Editorial Board
Publication date: 1 December 2018Source: Methods, Volume 151Author(s): (Source: Methods)
Source: Methods - December 7, 2018 Category: Molecular Biology Source Type: research

The age of data analytics: converting biomedical data into actionable insights
Publication date: 1 December 2018Source: Methods, Volume 151Author(s): Kirill Veselkov, Bjoern Schuller (Source: Methods)
Source: Methods - December 7, 2018 Category: Molecular Biology Source Type: research

Global analysis of RNA metabolism using bio-orthogonal labeling coupled with next-generation RNA sequencing
Publication date: Available online 6 December 2018Source: MethodsAuthor(s): Michael B. Wolfe, Aaron C. Goldstrohm, Peter L. FreddolinoAbstractMany open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the ...
Source: Methods - December 7, 2018 Category: Molecular Biology Source Type: research

Practical considerations on performing and analyzing CLIP-seq experiments to identify transcriptomic-wide RNA-Protein interactions
Publication date: Available online 6 December 2018Source: MethodsAuthor(s): Xiaoli Chen, Sarah A. Castro, Qiuying Liu, Wenqian Hu, Shaojie ZhangAbstractRNA-binding proteins are important players in post-transcriptional regulation, such as modulating mRNA splicing, translation, and degradation under diverse biological settings. Identifying and characterizing the RNA substrates is a critical step in deciphering the function and molecular mechanisms of the target RNA-binding proteins. High-throughput sequencing of the RNA fragments isolated by crosslinking immunoprecipitation (CLIP-seq) is one of the standard techniques to id...
Source: Methods - December 6, 2018 Category: Molecular Biology Source Type: research

Engineered Viral RNA Decay Intermediates to Assess XRN1-mediated decay
Publication date: Available online 3 December 2018Source: MethodsAuthor(s): Joseph Russo, Cary T. Mundell, Phillida A. Charley, Carol Wilusz, Jeffrey WiluszAbstractBoth RNA synthesis and decay must be balanced within a cell to achieve proper gene expression. Additionally, modulation of RNA decay specifically offers the cell an opportunity to rapidly reshape the transcriptome in response to specific stimuli or cues. Therefore, it is critical to understand the underlying mechanisms through which RNA decay contribute to gene expression homeostasis. Cell-free reconstitution approaches have been used successfully to reveal mech...
Source: Methods - December 4, 2018 Category: Molecular Biology Source Type: research

Genome-wide probing RNA structure with the modified DMS-MaPseq in Arabidopsis
Publication date: Available online 29 November 2018Source: MethodsAuthor(s): Zhiye Wang, Meiyue Wang, Tian Wang, Yijing Zhang, Xiuren ZhangAbstractTranscripts have intrinsic propensity to form stable secondary structure that is fundamental to regulate RNA transcription, splicing, translation, RNA localization and turnover. Numerous methods that integrate chemical reactions with next-generation sequencing (NGS) have been applied to study in vivo RNA structure, providing new insights into RNA biology. Dimethyl sulfate (DMS) probing coupled with mutational profiling through NGS (DMS-MaPseq) is a newly developed method for rev...
Source: Methods - November 30, 2018 Category: Molecular Biology Source Type: research

Sequencing-based methods for detection and quantitation of ribose methylations in RNA
Publication date: Available online 29 November 2018Source: MethodsAuthor(s): Nicolai Krogh, Henrik NielsenAbstractRibose methylation is one of the most abundant RNA modifications and is found in all domains of life and all major classes of RNA (rRNA, tRNA, and mRNA). Ribose methylations are introduced by stand-alone enzymes or by generic enzymes guided to the target by small RNA guides. Recent years have seen the development of several sequencing-based methods for RNA modifications relying on different principles. In this review, we compare mapping and quantitation studies of ribose methylations from yeast and human cultur...
Source: Methods - November 30, 2018 Category: Molecular Biology Source Type: research

