Nucleic Acids Research

Soler-Vila, Paula; Cuscó, Pol; Farabella, Irene; Di Stefano, Marco; Marti-Renom, Marc A

doi: 10.1093/nar/gkaa087pmid: 32083658

The rapid development of Chromosome Conformation Capture (3C-based techniques), as well as imaging together with bioinformatics analyses, has been fundamental for unveiling that chromosomes are organized into the so-called topologically associating domains or TADs. While TADs appear as nested patterns in the 3C-based interaction matrices, the vast majority of available TAD callers are based on the hypothesis that TADs are individual and unrelated chromatin structures. Here we introduce TADpole, a computational tool designed to identify and analyze the entire hierarchy of TADs in intra-chromosomal interaction matrices. TADpole combines principal component analysis and constrained hierarchical clustering to provide a set of significant hierarchical chromatin levels in a genomic region of interest. TADpole is robust to data resolution, normalization strategy and sequencing depth. Domain borders defined by TADpole are enriched in main architectural proteins (CTCF and cohesin complex subunits) and in the histone mark H3K4me3, while their domain bodies, depending on their activation-state, are enriched in either H3K36me3 or H3K27me3, highlighting that TADpole is able to distinguish functional TAD units. Additionally, we demonstrate that TADpole's hierarchical annotation, together with the new DiffT score, allows for detecting significant topological differences on Capture Hi-C maps between wild-type and genetically engineered mouse.

Poulain, Stéphane; Arnaud, Ophélie; Kato, Sachi; Chen, Iris; Ishida, Hiro; Carninci, Piero; Plessy, Charles

doi: 10.1093/nar/gkaa079pmid: 32025730

The development of complex methods in molecular biology is a laborious, costly, iterative and often intuition-bound process where optima are sought in a multidimensional parameter space through step-by-step optimizations. The difficulty of miniaturizing reactions under the microliter volumes usually handled in multiwell plates by robots, plus the cost of the experiments, limit the number of parameters and the dynamic ranges that can be explored. Nevertheless, because of non-linearities of the response of biochemical systems to their reagent concentrations, broad dynamic ranges are necessary. Here we use a high-performance nanoliter handling platform and computer generation of liquid transfer programs to explore in quadruplicates 648 combinations of 4 parameters of a biochemical reaction, the reverse-transcription, which lead us to uncover non-linear responses, parameter interactions and novel mechanistic insights. With the increased availability of computer-driven laboratory platforms for biotechnology, our results demonstrate the feasibility and advantage of methods development based on reproducible, computer-aided exhaustive characterization of biochemical systems.

Rolando, Justin C; Jue, Erik; Barlow, Jacob T; Ismagilov, Rustem F

doi: 10.1093/nar/gkaa099pmid: 32103255

Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.

Hagelskamp, Felix; Borland, Kayla; Ramos, Jillian; Hendrick, Alan G; Fu, Dragony; Kellner, Stefanie

doi: 10.1093/nar/gkaa091pmid: 32083657

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.

DiNapoli, Sara E; Martinez-McFaline, Raul; Gribbin, Caitlin K; Wrighton, Paul J; Balgobin, Courtney A; Nelson, Isabel; Leonard, Abigail; Maskin, Carolyn R; Shwartz, Arkadi; Quenzer, Eleanor D; Mailhiot, Darya; Kao, Clara; McConnell, Sean C; de Jong, Jill L O; Goessling, Wolfram; Houvras, Yariv

doi:

