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Methods: a Companion to Methods in Enzymology

Leonid A Serebryannyy, Tom Misteli
Protein-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 systematically visualize changes to protein interactomes...
November 9, 2018: Methods: a Companion to Methods in Enzymology
Joshua B Kelley, Bryce M Paschal
The 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 present some important technical considerations for studying nuclear and cytoplasmic localization, and provide guidance for quantifying protein levels using fluorescence microscopy and ImageJ software...
November 9, 2018: Methods: a Companion to Methods in Enzymology
Daniel J Luciano, Joel G Belasco
The 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 capping assay that reveals the percentage of 5' ends that are diphosphorylated...
November 9, 2018: Methods: a Companion to Methods in Enzymology
Luke T Oostdyk, Leonard Shank, Kasey Jividen, Natalia Dworak, Nicholas E Sherman, Bryce M Paschal
The 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. The most widely used proximity labeling method is BioID, which features a mutant biotin ligase BirA(Arg118Gly), termed BirA*, fused to a protein of interest...
November 9, 2018: Methods: a Companion to Methods in Enzymology
Christopher E Holmquist, William F Marzluff
The 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' degradation. Characterizing these intermediates requires not only determining the precise 3' end of the molecule to a single nucleotide resolution, but also the ability to detect and characterize any untemplated nucleotides present on the intermediates...
November 5, 2018: Methods: a Companion to Methods in Enzymology
Mehdi Pirouz, Aref G Ebrahimi, Richard I Gregory
Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases...
November 2, 2018: Methods: a Companion to Methods in Enzymology
Valentin F Reichle, Steffen Kaiser, Matthias Heiss, Felix Hagelskamp, Kayla Borland, Stefanie Kellner
Ribonucleic acids (RNA) are extensively modified. These modifications are quantified by mass spectrometry (LC-MS/MS) to determine the abundance of a modification under certain conditions or in various genetic backgrounds. With LC-MS/MS the steady state of modifications is determined, and thus we only have a static view of the dynamics of RNA modifications. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) we overcome this limitation and get access to the dynamics of RNA modifications. We describe labeling techniques for E...
November 2, 2018: Methods: a Companion to Methods in Enzymology
Diego Ellerman
Bispecific 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 engagers...
November 2, 2018: Methods: a Companion to Methods in Enzymology
Poonam R Pandey, Pranita K Rout, Aniruddha Das, Myriam Gorospe, Amaresh C Panda
Recent 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 deep-sequencing is widely used for the systematic identification of circRNAs...
November 1, 2018: Methods: a Companion to Methods in Enzymology
Prabuddha Waduge, Yogo Sakakibara, Christine S Chow
Among 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 nucleosides on conformational dynamics of the ribosome under solution conditions that mimic the cellular environment...
November 1, 2018: Methods: a Companion to Methods in Enzymology
Andrea Tanzer, Ivo L Hofacker, Ronny Lorenz
Chemical 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 this so called epitranscriptome...
October 29, 2018: Methods: a Companion to Methods in Enzymology
Kaouthar Slama, Aurellia Galliot, Franziska Weichmann, Jasmin Hertler, Regina Feederle, Gunter Meister, Mark Helm
In 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 (m6 A). Testing different pulldown and elution conditions, we measure enrichment factors of 4-5 using m6 A-containing mRNAs against an unmodified control of identical sequence...
October 28, 2018: Methods: a Companion to Methods in Enzymology
Joseph Hwang, Munmun Banerjee, Adam S Venable, Zara Walden, John Jolly, Cathleen Zimmerman, Elizabeth Adkisson, Qiang Xiao
Quantitation 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 uncovered numerous analytes in blood that were once undetectable using traditional ELISAs. To meet this new challenge for quantifying low abundant proteins in biofluids, Single Molecule Counting (SMCTM ) technology was developed...
October 27, 2018: Methods: a Companion to Methods in Enzymology
Felix Bormann, Francesca Tuorto, Cansu Cirzi, Frank Lyko, Carine Legrand
RNA cytosine-5 methylation (m5 C) has emerged as a key epitranscriptomic mark, which fulfills multiple roles in structural modulation, stress signaling and the regulation of protein translation. Bisulfite sequencing is currently the most accurate and reliable method to detect m5 C marks at nucleotide resolution. Targeted bisulfite sequencing allows m5 C detection at single base resolution, by combining the use of tailored primers with bisulfite treatment. A number of computational tools currently exist to analyse m5 C marks in DNA bisulfite sequencing...
October 24, 2018: Methods: a Companion to Methods in Enzymology
Peter A Lobue, Ningxi Yu, Manasses Jora, Scott Abernathy, Patrick A Limbach
Research 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 throughput of LC-MS/MS modification mapping experiments continues to be impeded by tedious and time-consuming spectral interpretation, particularly during for the analysis of complex RNA samples...
October 24, 2018: Methods: a Companion to Methods in Enzymology
Gregory L Moore, Matthew J Bernett, Rumana Rashid, Erik W Pong, Duc-Hanh T Nguyen, Jonathan Jacinto, Araz Eivazi, Alex Nisthal, Juan E Diaz, Seung Y Chu, Umesh S Muchhal, John R Desjarlais
Bispecific monoclonal antibodies can bind two protein targets simultaneously and enable therapeutic modalities inaccessible by traditional mAbs. Bispecific formats containing a heterodimeric Fc region are of particular interest, as a heterodimeric Fc empowers both bispecificity and altered valencies while retaining the developability and druggability of a monoclonal antibody. 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...
October 23, 2018: Methods: a Companion to Methods in Enzymology
Aldema Sas-Chen, Schraga Schwartz
Post-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 artifacts, which can lead to substantially inflated estimates of the abundance of diverse modifications...
October 23, 2018: Methods: a Companion to Methods in Enzymology
Jessica F Williams, Simon G J Mochrie, Megan C King
Nuclear 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 image analysis modality, we have developed a software platform that faithfully reconstructs membrane surfaces in three dimensions with sub-pixel resolution...
October 22, 2018: Methods: a Companion to Methods in Enzymology
Jeanesse Scerri, Shawn Baldacchino, Christian Saliba, Christian Scerri, Godfrey Grech
Patient 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 has shifted to the use of molecular tumour signatures as specific biomarkers. Multiplex microsphere-based panels are robust and cost-effective, high throughput molecular assays, which can accurately characterize tumours even from small amounts of poor quality nucleic acids...
October 22, 2018: Methods: a Companion to Methods in Enzymology
Giulia Fabozzi, Amarendra Pegu, Richard A Koup, Constantinos Petrovas
Bispecific (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 functional compromise of the adaptive CTL response complicate the potential usefulness of some immunotherapeutic strategies...
October 22, 2018: Methods: a Companion to Methods in Enzymology
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