Current Strategies for Site-Directed RNA editing using ADARs
Publication date: Available online 29 November 2018Source: MethodsAuthor(s): Maria Fernanda Montiel-Gonzalez, Juan Felipe Diaz Quiroz, Joshua J.C. RosenthalAbstractAdenosine Deaminases that Act on RNA (ADARs) are a group of enzymes that catalyze the conversion of adenosines (A’s) to inosines (I’s) in a process known as RNA editing. Though ADARs can act on different types of RNA, editing events in coding regions of mRNA are of particular interest as I’s base pair like guanosines (G’s). Thus, every A-to-I change catalyzed by ADAR is read as an A-to-G change during translation, potentially altering pro...
Source: Methods - November 30, 2018 Category: Molecular Biology Source Type: research

Cell-type specific polysome profiling from mammalian tissues
Publication date: Available online 27 November 2018Source: MethodsAuthor(s): Joseph Seimetz, Waqar Arif, Sushant Bangru, Mikel Hernaez, Auinash KalsotraAbstractThe regulation of gene expression occurs through complex relationships between transcription, processing, turnover, and translation, which are only beginning to be elucidated. We know that at least for certain messenger (m) RNAs, processing, modifications, and sequence elements can greatly influence their translational output through recognition by translation and turn-over machinery. Recently, we and others have combined high-throughput sequencing technologies with...
Source: Methods - November 28, 2018 Category: Molecular Biology Source Type: research

Single base resolution mapping of 2′-O-methylation sites in human mRNA and in 3′ terminal ends of small RNAs
Publication date: Available online 22 November 2018Source: MethodsAuthor(s): Phillip J. Hsu, Qili Fei, Qing Dai, Hailing Shi, Dan Dominissini, Lijia Ma, Chuan HeAbstractThe post-transcriptional modification 2′-O-Methyl (2′OMe) could be present on the ribose of all four ribonucleosides, and is highly prevalent in a wide variety of RNA species, including the 5′ RNA cap of viruses and higher eukaryotes, as well as internally in transfer RNA and ribosomal RNA. Recent studies have suggested that 2′OMe is also located internally in low-abundance RNA species such as viral RNA and mRNA. To profile 2′O...
Source: Methods - November 27, 2018 Category: Molecular Biology Source Type: research

Combining Single Molecule Counting with Bead-Based Multiplexing to Quantify Biological Inflammation Time Course Following Skeletal Muscle Injury
Publication date: Available online 22 November 2018Source: MethodsAuthor(s): Elizabeth A. Tanner, Melody A. Gary, Asheal A. Davis, Brian K. McFarlinAbstractBead-based analysis methods allow for the exploration of a variety of complex biological processes. In particular, these techniques can be applied to better understand how peripheral muscle injury contributes to systemic inflammation. Understanding how these two processes affect one another can give additional insight concerning how changes in inflammation effect readiness to perform in exercise and work environments. The present method sought to combine the strengths o...
Source: Methods - November 23, 2018 Category: Molecular Biology Source Type: research

Ancestral Transcriptome Inference Based on RNA-Seq and ChIP-seq Data
Publication date: Available online 22 November 2018Source: MethodsAuthor(s): Jingwen Yang, Hang Ruan, Yangyun Zou, Zhixi Su, Xun GuAbstractWith the help of high-throughput NGS (next-generation sequencing) technologies, ancestral transcriptome reconstruction is helpful to understand the complexity of transcriptional regulatory systems that underlies the evolution of multiple cellular metazoans with sophisticated functions and distinctive morphologies. To this end, we report a new method of ancestral state inference. The new method used Ornstein-Uhlenbeck (OU) model, which is more biologically realistic, to replace the Brown...
Source: Methods - November 23, 2018 Category: Molecular Biology Source Type: research

