Identifying and characterizing functional 3’ nucleotide addition in the miRNA pathway
Publication date: Available online 20 August 2018Source: MethodsAuthor(s): A. Maxwell Burroughs, Yoshinari AndoAbstractOver the past decade, modifications to microRNAs (miRNAs) via 3’ end nucleotide addition have gone from a deep-sequencing curiosity to experimentally confirmed drivers of a range of regulatory activities. Here we overview the methods that have been deployed by researchers seeking to untangle these diverse functional roles and include characterizing not only the nucleotidyl transferases catalyzing the additions but also the nucleotides being added, and the timing of their addition during the miRNA pat...
Source: Methods - August 20, 2018 Category: Molecular Biology Source Type: research

Nuclear export of mRNA molecules studied by SPEED microscopy
Publication date: Available online 18 August 2018Source: MethodsAuthor(s): Yichen Li, Samuel L. Junod, Andrew Ruba, Joseph M. Kelich, Weidong YangAbstractThe nuclear exit of messenger RNA (mRNA) molecules through the nuclear pore complex (NPC) is an essential step in the translation process of all proteins. The current limitations of conventional fluorescence and electron microscopy have prevented elucidation of how mRNA exports through the NPCs of live cells. In the recent years, various single-molecule fluorescence (SMF) microscopy techniques have been developed to improve the temporal and spatial resolutions of live-cel...
Source: Methods - August 18, 2018 Category: Molecular Biology Source Type: research

Editorial Board
Publication date: 15 August 2018Source: Methods, Volume 146Author(s): (Source: Methods)
Source: Methods - August 15, 2018 Category: Molecular Biology Source Type: research

Affinity-Based Separation Methods for the Study of Biological Interactions
Publication date: 15 August 2018Source: Methods, Volume 146Author(s): David S. Hage (Source: Methods)
Source: Methods - August 15, 2018 Category: Molecular Biology Source Type: research

Determination of the conformational states of strychnine in solution using NMR residual dipolar couplings in a tensor-free approach
Publication date: Available online 21 July 2018Source: MethodsAuthor(s): Giulia Tomba, Carlo Camilloni, Michele VendruscoloAbstractSmall molecules with rotatable bonds can occupy different conformational states in solution as a consequence of their thermal fluctuations. The accurate determination of the structures of such states, as well as of their statistical weights, has been challenging because of the technical difficulties in extracting information from experimental measurements, which are normally averaged over the conformational space available. Here, to achieve this objective, we present an approach based on a rece...
Source: Methods - August 11, 2018 Category: Molecular Biology Source Type: research

Corrigendum to “Contemporary hydrogen deuterium exchange mass spectrometry” [144 (2018) 27–42]
Publication date: Available online 10 August 2018Source: MethodsAuthor(s): Irina Oganesyan, Cristina Lento, Derek J. Wilson (Source: Methods)
Source: Methods - August 11, 2018 Category: Molecular Biology Source Type: research

Molecular Engineering Strategies and Methods for the Expression and Purification of IgG1-based Bispecific Bivalent Antibodies
Publication date: Available online 11 August 2018Source: MethodsAuthor(s): N. Dimasi, R. Fleming, H. Wu, C. GaoAbstractIn recent years, bispecific antibodies (BisAbs) have emerged as novel pharmaceutical candidates owing to their ability to engage two disease mediators simultaneously, thus providing a possible alternative therapeutic approach in complex diseases such as cancer and inflammation. Here we provide an overview of the molecular design, recombinant expression in mammalian cells and purification of BisAbs based on full-length IgG-scFv formats. Practical considerations and strategies to optimize transient expressio...
Source: Methods - August 11, 2018 Category: Molecular Biology Source Type: research

