A rapid cloning method employing orthogonal end protection

We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors.     

Single-molecule Förster resonance energy transfer studies of RNA structure, dynamics and function

Single-molecule fluorescence microscopy experiments on RNA molecules brought to light the highly complex dynamics of key biological processes, including RNA folding, catalysis of ribozymes, ligand sensing of riboswitches and aptamers, and protein synthesis in the ribosome. By using highly advanced biophysical spectroscopy techniques in combination with sophisticated biochemical synthesis approaches, molecular dynamics of individual RNA molecules can be observed in real time and under physiological conditions in unprecedented detail that cannot be achieved with bulk experiments. Here, we review recent advances in RNA folding and functional studies of RNA and RNA-protein complexes addressed by using single-molecule Förster (fluorescence) resonance energy transfer (smFRET) technique.     

Biophysical nanotools for single-molecule dynamics

The focus of the cell biology field is now shifting from characterizing cellular activities to organelle and molecular behaviors. This process accompanies the development of new biophysical visualization techniques that offer high spatial and temporal resolutions with ultra-sensitivity and low cell toxicity. They allow the biology research community to observe dynamic behaviors from scales of single molecules, organelles, cells to organoids, and even live animal tissues. In this review, we summarize these biophysical techniques into two major classes: the mechanical nanotools like dynamic force spectroscopy (DFS) and the optical nanotools like single-molecule and super-resolution microscopy. We also discuss their applications in elucidating molecular dynamics and functionally mapping of interactions between inter-cellular networks and intra-cellular components, which is key to understanding cellular processes such as adhesion, trafficking, inheritance, and division.     

Massively Parallel Single-Molecule Manipulation Using Centrifugal Force

Precise manipulation of single molecules has already led to remarkable insights in physics, chemistry, biology, and medicine. However, widespread adoption of single-molecule techniques has been impeded by equipment cost and the laborious nature of making measurements one molecule at a time. We have solved these issues by developing an approach that enables massively parallel single-molecule force measurements using centrifugal force. This approach is realized in an instrument that we call the centrifuge force microscope in which objects in an orbiting sample are subjected to a calibration-free, macroscopically uniform force-field while their micro-to-nanoscopic motions are observed. We demonstrate high-throughput single-molecule force spectroscopy with this technique by performing thousands of rupture experiments in parallel, characterizing force-dependent unbinding kinetics of an antibody-antigen pair in minutes rather than days. Additionally, we verify the force accuracy of the instrument by measuring the well-established DNA overstretching transition at 66 ± 3 pN. With significant benefits in efficiency, cost, simplicity, and versatility, single-molecule centrifugation has the potential to expand single-molecule experimentation to a wider range of researchers and experimental systems.     

Characterisation of bacterial polysaccharides: steps towards single-molecular studies

Techniques used in studies of polysaccharides, including chemical composition, linkage pattern, and higher order structures are in constant development. They provide information necessary for understanding of the polysaccharide properties and functions. Here, recent advancements in studies of the polysaccharides at the single-molecule level are highlighted. Over the last few years, single-molecule techniques such as force spectroscopy have improved in sensitivity and can today be used to detect forces in the pN range. In addition, these techniques can be used to investigate properties of single molecules close to physiological conditions. The challenges in the interpretation of the observations are aided by control experiments using well-characterised polysaccharides and by data provided by complementary methods. This field is expected to have increasing impact on the further advancement of the molecular understanding of the role of polysaccharides in various biological processes such as recognition and cell adhesion.     

Forcing a connection: impacts of single-molecule force spectroscopy on in vivo tension sensing

Mechanical tension plays a large role in cell development ranging from morphology to gene expression. On the molecular level, the effects of tension can be seen in the dynamic arrangement of membrane proteins as well as the recruitment and activation of intracellular proteins. Forces applied to biopolymers during in vitro force measurements offer greater understanding of the effects of tension on molecules in live cells, and experimental techniques involving test tubes and live cells can often overlap. Indeed, when forces exerted on cellular components can be calibrated ex vivo with force spectroscopy, a powerful tool is available for researchers in probing cellular mechanotransduction on the molecular scale. This review will discuss the techniques used in measuring both cellular traction forces and single-molecule force spectroscopy. Emphasis will be placed on the use of fluorescence reporter systems for the development of in vivo tension sensors that can be used for calibration with single molecule force methods.     

Do-it-yourself guide: how to use the modern single-molecule toolkit

Single-molecule microscopy has evolved into the ultimate-sensitivity toolkit to study systems from small molecules to living cells, with the prospect of revolutionizing the modern biosciences. Here we survey the current state of the art in single-molecule tools including fluorescence spectroscopy, tethered particle microscopy, optical and magnetic tweezers, and atomic force microscopy. We also provide guidelines for choosing the right approach from the available single-molecule toolkit for applications as diverse as structural biology, enzymology, nanotechnology and systems biology.     

Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly

MicroRNAs (miRNAs) associate with components of the RNA-induced silencing complex (RISC) to assemble on mRNA targets and regulate protein expression in higher eukaryotes. Here we describe a method for the intracellular single-molecule, high-resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore-labelled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules. Mobility and mRNA-dependent assembly changes suggest the existence of two kinetically distinct pathways for miRNA assembly, revealing the dynamic nature of this important gene regulatory pathway. iSHiRLOC achieves an unprecedented resolution in the visualization of functional miRNAs, paving the way to understanding RNA silencing through single-molecule systems biology.     