Single base resolution mapping of 2’-O-methylation sites in human mRNA and in 3’ terminal ends of small RNAs
Publication date: Available online 22 November 2018Source: MethodsAuthor(s): Phillip J. Hsu, Qili Fei, Qing Dai, Hailing Shi, Dan Dominissini, Lijia Ma, Chuan HeAbstractThe post-transcriptional modification 2’-O-Methyl (2’OMe) could be present on the ribose of all four ribonucleosides, and is highly prevalent in a wide variety of RNA species, including the 5’ RNA cap of viruses and higher eukaryotes, as well as internally in transfer RNA and ribosomal RNA. Recent studies have suggested that 2’OMe is also located internally in low-abundance RNA species such as viral RNA and mRNA. To profile 2’O...
Source: Methods - November 23, 2018 Category: Molecular Biology Source Type: research

Combined Bead-Based Multiplex Detection of RNA and Protein Biomarkers: Implications for Understanding the Time Course of Skeletal Muscle Injury and Repair
Publication date: Available online 22 November 2018Source: MethodsAuthor(s): Melody A. Gary, Elizabeth A. Tanner, Asheal A. Davis, Brian K. McFarlinAbstractBiological response to skeletal muscle injury time course is generally classified as initial (elevated within first 4-h), delayed (elevated at 24-h), and/or prolonged (elevated at 4-h and sustained to 24-h). Accurate description of this process requires the ability to measure a robust set of RNA and protein biomarkers, yet such an approach is not common and not always feasible. This method proposes a novel experimental approach that focuses on the use of bead-based mult...
Source: Methods - November 23, 2018 Category: Molecular Biology Source Type: research

Analysis of Spliceosome Dynamics by Maximum Likelihood Fitting of Dwell Time Distributions
Publication date: Available online 23 November 2018Source: MethodsAuthor(s): Harpreet Kaur, Fatemehsadat Jamalidinan, Samson G.F. Condon, Alessandro Senes, Aaron A. HoskinsAbstractColocalization single-molecule methods can provide a wealth of information concerning the ordering and dynamics of biomolecule assembly. These have been used extensively to study the pathways of spliceosome assembly in vitro. Key to these experiments is the measurement of binding times—either the dwell times of a multi-molecular interaction or times in between binding events. By analyzing hundreds of these times, many new insights into the ...
Source: Methods - November 23, 2018 Category: Molecular Biology Source Type: research

Computational approaches for detection and quantification of A-to-I RNA-editing
Publication date: Available online 20 November 2018Source: MethodsAuthor(s): Yishay Pinto, Erez Y. LevanonAbstractAdenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine (A-to-I) RNA editing in double-stranded RNA. Such editing is important for protection against false activation of the immune system, but also confers plasticity on the transcriptome by generating several versions of a transcript from a single genomic locus. Recently, great efforts were made in developing computational methods for detecting editing events directly from RNA-sequencing (RNA-seq) data. These efforts have led to an improved u...
Source: Methods - November 21, 2018 Category: Molecular Biology Source Type: research

Engineering therapeutic bispecific antibodies using CrossMab technology
Publication date: Available online 16 November 2018Source: MethodsAuthor(s): Christian Klein, Wolfgang Schaefer, Joerg T Regula, Charles Dumontet, Ulrich Brinkmann, Marina Bacac, Pablo UmañaAbstractBispecific antibodies have recently gained major interest as they allow novel mechanisms-of-action and/or therapeutic applications that cannot be achieved using conventional IgG-based antibodies. A major issue in engineering IgG-based bispecific antibodies has been to enable the correct association of heavy and light chains resulting in correct assembly of the desired bispecific antibody in sufficient yield. Various appro...
Source: Methods - November 17, 2018 Category: Molecular Biology Source Type: research

Determining degradation intermediates and the pathway of 3′ to 5′ degradation of histone mRNA using high-throughput sequencing
Publication date: Available online 5 November 2018Source: MethodsAuthor(s): Christopher E. Holmquist, William F. MarzluffAbstractThe half-life of an mRNA is an important parameter contributing to the steady-state level of the mRNA. Rapid changes in mRNA levels can result from decreasing the half-life of an mRNA. Establishing the detailed pathway of mRNA degradation for a particular class of mRNAs requires the ability to isolate mRNA degradation intermediates. High-throughput sequencing provides a method for detecting these intermediates. Here we describe a method for determining the intermediates in 3′ to 5′ de...
Source: Methods - November 16, 2018 Category: Molecular Biology Source Type: research