Using Bioreactors to Study the Effects of Drugs on the Human Microbiota
Publication date: Available online 11 August 2018Source: MethodsAuthor(s): Mabel Guzman-Rodriguez, Julie A.K. McDonald, Richard Hyde, Emma Allen-Vercoe, Erika C. Claud, Prameet M. Sheth, Elaine O. PetrofAbstractThe study of complex microbial communities has become a major research focus as mounting evidence suggests the pivotal role microbial communities play in host health and disease. Microbial communities of the gastrointestinal tract, known as the gut microbiota, have been implicated in aiding the host with vitamin biosynthesis, regulation of host energy metabolism, immune system development, and resistance to pathogen...
Source: Methods - August 11, 2018 Category: Molecular Biology Source Type: research

A guide to nucleic acid detection by single-molecule kinetic fingerprinting
Publication date: Available online 10 August 2018Source: MethodsAuthor(s): Alexander Johnson-Buck, Jieming Li, Muneesh Tewari, Nils G. WalterAbstractConventional methods for detecting small quantities of nucleic acids require amplification by the polymerase chain reaction (PCR), which necessitates prior purification and introduces copying errors. While amplification-free methods do not have these shortcomings, they are generally orders of magnitude less sensitive and specific than PCR-based methods. In this review, we provide a practical guide to a novel amplification-free method, single-molecule recognition through equili...
Source: Methods - August 10, 2018 Category: Molecular Biology Source Type: research

Speech analysis for health: current state-of-the-art and the increasing impact of deep learning
Publication date: Available online 10 August 2018Source: MethodsAuthor(s): Nicholas Cummins, Alice Baird, Björn SchullerAbstractDue to the complex and intricate nature associated with their production, the acoustic-prosodic properties of a speech signal are modulated with a range of health related effects. There is an active and growing area of machine learning research in this speech and health domain, focusing on developing paradigms to objectively extract and measure such effects. Concurrently, deep learning is transforming intelligent signal analysis, such that machines are now reaching near human capabilities in ...
Source: Methods - August 10, 2018 Category: Molecular Biology Source Type: research

Generation of Fabs-in-Tandem Immunoglobulin Molecules for Dual-Specific Targeting
Publication date: Available online 3 August 2018Source: MethodsAuthor(s): Shiyong Gong, Chengbin WuAbstractBispecific antibody (BsAb) has become an important trend in developing next generation biologics therapies. By simultaneously engaging two molecular targets, BsAbs show distinctive mechanism of actions that could lead to clinical benefits unattainable by conventional monoclonal antibodies (mAbs). Successful launch provided clinical validation and encourage more BsAb development in the pipeline of pharmaceutical companies. Fabs-in-tandem immunoglobulin (FIT-IgTM) format was initially described in 2017. This unique desi...
Source: Methods - August 4, 2018 Category: Molecular Biology Source Type: research

Preparation of bispecific antibody-protein adducts by site-specific chemo-enzymatic conjugation
We present an alternative method for the introduction of additional functional domains to an antibody: site-specific chemo-enzymatic conjugation. This method relies on the combination of site-specific transpeptidases and bioorthogonal chemistry. Transpeptidases are used to site-specifically introduce chemical handles, which can then be used to couple new functional groups by means of a bioorthogonal chemical reaction. We demonstrate site-specific chemo-enzymatic linkage using the transpeptidase sortase (hereafter: sortase) and either a strain-promoted alkyne-azide cycloaddition (SPAAC) or an inverse-electron demand Diels-A...
Source: Methods - August 4, 2018 Category: Molecular Biology Source Type: research

Fishing for understanding: Unlocking the zebrafish gene editor’s toolbox
Publication date: Available online 2 August 2018Source: MethodsAuthor(s): Brandon W. Simone, Gabriel Martínez-Gálvez, Zachary WareJoncas, Stephen C. EkkerAbstractThe rapid growth of the field of gene editing can largely be attributed to the discovery and optimization of designer endonucleases. These include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regular interspersed short palindromic repeat (CRISPR) systems including Cas9, Cas12a, and structure-guided nucleases. Zebrafish (Danio rerio) have proven to be a powerful model system for genome engineeri...
Source: Methods - August 2, 2018 Category: Molecular Biology Source Type: research