Moffitt, Andrea B; Spector, Mona S; Andrews, Peter; Kendall, Jude; Alexander, Joan; Stepansky, Asya; Ma, BeiCong; Kolitz, Jonathan; Chiorazzi, Nicholas; Allen, Steven L; Krasnitz, Alex; Wigler, Michael; Levy, Dan; Wang, Zihua

doi: 10.1093/nar/gkaa090pmid: 32083660

Measuring minimal residual disease in cancer has applications for prognosis, monitoring treatment and detection of recurrence. Simple sequence-based methods to detect nucleotide substitution variants have error rates (about 10−3) that limit sensitive detection. We developed and characterized the performance of MASQ (multiplex accurate sensitive quantitation), a method with an error rate below 10−6. MASQ counts variant templates accurately in the presence of millions of host genomes by using tags to identify each template and demanding consensus over multiple reads. Since the MASQ protocol multiplexes 50 target loci, we can both integrate signal from multiple variants and capture subclonal response to treatment. Compared to existing methods for variant detection, MASQ achieves an excellent combination of sensitivity, specificity and yield. We tested MASQ in a pilot study in acute myeloid leukemia (AML) patients who entered complete remission. We detect leukemic variants in the blood and bone marrow samples of all five patients, after induction therapy, at levels ranging from 10−2 to nearly 10−6. We observe evidence of sub-clonal structure and find higher target variant frequencies in patients who go on to relapse, demonstrating the potential for MASQ to quantify residual disease in AML.