Analysis of post-transcriptional RNA metabolism in prokaryotes
Publication date: Available online 15 November 2018Source: MethodsAuthor(s): Bijoy K. Mohanty, Sidney R. KushnerAbstractPost-transcriptional RNA metabolic pathways play important roles in permitting prokaryotes to operate under a variety of environmental conditions. Although significant progress has been made during the last decade in deciphering RNA processing pathways in a number of bacteria, a complete understanding of post-transcriptional RNA metabolism in any single microorganism is far from reality. Here we describe multiple experimental approaches that can be used to study mRNA stability, tRNA and rRNA processing, s...
Source: Methods - November 16, 2018 Category: Molecular Biology Source Type: research

Round Optimization for Improved Discovery of Native Bispecific Antibodies
Publication date: Available online 16 November 2018Source: MethodsAuthor(s): F. Gueneau, U. Ravn, N. FischerAbstractThe assembly of bispecific antibodies (bsAb) that retain the structure of a standard IgG can be challenging as the correct pairing of the different heavy and light chains has to be ensured while unwanted side products kept to a minimum. The use of antibodies sharing a common chain facilitates assembly of such bsAb formats but requires additional efforts during the initial discovery phase. We have developed a native bsAb format called κλ body based on antibodies that, while being specific for diff...
Source: Methods - November 16, 2018 Category: Molecular Biology Source Type: research

Optimization of DamID for use in primary cultures of mouse hepatocytes
Publication date: Available online 13 November 2018Source: MethodsAuthor(s): Leonardo Gatticchi, Jose I. de las Heras, Rita Roberti, Eric C. SchirmerAbstractDamID, a method to identify DNA associating with a particular protein, was originally developed for use in immortalized tissue culture lines. The power of this technique has led to its adaptation for a number of additional systems. Here we report adaptations for its use in primary cells isolated from rodents with emphasis on the challenges this presents. Specifically, we present several modifications that allow the method to be performed in mouse acutely isolated prima...
Source: Methods - November 14, 2018 Category: Molecular Biology Source Type: research

RNA modifications in structure prediction – Status quo and future challenges
Publication date: Available online 30 October 2018Source: MethodsAuthor(s): Andrea Tanzer, Ivo L. Hofacker, Ronny LorenzAbstractChemical modifications of RNA nucleotides change their identity and characteristics and thus alter genetic and structural information encoded in the genomic DNA. tRNA and rRNA are probably the most heavily modified genes, and often depend on derivatization or isomerization of their nucleobases in order to correctly fold into their functional structures. Recent RNomics studies, however, report transcriptome wide RNA modification and suggest a more general regulation of structuredness of RNAs by thi...
Source: Methods - November 11, 2018 Category: Molecular Biology Source Type: research

Analysis of RNA 5′ ends: phosphate enumeration and cap characterization
Publication date: Available online 10 November 2018Source: MethodsAuthor(s): Daniel J. Luciano, Joel G. BelascoAbstractThe function and fate of cellular RNAs are often governed by the phosphorylation state at the 5′ end or the identity of whatever cap may be present there. Here we described methods for examining these important 5′-terminal features on any cellular or synthetic RNA of interest that can be detected by Northern blotting. One such method, PABLO, is a splinted ligation assay that makes it possible to accurately quantify the percentage of 5′ ends that are monophosphorylated. Another, PACO, is a...
Source: Methods - November 11, 2018 Category: Molecular Biology Source Type: research