Programmable base editing in zebrafish using a modified CRISPR-Cas9 system
Publication date: Available online 2 August 2018Source: MethodsAuthor(s): Wei Qin, Xiaochan Lu, Shuo LinAbstractThe use of CRISPR/Cas9 to knockout genes in zebrafish has been well established. However, to better model many human diseases that are caused by point mutations, a robust methodology for generating desirable DNA base changes is still needed. Recently, Cas9-linked cytidine deaminases (base editors) evolved as a strategy to introduce single base mutations in model organisms. They have been used to convert cytidine to thymine at specific genomic loci. Here we describe a protocol for using the base editing system in ...
Source: Methods - August 2, 2018 Category: Molecular Biology Source Type: research

Biotagging, an in vivo biotinylation approach for cell-type specific subcellular profiling in zebrafish
Publication date: Available online 2 August 2018Source: MethodsAuthor(s): Le A. Trinh, Vanessa Chong-Morrison, Tatjana Sauka-SpenglerAbstractInterrogation of gene regulatory circuits in complex organisms requires precise and robust methods to label cell-types for profiling of target proteins in a tissue-specific fashion as well as data analysis to understand interconnections within the circuits. There are several strategies for obtaining cell-type and subcellular specific genome-wide data. We have developed a methodology, termed “biotagging” that uses tissue-specific, genetically encoded components to biotinyla...
Source: Methods - August 2, 2018 Category: Molecular Biology Source Type: research

Imaging Cell-Type-Specific Dynamics of mRNAs in Living Mouse Brain
We describe a method for visualizing mRNAs in living mouse. Nascent transcripts and cytoplasmic mRNAs were labeled via lentiviral expression of MS2 coat protein (MCP) tagged with fluorescent protein (MCP-XFP) in knock-in mice whose β-actin mRNAs contained MCP binding stem loops (MBS). Then the mRNA molecules were imaged in the live cerebral cortex through an optical cranial window by intravital two-photon microscopy. By means of the controlled expression of MCP-XFP, single mRNA particles could be detected differentially in the nucleus and cytoplasm of a specific cell type. Consequently, this method is useful for inves...
Source: Methods - July 29, 2018 Category: Molecular Biology Source Type: research

Time-lapse imaging beyond the diffraction limit
Publication date: Available online 27 July 2018Source: MethodsAuthor(s): Ajay Chitnis, Damian Dalle NogareAbstractThe zebrafish, with its rapid external development, optical transparency, and the relative ease with which transgenic lines can be created, is rapidly becoming the model of choice for examining developmental processes via time-lapse microscopy. The recent proliferation of techniques for super-resolution imaging now allows for an unprecedented view of embryonic development at high spatial and temporal resolution in live tissues. This review examines both the theoretical basis and practical application of a numbe...
Source: Methods - July 27, 2018 Category: Molecular Biology Source Type: research

Systematic optimization of cell-free synthesized human endothelin B receptor folding
Publication date: Available online 2 February 2018Source: MethodsAuthor(s): Ralf-Bernhardt Rues, Fang Dong, Volker Dötsch, Frank BernhardAbstractCell-free production of G-protein coupled receptors is becoming attractive for biochemical characterization, ligand screening or even structural purposes. However, despite high production levels within the range of mg/mL, the fraction of functionally folded receptor is frequently below 1%. In synthetic cell-free reactions, numerous factors that affect the efficient folding and stability of translated membrane proteins can be addressed by the appropriate design of the syntheti...
Source: Methods - July 25, 2018 Category: Molecular Biology Source Type: research

Editorial Board
Publication date: 1 August 2018Source: Methods, Volume 145Author(s): (Source: Methods)
Source: Methods - July 25, 2018 Category: Molecular Biology Source Type: research

Data mining methods for analyzing biological data in terms of phenotypes
Publication date: 1 August 2018Source: Methods, Volume 145Author(s): Sun Kim (Source: Methods)
Source: Methods - July 25, 2018 Category: Molecular Biology Source Type: research