doi: 10.1093/nar/gkaa152pmid: 32991676

Hans J. Gross, Prof. Emeritus and former Head of Biochemistry at the University of Würzburg died 6 August 2019 at the age of 83. Hans was involved with NAR from the journal's beginning in 1974, and published in the first issue (Nucleic Acids Res., (1974) 1, 35–44). In 1983, Hans joined NAR’s Editorial Board before becoming an Executive Editor in 1992 until 2010, and sensitively handled well over a thousand papers, dealing with authors and referees with his characteristic charm and gentle good humor. Trained in chemistry, Hans switched to biochemistry and made major contributions to RNA structure, enzymology and function. Hans was born on 11 April 1936 in the German province of Silesia, which is now part of Poland. His childhood was a difficult one. When he was 7 years old, his father was killed during the Second World War in Russia. After the war ended, Silesia was ceded back to Poland and Hans’s mother moved to a small town in Bavaria, Germany. School was 12 km away and Hans rode his bicycle each way to school every day of the week. In spite of this arduous daily commute, he did well in school to get into the ‘Ludwig Maximillians University’ in Munich and to obtain a diploma in Chemistry in 1962. Hans did his PhD research with Heinz Dannenberg at the Max Planck Institute of Biochemistry, also in Munich, on aromatic reactions of steroids. The director of the Institute was Adolf Butenandt, a mentor of Dannenberg, known for his discovery of the steroid hormones estrogen, progesterone and androsterone, for which he was awarded the Nobel Prize in Chemistry. Hans stayed on at the Max Planck Institute for postdoctoral research with Dannenberg for two more years and then went to work with H. Gobind Khorana at the University of Wisconsin in the USA. His stay in Wisconsin was to have a lasting effect; Hans was introduced to tRNAs and he developed a lifelong interest in tRNAs and nucleic acids, an area in which he worked for the rest of his scientific career. In 1968, Hans returned to the Max Planck Institute, now in Martinsried outside of Munich, as an independent investigator and in 1980 moved to the University of Würzburg as Professor and Head of the Institute of Biochemistry, a position he would hold until 2003, when he retired. Elected to the European Molecular Biology Organization in 1980, Hans was prolific as a scientist and made many important contributions. His first major contribution was the sequence and structural analysis in 1978 of viroid RNA. Viroids are plant pathogens, which are small RNA molecules (246–371-nt long) and are of much interest since they are different from plant viruses, which have much longer RNAs and code for many proteins. Hans’ work published in collaboration with Heinz Sänger established the sequence for the first time of a 359 nt long viroid RNA, at a time when it was extremely difficult to sequence RNA molecules of this size, particularly those RNAs obtainable only in miniscule amounts and not available in radioactively labeled form. Hans’ work also showed that the viroid RNA was circular with a very tight rod like secondary structure and with no potential for coding proteins of reasonable size. Hans’ next major contribution was on RNA splicing. He discovered in wheat germ extracts an RNA ligase with novel and—at the time—unexpected properties. His work showed that the ligase could circularize RNA molecules carrying specific 5′- and 3′- termini and the ligase turned out to be involved in tRNA splicing in plants, yeasts and other organisms. The same ligase could also convert a linear viroid RNA into a circular one, suggesting its possible involvement also in the circularization step of viroid RNA replication. Hans also published many important papers on tRNA biochemistry, structure and function including identity elements on the tRNA that inserts selenocysteine into proteins in mammalian cells. tRNAs were in many ways his first love in science. One of his students said that the mantra in Hans’s lab was ‘the most popular four-letter word in science is the word tRNA’. Some of Hans’ work on tRNA was also carried out in collaboration with his longtime collaborator and scientific partner Hildburg Beier, a faculty colleague in Würzburg. Hans had a broad interest in science. After retiring and closing his lab, he continued to work with another research group in Würzburg, this time on the innate ability of honeybees for number recognition without counting. Over years in teaching and research, Hans trained many scientists, many of whom are active in academia and in biotech industry. He was a great mentor to students and postdocs in his lab. He supported them wholeheartedly and took great satisfaction in their accomplishments. They, in turn, admired him and were most thankful to him for his support and for his guidance. Hans often used subtle humor and storytelling effectively to make a point in scientific communication and in teaching. An example comes from his description of work on sequence analysis of viroid RNAs. This required the development of methods for purification of viroid RNAs, which are present in miniscule amounts in infected plants and assays for infectivity of viroid RNAs took weeks. One of us still remembers a slide that Hans showed at a meeting over 45 years ago, consisting of a row of tomato plants, most of them vibrant green, tall and healthy looking, except for those toward the middle inoculated with viroid RNA, which were shriveled, short and sad looking. The slide left no doubt about infectivity of the short viroid RNAs on their own. One of us also remembers Hans talking about mushrooms at a family dinner event, and Hans saying ‘you can eat any type of mushroom (a pause), but (another pause), some only once’. This led to the question of why and Hans would go on to the topic of toxins, the nature of the toxins and how they worked. As a youngster, Hans loved hiking in the woods and learnt to forage for edible mushrooms. Outside of research and teaching, Hans also made important contributions toward publication of scientific journals and books. He was an Executive Editor of Nucleic Acids Research for close to two decades and played an important role in the development of the journal. Not only did he pursue the normal responsibilities of making decisions on acceptance or rejection of manuscripts, but he was always helpful in suggesting improvements and notably in telling authors when they could ignore the remarks of a reviewer, who seemed unnecessarily partisan. At the Annual Editorial Board Meetings, Hans would often come up with ideas aimed at improving the journal, which others had not considered previously. One of us remembers many occasions when Hans’s sense of humor and fun greatly enlivened the meetings and encouraged an irreverence that might not have otherwise surfaced and also what a pleasure it was to interact with Hans at these meetings. From 2001 to 2010, Hans was also the editor of the series of topical monographs on Nucleic Acids and Molecular Biology published by Springer Verlag. As a person, Hans was physically strong, perhaps from his days as a lumberjack in the forests of Finland during holiday periods from the University. He was also an avid gardener (he taught one of us how to graft one branch of a fruit tree to another tree), an excellent cook and he was always full of life. He was a loving father to his daughters. Above all, Hans was a most caring, kind and a compassionate person, always willing to help those in need. Hans was also an avid fisherman, almost quirky at times. While a postdoc at Wisconsin, during heavy thunderstorms, when most rush to shelters underground, Hans was known to tie an old wooden boat to the top of his small VW beetle car, and race to a deep lake about an hour drive outside of Madison to fish. He was a strong believer in the fish going for the bait during stormy weather. To both of us, who knew him for so long (one over a period of more than 50 years), and admired him for his accomplishments in science and for who he was as a person, Hans was a dear friend and a wonderful colleague to be around and we miss him deeply. The community at large has also lost a scientist, who made many important contributions in science and in education. Uttam L. RajBhandary Richard J. Roberts Uttam L. RajBhandary is in the Department of Biology at the Massachusetts Institute of Technology, Cambridge, MA 02139, USA and Richard J. Roberts is in New England Biolabs, Inc; Ipswich, MA 01938, USA. © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Debiais, Mégane; Lelievre, Amandine; Smietana, Michael; Müller, Sabine