Fluorescence-based Quantification of Nucleocytoplasmic Transport
Publication date: Available online 10 November 2018Source: MethodsAuthor(s): Joshua B. Kelley, Bryce M. PaschalAbstractThe sequestration of DNA within the membrane-bound nucleus is a defining characteristic of eukaryotic cells. Replication and transcription are therefore restricted to the nucleus, however, the regulation of these events relies on cytoplasmic processes including protein synthesis and signal transduction pathways. Because a variety of cellular activities depend on nuclear transport, researchers from diverse fields have found it useful to examine the nuclear localization of proteins of interest. Here we prese...
Source: Methods - November 11, 2018 Category: Molecular Biology Source Type: research

Towards Improving Proximity Labeling by the Biotin Ligase BirA
Publication date: Available online 10 November 2018Source: MethodsAuthor(s): Luke T. Oostdyk, Leonard Shank, Kasey Jividen, Natalia Dworak, Nicholas E. Sherman, Bryce M. PaschalAbstractThe discovery and validation of protein-protein interactions provides a knowledge base that is critical for defining protein networks and how they underpin the biology of the cell. Identification of protein interactions that are highly transient, or sensitive to biochemical disruption, can be very difficult. This challenge has been met by proximity labeling methods which generate reactive species that chemically modify neighboring proteins. ...
Source: Methods - November 11, 2018 Category: Molecular Biology Source Type: research

HiPLA: High-throughput imaging Proximity Ligation Assay
Publication date: Available online 10 November 2018Source: MethodsAuthor(s): Leonid A. Serebryannyy, Tom MisteliAbstractProtein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the antibody-based proximity ligation assay in a high-throughput imaging screening format as a novel means to systemat...
Source: Methods - November 11, 2018 Category: Molecular Biology Source Type: research

Unraveling 3′-end RNA uridylation at nucleotide resolution
We describe the detailed methods used to precisely identify 3′-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3′-end modifications. (Source: Methods)
Source: Methods - November 10, 2018 Category: Molecular Biology Source Type: research

Determining degradation intermediates and the pathway of 3’ to 5’ degradation of histone mRNA using high-throughput sequencing
Publication date: Available online 5 November 2018Source: MethodsAuthor(s): Christopher E. Holmquist, William F. MarzluffAbstractThe half-life of an mRNA is an important parameter contributing to the steady-state level of the mRNA. Rapid changes in mRNA levels can result from decreasing the half-life of an mRNA. Establishing the detailed pathway of mRNA degradation for a particular class of mRNAs requires the ability to isolate mRNA degradation intermediates. High-throughput sequencing provides a method for detecting these intermediates. Here we describe a method for determining the intermediates in 3’ to 5’ de...
Source: Methods - November 6, 2018 Category: Molecular Biology Source Type: research

Chemical Probing for Examining the Structure of Modified RNAs and Ligand Binding to RNA
Publication date: Available online 2 November 2018Source: MethodsAuthor(s): Prabuddha Waduge, Yogo Sakakibara, Christine S. ChowAbstractAmong different RNA modifications, the helix 69 (H69) region of the bacterial ribosomal RNA (rRNA) contains three pseudouridines (Ψs). H69 is functionally important due to its location in the heart of the ribosome. Several structural and functional studies have shown the importance of Ψ modifications in influencing the H69 conformation as well as maintaining key interactions in the ribosome during protein synthesis. Therefore, a need exists to understand the influence of modified n...
Source: Methods - November 3, 2018 Category: Molecular Biology Source Type: research

RPAD (RNase R Treatment, Polyadenylation, and Poly(A)+ RNA Depletion) Method to Isolate Highly Pure Circular RNA
Publication date: Available online 2 November 2018Source: MethodsAuthor(s): Poonam R. Pandey, Pranita K. Rout, Aniruddha Das, Myriam Gorospe, Amaresh C. PandaAbstractRecent developments in high-throughput RNA sequencing methods coupled with innovative bioinformatic tools have uncovered thousands of circular (circ)RNAs. CircRNAs have emerged as a vast and novel class of regulatory RNAs with potential to modulate gene expression by acting as sponges for microRNAs (miRNAs) and RNA-binding proteins (RBPs). The biochemical enrichment of circRNAs by exoribonuclease treatment or by depletion of polyadenylated RNAs coupled with de...
Source: Methods - November 3, 2018 Category: Molecular Biology Source Type: research