Toward Comprehensive Measurement of Protein Hydration Dynamics: Facilitation of NMR-based Methods by Reverse Micelle Encapsulation
Publication date: Available online 24 July 2018Source: MethodsAuthor(s): Pamela N. Gallo, Joseph C. Iovine, Nathaniel V. NucciAbstractProtein-water interactions are a fundamental determinant of protein structure and function. Despite their importance, the molecular details of water orientations and dynamics near protein surfaces remain poorly understood, largely due to the difficulty of measuring local water mobility near the protein in a site-resolved fashion. Solution NMR-based measurement of water mobility via the nuclear Overhauser effect was presented as a method for performing comprehensive, site-resolved measurement...
Source: Methods - July 24, 2018 Category: Molecular Biology Source Type: research

Probing membrane protein ground and conformationally excited states using dipolar- and J-coupling mediated MAS solid state NMR experiments
Publication date: Available online 23 July 2018Source: MethodsAuthor(s): T. Gopinath, Gianluigi VegliaAbstractThe intrinsic conformational plasticity of membrane proteins directly influences the magnitude of the orientational-dependent NMR interactions such as dipolar couplings (DC) and chemical shift anisotropy (CSA). As a result, the conventional cross-polarization (CP)-based techniques mainly capture the more rigid regions of membrane proteins, while the most dynamic regions are essentially invisible. Nonetheless, dynamic regions can be detected using experiments in which polarization transfer takes place via J-coupling...
Source: Methods - July 23, 2018 Category: Molecular Biology Source Type: research

Determination of the conformational states of strychnine using NMR residual dipolar couplings in a tensor-free approach
Publication date: Available online 21 July 2018Source: MethodsAuthor(s): Giulia Tomba, Carlo Camilloni, Michele VendruscoloAbstractSmall molecules with rotatable bonds can occupy different conformational states as a consequence of their thermal fluctuations. The accurate determination of the structures of such states, as well as of their statistical weights, has been challenging because of the technical difficulties in extracting information from experimental measurements, which are normally averaged over the conformational space available. Here, to achieve this objective, we present an approach based on a recently propose...
Source: Methods - July 21, 2018 Category: Molecular Biology Source Type: research