doi: 10.1093/nar/gkaa132pmid: 32112111

In analogy to split-protein systems, which rely on the appropriate fragmentation of protein domains, split aptamers made of two or more short nucleic acid strands have emerged as novel tools in biosensor set-ups. The concept relies on dissecting an aptamer into a series of two or more independent fragments, able to assemble in the presence of a specific target. The stability of the assembled structure can further be enhanced by functionalities that upon folding would lead to covalent end-joining of the fragments. To date, only a few aptamers have been split successfully, and application of split aptamers in biosensing approaches remains as promising as it is challenging. Further improving the stability of split aptamer target complexes and with that the sensitivity as well as efficient working modes are important tasks. Here we review functional nucleic acid assemblies that are derived from aptamers and ribozymes/DNAzymes. We focus on the thrombin, the adenosine/ATP and the cocaine split aptamers as the three most studied DNA split systems and on split DNAzyme assemblies. Furthermore, we extend the subject into split light up RNA aptamers used as mimics of the green fluorescent protein (GFP), and split ribozymes.

Shaban, Haitham A; Seeber, Andrew

doi: 10.1093/nar/gkaa135pmid: 32123910

The spatio-temporal organization of chromatin in the eukaryotic cell nucleus is of vital importance for transcription, DNA replication and genome maintenance. Each of these activities is tightly regulated in both time and space. While we have a good understanding of chromatin organization in space, for example in fixed snapshots as a result of techniques like FISH and Hi-C, little is known about chromatin dynamics in living cells. The rapid development of flexible genomic loci imaging approaches can address fundamental questions on chromatin dynamics in a range of model organisms. Moreover, it is now possible to visualize not only single genomic loci but the whole genome simultaneously. These advances have opened many doors leading to insight into several nuclear processes including transcription and DNA repair. In this review, we discuss new chromatin imaging methods and how they have been applied to study transcription.

Pande, Amit; Makalowski, Wojciech; Brosius, Jürgen; Raabe, Carsten A

doi: 10.1093/nar/gkaa026pmid: 32133533

Analysis of ENCODE long RNA-Seq and ChIP-seq (Chromatin Immunoprecipitation Sequencing) datasets for HepG2 and HeLa cell lines uncovered 1647 and 1958 transcripts that interfere with transcription factor binding to human enhancer domains. TFBSs (Transcription Factor Binding Sites) intersected by these ‘Enhancer Occlusion Transcripts’ (EOTrs) displayed significantly lower relative transcription factor (TF) binding affinities compared to TFBSs for the same TF devoid of EOTrs. Expression of most EOTrs was regulated in a cell line specific manner; analysis for the same TFBSs across cell lines, i.e. in the absence or presence of EOTrs, yielded consistently higher relative TF/DNA-binding affinities for TFBSs devoid of EOTrs. Lower activities of EOTr-associated enhancer domains coincided with reduced occupancy levels for histone tail modifications H3K27ac and H3K9ac. Similarly, the analysis of EOTrs with allele-specific expression identified lower activities for alleles associated with EOTrs. ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag Sequencing) and 5C (Carbon Copy Chromosome Conformation Capture) uncovered that enhancer domains associated with EOTrs preferentially interacted with poised gene promoters. Analysis of EOTr regions with GRO-seq (Global run-on) data established the correlation of RNA polymerase pausing and occlusion of TF-binding. Our results implied that EOTr expression regulates human enhancer domains via transcriptional interference.