Unraveling 3’-end RNA Uridylation at Nucleotide Resolution
We describe the detailed methods used to precisely identify 3’-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3’-end modifications. (Source: Methods)
Source: Methods - November 3, 2018 Category: Molecular Biology Source Type: research

Surpassing limits of static RNA modification analysis with dynamic NAIL-MS
We describe labeling techniques for E. coli, S. cerevisiae and human cell culture and the current instrumental limitations. We present the power of NAIL-MS but we also outline validation experiments, which are necessary for correct data interpretation.As an example, we apply NAIL-MS to study the demethylation of adenine and cytidine, which are methylated by the damaging agent methyl-methanesulfonate in E. coli. With NAIL-MS we exclude the concurrent processes for removal of RNA methylation, namely RNA degradation, turnover and dilution. We use our tool to study the speed and efficiency of 1-methyladenosine and 3-methylcyti...
Source: Methods - November 3, 2018 Category: Molecular Biology Source Type: research

Bispecific T-cell engagers: towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety
Publication date: Available online 3 November 2018Source: MethodsAuthor(s): Diego EllermanAbstractBispecific molecules redirecting the cytotoxicity of T-cells are a growing class of therapeutics with numerous molecules being tested in clinical trials. However, it has been a long way since the proof of concept studies in the mid 1980’s. In the process we have learnt about the impact of different variables related to the bispecific molecule and the target antigen on the potency of this type of drugs. This work reviews the insights gained and how that knowledge has been used to design more potent bispecific T-cell engag...
Source: Methods - November 3, 2018 Category: Molecular Biology Source Type: research

Zebrafish research: Rewriting the rulebook
Publication date: 1 November 2018Source: Methods, Volume 150Author(s): (Source: Methods)
Source: Methods - November 2, 2018 Category: Molecular Biology Source Type: research

Editorial Board
Publication date: 1 November 2018Source: Methods, Volume 150Author(s): (Source: Methods)
Source: Methods - November 2, 2018 Category: Molecular Biology Source Type: research

RNA modifications in structure prediction - status quo and future challenges
Publication date: Available online 30 October 2018Source: MethodsAuthor(s): Andrea Tanzer, Ivo L. Hofacker, Ronny LorenzAbstractChemical modifications of RNA nucleotides change their identity and characteristics and thus alter genetic and structural information encoded in the genomic DNA. tRNA and rRNA are probably the most heavily modified genes, and often depend on derivatization or isomerization of their nucleobases in order to correctly fold into their functional structures. Recent RNomics studies, however, report transcriptome wide RNA modification and suggest a more general regulation of structuredness of RNAs by thi...
Source: Methods - October 30, 2018 Category: Molecular Biology Source Type: research

Determination of enrichment factors for modified RNA in MeRIP experiments
Publication date: Available online 28 October 2018Source: MethodsAuthor(s): Kaouthar Slama, Aurellia Galliot, Franziska Weichmann, Jasmin Hertler, Regina Feederle, Gunter Meister, Mark HelmAbstractIn the growing field of RNA modification, precipitation techniques using antibodies play an important role. However, little is known about their specificities and protocols are missing to assess their effectiveness. Here we present a method to assess enrichment factors after MeRIP-type pulldown experiments, here exemplified with a commercial antibody against N6-methyladenosine (m6A). Testing different pulldown and elution conditi...
Source: Methods - October 29, 2018 Category: Molecular Biology Source Type: research

Quantitation of low abundant soluble biomarkers using high sensitivity Single Molecule Counting technology
Publication date: Available online 28 October 2018Source: MethodsAuthor(s): Joseph Hwang, Munmun Banerjee, Adam S. Venable, Zara Walden, John Jolly, Cathleen Zimmerman, Elizabeth Adkisson, Qiang XiaoAbstractQuantitation of biomarkers in biofluids plays a central role in basic research to management of patient care and is routinely used in clinical laboratories and academic institutions. Standard immunoassays, such as an enzyme-linked immunosorbent assay (ELISA), have provided understanding of both normal and pathological processes for many decades. However, in more recent decades, new immunoassay technologies have uncovere...
Source: Methods - October 29, 2018 Category: Molecular Biology Source Type: research