A reconstitution method for integral membrane proteins in hybrid lipid-polymer vesicles for enhanced functional durability
Publication date: Available online 2 February 2018Source: MethodsAuthor(s): Rashmi Seneviratne, Sanobar Khan, Ellen Moscrop, Michael Rappolt, Stephen P. Muench, Lars J.C. Jeuken, Paul A. BealesAbstractHybrid vesicles composed of lipids and block copolymers hold promise for increasing liposome stability and providing a stable environment for membrane proteins. Recently we reported the successful functional reconstitution of the integral membrane protein cytochrome bo3 (ubiquinol oxidase) into hybrid vesicles composed of a blend of phospholipids and a block copolymer (PBd-PEO). We demonstrated that these novel membrane envir...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Analysis of solute-protein interactions and solute-solute competition by zonal elution affinity chromatography
Publication date: Available online 2 February 2018Source: MethodsAuthor(s): Pingyang Tao, Saumen Poddar, Zuchen Sun, David S. Hage, Jianzhong ChenAbstractMany biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein bindi...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Corrigendum to “Molecular signatures in breast cancer” [Methods 131 (2017) 135–146]
Publication date: Available online 3 February 2018Source: MethodsAuthor(s): Samir Lal, Amy E. McCart Reed, Xavier M. de Luca, Peter T. Simpson (Source: Methods)
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Bi-clustering of metabolic data using matrix factorization tools
Publication date: Available online 10 February 2018Source: MethodsAuthor(s): Quan Gu, Kirill VeselkovAbstractMetabolic phenotyping technologies based on Nuclear Magnetic Spectroscopy (NMR) and Mass Spectrometry (MS) generate vast amounts of unrefined data from biological samples. Clustering strategies are frequently employed to provide insight into patterns of relationships between samples and metabolites. Here, we propose the use of a non-negative matrix factorization driven bi-clustering strategy for metabolic phenotyping data in order to discover subsets of interrelated metabolites that exhibit similar behaviour across ...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Temperature controlled ionic liquid aqueous two phase system combined with affinity capillary electrophoresis for rapid and precise pharmaceutical-protein binding measurements
Publication date: Available online 10 February 2018Source: MethodsAuthor(s): Deia Abd El-Hady, Hassan M. AlbishriAbstractTemperature controlled ionic liquid aqueous two phase system (ILATPS) was used to improve the precision of pharmaceutical-AGP (human alpha (α1)-acid glycoprotein) binding measurements by affinity capillary electrophoresis (ACE). The effect of different types of short-chain alkyl imidazolium ILs within the concentration range of 10.0–1000.0 μmol L−1 on a propranolol (PRO)-AGP model was firstly investigated by ILATPS/ACE system. Use of 100.0 μmol L−1 1-butyl-3-methylim...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Thread- paper, and fabric enzyme-linked immunosorbent assays (ELISA)
Publication date: Available online 10 February 2018Source: MethodsAuthor(s): Ariana Gonzalez, Michelle Gaines, Laura Y. Gallegos, Ricardo Guevara, Frank A. GomezAbstractEnzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins, and glycoproteins in biological samples. While the procedure is routine and straightforward, there are a number of variables (reagent selection, volume measurement, temperature, and time) that if not carefully considered, can affect the test outcome. Herein, we describe the development of microfluidic thread/paper-based analytical dev...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Thermophoresis for characterizing biomolecular interaction
Publication date: Available online 10 February 2018Source: MethodsAuthor(s): Mufarreh Asmari, Ratih Ratih, Hassan A. Alhazmi, Sami El DeebAbstractThe study of biomolecular interactions is crucial to get more insight into the biological system. The interactions of protein-protein, protein-nucleic acids, protein-sugars, nucleic acid-nucleic acids and protein-small molecules are supporting therapeutics and technological developments. Recently, the development in a large number of analytical techniques for characterizing biomolecular interactions reflect the promising research investments in this field.In this review, microsca...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Methods of reconstitution to investigate membrane protein function
Publication date: Available online 16 February 2018Source: MethodsAuthor(s): Ruth Skrzypek, Shagufta Iqbal, Richard CallaghanAbstractMembrane proteins are notoriously difficult to investigate in isolation. The focus of this chapter is the key step following extraction and purification of membrane proteins; namely reconstitution. The process of reconstitution re-inserts proteins into a lipid bilayer that partly resembles their native environment. This native environment is vital to the stability of membrane proteins, ensuring that they undergo vital conformational transitions and maintain optimal interaction with their subs...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Affinity-based separation methods for the study of biological interactions: The case of peroxisome proliferator-activated receptors in drug discovery
Publication date: Available online 17 February 2018Source: MethodsAuthor(s): Caterina Temporini, Gloria Brusotti, Giorgio Pochetti, Gabriella Massolini, Enrica CalleriAbstractAffinity-based methods using immobilized proteins are important approaches for understanding the interactions between small molecules and biological targets. This review is intended to provide an overview of different affinity based separation methods that have been applied to the study of peroxisome proliferator activated receptors (PPARs). The screening of compound to increase screening rates for synthetic and natural ligands of PPAR are reported. P...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Fragment screening for drug leads by weak affinity chromatography (WAC-MS)
Publication date: Available online 23 February 2018Source: MethodsAuthor(s): Sten Ohlson, Minh-Dao Duong-ThiAbstractFragment-based drug discovery is an important tool for design of small molecule hit-to-lead compounds against various biological targets. Several approved drugs have been derived from an initial fragment screen and many such candidates are in various stages of clinical trials. Finding fragment hits, that are suitable for optimisation by medicinal chemists, is still a challenge as the binding between the small fragment and its target is weak in the range of mM to µM of Kd and irrelevant non-specific inte...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

