a length of more than 200 nt. This could be validated by splicing inhibition experiments which uncovered potential links between the completion of splicing and poly(A) tail shortening. Measuring RNA deadenylation kinetics using metabolic labeling experiments hinted at a rapid shortening of tails within minutes. The analysis of subcellular fractions obtained from HeLa cells and a mouse brain showed that initial deadenylation is a nuclear process. Nuclear deadenylation is gene specific and poly(A) tails of lncRNAs retained in the nucleus were not shortened. To identify enzymes responsible for nuclear deadenylation, RNA targeting Cas-systems, siRNAs and shRNA cell lines were used to different deadenylase complexes. Despite efficient mRNA knockdown, subcellular analysis of poly(A) tail length by did not yield molecular phenotypes of changing nuclear poly(A) tail length

Whole genome experimental maps of DNA G-quadruplexes in multiple species.

Genomic maps of DNA G-quadruplexes (G4s) can help elucidate the roles that these secondary structures play in various organisms. Herein, we employ an improved version of a G-quadruplex sequencing method (G4-seq) to generate whole genome G4 maps for 12 species that include widely studied model organisms and also pathogens of clinical relevance. We identify G4 structures that form under physiological K+ conditions and also G4s that are stabilized by the G4-targeting small molecule pyridostatin (PDS). We discuss the various structural features of the experimentally observed G-quadruplexes (OQs), highlighting differences in their prevalence and enrichment across species. Our study describes diversity in sequence composition and genomic location for the OQs in the different species and reveals that the enrichment of OQs in gene promoters is particular to mammals such as mouse and human, among the species studied. The multi-species maps have been made publicly available as a resource to the research community. The maps can serve as blueprints for biological experiments in those model organisms, where G4 structures may play a role.The S.B. research group is supported by programme grant funding from Cancer Research UK (C9681/A18618), European Research Council Advanced Grant No. 339778, a Wellcome Trust Senior Investigator Award (grant 209441/z/17/z) and by core funding from Cancer Research UK (C14303/A17197). We are grateful to the Biotechnology and Biological Sciences Research Council (BBSRC) and Illumina for the CASE studentship supporting V.S.C. (BB/I015477/1)

From in vitro evolution to protein structure

In the nanoscale, the machinery of life is mainly composed by macromolecules and macromolecular complexes that through their shapes create a network of interconnected mechanisms of biological processes. The relationship between shape and function of a biological molecule is the foundation of structural biology, that aims at studying the structure of a protein or a macromolecular complex to unveil the molecular mechanism through which it exerts its function. What about the reverse: is it possible by exploiting the function for which a protein was naturally selected to deduce the protein structure? To this aim we developed a method, called CAMELS (Coupling Analysis by Molecular Evolution Library Sequencing), able to obtain the structural features of a protein from an artificial selection based on that protein function. With CAMELS we tried to reconstruct the TEM-1 beta lactamase fold exclusively by generating and sequencing large libraries of mutational variants. Theoretically with this method it is possible to reconstruct the structure of a protein regardless of the species of origin or the phylogenetical time of emergence when a functional phenotypic selection of a protein is available. CAMELS allows us to obtain protein structures without needing to purify the protein beforehand

Dynamique adaptative des virus hautement variables à un nouvel environnement réplicatif

La lutte pour les ressources est un phénomène qui a débuté dès l'apparition d'organismes reproductifs et dont la description a été initiée par Malthus puis remarquablement synthétisée et étendue à la biologie sous le terme d'évolution par Darwin en 1859 dans De l'origine des espèces . Si le concept est ancien à l'échelle des sciences biologiques, il continue à caractériser des domaines à l'époque insoupçonnés par son auteur tels que la virologie. En effet, les virus hautement variables tels que le virus de l'immunodéficience humaine (VIH), de l'hépatite B (VHB) et de l'hépatite C (VHC) sont présents sous forme de quasi espèce au sein de leur environnement réplicatif, c'est à dire qu'une multitude de virus génétiquement proche mais distincts coexistent au sein de cet espace qu'ils doivent partager selon les mêmes règles générales que les êtres vivants. Ainsi, lorsque des pressions de sélection s'exercent (immunitaires, antivirales ), une redistribution des variants majoritaires est observé au bénéfice de variants minoritaires mieux adaptés à cet environnement changeant. La modélisation mathématique et informatique de la capacité mutationnelle et la dynamique d'adaptation des variants minoritaires au travers de 6 études de cohortes de patients infectés, par la technique ultra-sensible de pyroséquençage haut débit associée à des logiciels originaux ont permis de mettre en évidence, caractériser et évaluer l'impact de marqueurs diagnostics permettant de prédire la résistance aux antiviraux mais aussi de caractériser de nouvelles cibles antivirales.Struggle for resources is a worldwide rule which has been first described by Malthus and extended to whole world of living organisms by Darwin in 1859 in Origin of species . Today, this concept has been enlarged to virological field, and is particularly adapted to describe highly variable viruses like Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) which have a quasispecies distribution in infected patients characterized by the co-existence of a number of distinct but related viral populations. Selection pressure on viral replicative environment (immune, antiviral drug treatment ), generally lead to a redistribution of the viral quasispecies with an increasing of the best adapted minor viral variants at the expense of major viral populations. Mathematical and bioinformatic modelization of this phenomenon through 6 infected patients cohorts by means of ultra-deep sequencing and an original bioinformatic package allowed discovery, characterization and evaluation of new diagnostic markers that could be used to prevent resistance emergence to antiviral drugs and to characterized new therapeutics antiviral targets.PARIS-EST-Université (770839901) / SudocPARIS12-Bib. électronique (940280011) / SudocSudocFranceF