Nucleoside Analogs in the Study of the Epitranscriptome
Publication date: Available online 26 October 2018Source: MethodsAuthor(s): Cody M. Palumbo, Peter A. BealAbstractOver 150 unique RNA modifications are now known including several nonstandard nucleotides present in the body of messenger RNAs. These modifications can alter a transcript’s function and are collectively referred to as the epitrancriptome. Chemically modified nucleoside analogs are poised to play an important role in the study of these epitranscriptomic marks. Introduced chemical features on nucleic acid strands provide unique structures or reactivity that can be used for downstream detection or quantific...
Source: Methods - October 27, 2018 Category: Molecular Biology Source Type: research

Using Mouse Models to Unlock the Secrets of Non-Synonymous RNA Editing
Publication date: Available online 26 October 2018Source: MethodsAuthor(s): Hussain Jinnah, Randi J. UlbrichtAbstractThe deamination of adenosine to inosine by RNA editing is a widespread post-transcriptional process that expands genetic diversity. Selective substitution of inosine for adenosine in pre-mRNA transcripts can alter splicing, mRNA stability, and the amino acid sequence of the encoded protein. The functional consequences of RNA editing-dependent amino acid substitution are known for only a handful of RNA editing substrates. Many of these studies began in heterologous mammalian expression systems; however, the g...
Source: Methods - October 27, 2018 Category: Molecular Biology Source Type: research

A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats
We present a robust heterodimeric Fc platform, called the XmAb® bispecific platform, engineered for efficient development of bispecific antibodies and Fc fusions of multiple formats. First, we engineer a purification solution for proteins containing a heterodimeric Fc using engineered isoelectric point differences in the Fc region that enable straightforward purification of the heterodimeric species. Then, we combine this purification solution with a novel set of Fc substitutions capable of achieving heterodimer yields over 95% with little change in thermostability. Next, we illustrate the flexibility of our heterodime...
Source: Methods - October 24, 2018 Category: Molecular Biology Source Type: research

Improved Application of RNAModMapper – an RNA Modification Mapping Software Tool – for Analysis of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Data
Publication date: Available online 24 October 2018Source: MethodsAuthor(s): Peter A. Lobue, Ningxi Yu, Manasses Jora, Scott Abernathy, Patrick A. LimbachAbstractResearch into post-transcriptional processing and modification of RNA continues to speed forward, as their ever-emerging role in the regulation of gene expression in biological systems continues to unravel. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has proven for over two decades to be a powerful ally in the elucidation of RNA modification identity and location, but the technique has not proceeded without its own unique technical challenges. The thr...
Source: Methods - October 24, 2018 Category: Molecular Biology Source Type: research

BisAMP: A web-based pipeline for targeted RNA cytosine-5 methylation analysis
We describe a pipeline designed specifically for RNA cytosine-5 methylation analysis in targeted bisulfite sequencing experiments. The pipeline is directly applicable to Illumina MiSeq (or equivalent) sequencing datasets using a web interface (https://bisamp.dkfz.de), and is defined by optimized mapping parameters and the application of tailored filters for the removal of artifacts. We provide examples for the application of this pipeline in the unambiguous detection of m5C marks in tRNAs from mouse embryonic stem cells and neuron-differentiated stem cells as well as in 28S rRNA from human fibroblasts. Finally, we also dis...
Source: Methods - October 24, 2018 Category: Molecular Biology Source Type: research