The yin and yang of solubilization and stabilization for wild-type and full-length membrane protein
Publication date: Available online 23 February 2018Source: MethodsAuthor(s): David Hardy, Elodie Desuzinges Mandon, Alice Rothnie, Anass JawhariAbstractMembrane proteins (MP) are stable in their native lipid environment. To enable structural and functional investigations, MP need to be extracted from the membrane. This is a critical step that represents the main obstacle for MP biochemistry and structural biology. General guidelines and rules for membrane protein solubilization remain difficult to establish. This review aims to provide the reader with a comprehensive overview of the general concepts of MP solubilization an...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Advances in enzyme substrate analysis with capillary electrophoresis
Publication date: Available online 27 February 2018Source: MethodsAuthor(s): Srikanth Gattu, Cassandra L. Crihfield, Grace Lu, Lloyd Bwanali, Lindsay M. Veltri, Lisa A. HollandAbstractCapillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with vari...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction
This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha1-acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyz...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Functional characterisation of G protein-coupled receptors
Publication date: Available online 3 March 2018Source: MethodsAuthor(s): Romez Uddin, John Simms, David PoynerAbstractCharacterisation of receptors can involve either assessment of their ability to bind ligands or measure receptor activation as a result of agonist or inverse agonist interactions. This review focuses on G protein-coupled receptors (GPCRs), examining techniques that can be applied to both receptors in membranes and after solubilisation. Radioligand binding remains a widely used technique, although there is increasing use of fluorescent ligands. These can be used in a variety of experimental designs, either d...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Studying structure and function of membrane proteins with PELDOR/DEER spectroscopy – A crystallographers’ perspective
Publication date: Available online 3 March 2018Source: MethodsAuthor(s): Janin Glaenzer, Martin F. Peter, Gregor HageluekenAbstractIn 1985, the first X-ray structure of a membrane protein was determined. Today, more than 30 years later, many more structures have been solved. Nevertheless, studying the structure of membrane proteins remains a very challenging task. Due to their inherent conformational flexibility, having a single X-ray structure is usually only the first step towards truly understanding the function of these dynamic molecules. For this reason, additional methods are needed that can provide complementary i...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Mass spectrometry-enabled structural biology of membrane proteins
Publication date: Available online 3 March 2018Source: MethodsAuthor(s): Antonio N. Calabrese, Sheena E. RadfordAbstractThe last ∼25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based s...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Planar lipid bilayers in recombinant ion channel research
Publication date: Available online 9 March 2018Source: MethodsAuthor(s): Jacqueline Maher, Marcus AllenAbstractThere are a number of methods of investigating the function of recombinant proteins such as ion channels. However, after channel purification there are few methods to guarantee that the protein still functions. For ion channels, reconstituting back into planar lipid bilayers and demonstrating preserved function is a convenient and trusted method. It is cell free and even inaccessible, intracellular ion channels can be studied. We have used this method to study the function of recombinant channels of known subunit ...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Studies of antibody-antigen interactions by capillary electrophoresis: A review
The objective of this review is to identify and describe various CE techniques which have been used to study antibody-antigen interactions. A brief discussion of linear and nonlinear curve fitting is also included. (Source: Methods)
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Purification of membrane proteins free from conventional detergents: SMA, new polymers, new opportunities and new insights
Publication date: Available online 31 March 2018Source: MethodsAuthor(s): Zoe Stroud, Stephen C.L. Hall, Tim R. DaffornAbstractMembrane proteins remain a somewhat enigmatic group of biomolecules. On the one hand they mediate some of the most important processes in biology with molecular mechanisms that are often elegantly complex. On the other hand they are exceptionally challenging to produce, making studies of membrane protein structure and function among the most difficult projects undertaken by biochemists. The central issue with studies of a membrane protein has been the need to extract them from their native lipid en...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Peak decay analysis and biointeraction studies of immunoglobulin binding and dissociation on protein G affinity microcolumns
This report used the method of peak decay analysis along with frontal analysis and zonal elution studies to characterize the binding, elution and regeneration properties of affinity microcolumns that contained immobilized protein G. Frontal analysis was employed with rabbit immunoglobulin G (IgG) to characterize the binding capacity of these affinity microcolumns. Zonal elution experiments looking at the retained peaks for small injections of labeled rabbit IgG were used to optimize the column regeneration conditions. Peak decay analysis was then used to look at the effects of flow rate and elution pH on the release of sev...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Designing fluorescent biosensors using circular permutations of riboswitches
Publication date: 1 July 2018Source: Methods, Volume 143Author(s): Johnny Truong, Yu-Fang Hsieh, Lynda Truong, Guifang Jia, Ming C. HammondAbstractRNA-based fluorescent (RBF) biosensors have been applied to detect a variety of metabolites in vitro and in live cells. They are designed by combining the ligand sensing domain of natural riboswitches with in vitro selected fluorogenic aptamers. Different biosensor topologies have been developed to accommodate the diversity of riboswitch structures. Here we show that circular permutation of the riboswitch ligand sensing domain also gives functional biosensors, using the SAM-I ri...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Editorial Board
Publication date: 1 July 2018Source: Methods, Volume 143Author(s): (Source: Methods)
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Methods and advances in RNA characterization and design
Publication date: 1 July 2018Source: Methods, Volume 143Author(s): Lydia M. Contreras (Source: Methods)
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Methods for construction and characterization of simple or special multifunctional RNA nanoparticles based on the 3WJ of phi29 DNA packaging motor
This article is intended to serve as a general reference aimed at both apprentices and senior scientists for their future design, construction and characterization of RNA nanoparticles based on the pRNA-3WJ of phi29 DNA packaging motor. (Source: Methods)
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