Misincorporation signatures for detecting modifications in mRNA: Not as simple as it sounds
Publication date: Available online 23 October 2018Source: MethodsAuthor(s): Aldema Sas-Chen, Schraga SchwartzAbstractPost-transcriptional modification on mRNA has become a field of intense interest in recent years, and next-generation sequencing based technologies are constantly emerging to detect an increasing number of modifications at a transcriptome-wide level. Some of these approaches are based on identification of misincorporation events induced by reverse transcriptase at modified sites. Although conceptually trivial, sensitive and specific identification of such events is a challenge prone to a surprising number of...
Source: Methods - October 23, 2018 Category: Molecular Biology Source Type: research

A versatile image analysis platform for three-dimensional nuclear reconstruction
Publication date: Available online 22 October 2018Source: MethodsAuthor(s): Jessica F. Johnston, Simon G.J. Mochrie, Megan C. KingAbstractNuclear morphology is indicative of cellular health in many contexts. In order to robustly and quantitatively measure nuclear size and shape, numerous experimental methods leveraging fluorescence microscopy have been developed. While these methods are useful for quantifying two-dimensional morphology, they often fail to accurately represent the three-dimensional structure of the nucleus, thus omitting important spatial and volumetric information. To address the need for a more accurate i...
Source: Methods - October 23, 2018 Category: Molecular Biology Source Type: research

Bispecific antibodies: potential immunotherapies for HIV treatment
Publication date: Available online 22 October 2018Source: MethodsAuthor(s): Giulia Fabozzi, Amarendra Pegu, Richard A. Koup, Constantinos PetrovasAbstractBispecific (bs) antibodies (Abs, bsAbs) are engineered immunoglobulins that contain two different antigen-binding sites in one molecule. bsAbs can be divided in two molecular formats; the IgG-like and non-IgG like. The structural elements of each format have implications for engaging the immune system. Elimination of HIV will need sophisticated approaches with immunotherapies being one of the strategies under investigation. Furthermore, HIV genetic variability and functio...
Source: Methods - October 23, 2018 Category: Molecular Biology Source Type: research

Bead-based RNA multiplex panels for biomarker detection in oncology samples
Publication date: Available online 22 October 2018Source: MethodsAuthor(s): Jeanesse Scerri, Shawn Baldacchino, Christian Saliba, Christian Scerri, Godfrey GrechAbstractPatient stratification, prognosis and disease monitoring are three important aspects of personalized cancer medicine. With traditional serum tumour protein biomarkers showing lack of specificity and sensitivity, and tumour heterogeneity affecting the response to targeted therapy based on tissue biomarkers, the focus shifted to the use of molecular tumour signatures as specific biomarkers. Multiplex microsphere-based panels are robust and cost-effective, hig...
Source: Methods - October 23, 2018 Category: Molecular Biology Source Type: research

Engineering a bispecific antibody with a common light chain: Identification and optimization of an anti-CD3 epsilon and anti-GPC3 bispecific antibody, ERY974
Publication date: Available online 13 October 2018Source: MethodsAuthor(s): Hirotake Shiraiwa, Atsushi Narita, Mika Kamata-Sakurai, Takahiro Ishiguro, Yuji Sano, Naoka Hironiwa, Takashi Tsushima, Hiroaki Segawa, Toshiaki Tsunenari, Yosuke Ikeda, Yoko Kayukawa, Mizuho Noguchi, Tetsuya Wakabayashi, Akihisa Sakamoto, Hiroko Konishi, Taichi Kuramochi, Mika Endo, Kunihiro Hattori, Junichi Nezu, Tomoyuki IgawaAbstractThe antibody drug market is rapidly expanding, and various antibody engineering technologies are being developed to create antibodies that can provide better benefit to patients. Although bispecific antibody drugs h...
Source: Methods - October 14, 2018 Category: Molecular Biology Source Type: research

Editorial Board
Publication date: 1 October 2018Source: Methods, Volume 149Author(s): (Source: Methods)
Source: Methods - October 13, 2018 Category: Molecular Biology Source Type: research

Functional Microbiomics
Publication date: 1 October 2018Source: Methods, Volume 149Author(s): Jia V. Li (Source: Methods)
Source: Methods - October 13, 2018 Category: Molecular Biology Source Type: research