StructureFold2: Bringing chemical probing data into the computational fold of RNA structural analysis
Publication date: 1 July 2018Source: Methods, Volume 143Author(s): David C. Tack, Yin Tang, Laura E. Ritchey, Sarah M. Assmann, Philip C. BevilacquaAbstractThe secondary structure of an RNA is often implicit to its function. Recently, various high-throughput RNA structure probing techniques have been developed to elucidate important RNA structure–function relationships genome-wide. These techniques produce unwieldy experimental data sets that require evaluation with unique computational pipelines. Herein, we present StructureFold2, a user-friendly set of analysis tools that makes precise data processing and detailed ...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

Adventures with RNA graphs
Publication date: 1 July 2018Source: Methods, Volume 143Author(s): Tamar SchlickAbstractThe structure of RNA has been a natural subject for mathematical modeling, inviting many innovative computational frameworks. This single-stranded polynucleotide chain can fold upon itself in numerous ways to form hydrogen-bonded segments, imperfect with single-stranded loops. Illustrating these paired and non-paired interaction networks, known as RNA’s secondary (2D) structure, using mathematical graph objects has been illuminating for RNA structure analysis. Building upon such seminal work from the 1970s and 1980s, graph models ...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research

A high-throughput and rapid computational method for screening of RNA post-transcriptional modifications that can be recognized by target proteins
Publication date: 1 July 2018Source: Methods, Volume 143Author(s): Asuka A. Orr, Juan C. Gonzalez-Rivera, Mark Wilson, P. Reena Bhikha, Daiqi Wang, Lydia M. Contreras, Phanourios TamamisAbstractThere are over 150 currently known, highly diverse chemically modified RNAs, which are dynamic, reversible, and can modulate RNA-protein interactions. Yet, little is known about the wealth of such interactions. This can be attributed to the lack of tools that allow the rapid study of all the potential RNA modifications that might mediate RNA-protein interactions. As a promising step toward this direction, here we present a computati...
Source: Methods - July 15, 2018 Category: Molecular Biology Source Type: research