Direct Observation of Single DNA Structural Alterations at Low Forces with Surface-Enhanced Raman Scattering

DNA experiences numerous mechanical events, necessitating single-molecule force spectroscopy techniques to provide insight into DNA mechanics as a whole system. Inherent Brownian motion limits current force spectroscopy methods from observing possible bond level structural changes. We combine optical trapping and surface-enhanced Raman scattering to establish a direct relationship between DNA’s extension and structure in the low force, entropic regime. A DNA molecule is trapped close to a surface-enhanced Raman scattering substrate to facilitate a detectable Raman signal. DNA Raman modes shift in response to applied force, indicating phosphodiester mechanical alterations. Molecular dynamic simulations confirm the local structural alterations and the Raman sensitive band identified experimentally. The combined Raman and force spectroscopy technique, to our knowledge, is a novel methodology that can be generalized to all single-molecule studies.     

Atomic force microscopy-based force spectroscopy--biological and biomedical applications

The use of atomic force microscopy (AFM) applied to biological systems to generate high resolution images is gaining a wider acceptance. However, the most remarkable advances are being achieved on the use of the AFM to measure inter- and intramolecular interaction forces with piconewton resolution, not only to demonstrate this ability but also actually to solve biological and biomedical relevant questions. Single-molecule force spectroscopy recognition studies enable the detection of specific interaction forces, based on the AFM sensitivity and the possibility of manipulating individual molecules. In this review, we describe the basic principles of this methodology and some of the practical aspects involved. The ability to measure interactions at the single-molecule level is illustrated by some relevant examples. A special focus is given to the study of the fibrinogen-erythrocyte binding and its relevance as a cardiovascular risk factor. An approach to the latter problem by single-molecule force spectroscopy allowed the molecular recognition, characterization, and partial identification of a previously unknown receptor for fibrinogen on human erythrocytes.     

Facile tethering of stable and unstable proteins for optical tweezers experiments

Single-molecule force spectroscopy with optical tweezers has emerged as a powerful tool for dissecting protein folding. The requirement to stably attach “molecular handles” to specific points in the protein of interest by preparative biochemical techniques is a limiting factor in applying this methodology, especially for large or unstable proteins that are difficult to produce and isolate. Here, we present a streamlined approach for creating stable and specific attachments using autocatalytic covalent tethering. The high specificity of coupling allowed us to tether ribosome-nascent chain complexes, demonstrating its suitability for investigating complex macromolecular assemblies. We combined this approach with cell-free protein synthesis, providing a facile means of preparing samples for single-molecule force spectroscopy. The workflow eliminates the need for biochemical protein purification during sample preparation for single-molecule measurements, making structurally unstable proteins amenable to investigation by this powerful single-molecule technique. We demonstrate the capabilities of this approach by carrying out pulling experiments with an unstructured domain of elongation factor G that had previously been refractory to analysis. Our approach expands the pool of proteins amenable to folding studies, which should help to reduce existing biases in the currently available set of protein folding models.     

单分子荧光检测技术的发展及其在植物生物学研究中的应用

单分子荧光检测技术是利用荧光基团对目的分子标记后,在单分子水平成像并追踪分子的构象变化、动力学特征以及分子之间相互作用的研究方法.相较于传统分子生物学和遗传学的研究手段,单分子检测技术可以对单个分子的动态和特性进行分析,特别是瞬时或偶发性的事件,从而更加深入地挖掘在群体测量中被掩盖的信息.该技术已广泛应用于动物细胞生物...     

Easy In Vitro Synthesis of Optimised Functioning Reporter mRNA from Common eGFP Plasmid

The extensive growth in number and importance of experiments and clinical-aimed techniques based solely or majorly on the activity of RNA strands, e.g. CRSPR/Cas9 and siRNA, has put emphasis on the necessity of standardisation of experiments with RNA. Considering RNA degradation during its handling seems to be a major hindrance in all RNA-based tools, the assessment of its integrity is of utmost importance. Furthermore, evaluating whether the RNA to be transfected is intact requires time-consuming electrophoresis protocol. In view of the RNA lability and the necessity for controlling experiments performed with this molecule, the transfection of a reporter mRNA may be of aid in optimising experiments. Nevertheless, commercial reporter mRNAs are far less available than plasmids for such purpose. Thus, in this work, we aimed at the optimisation of an easily performed protocol to produce a suitable eGFP mRNA. By utilising molecular biology kits customarily employed in molecular biology laboratories working with RNA-based techniques and starting from any eGFP coding vector, we produced four mRNA molecules: (1) eGFP mRNA (non-polyadenylated); (2) Kozak-eGFP mRNA (non-polyadenylated, produced from the Kozak-containing amplicon); (3) eGFP-PolyA mRNA (polyadenylated); (4) Kozak-eGFP-PolyA mRNA (containing both signals, Kozak sequence and poly(A) tail). These mRNA molecules were transfected into HEK 293 FT cells, readily transfectable, and into the MDBK bovine lineage, which has been observed as difficult-to-transfect DNA constructs. eGFP expression could be detected both by flow cytometry and by fluorescence microscopy after transfection with the polyadenylated mRNAs. Upon cytometric analysis, we noted a marked difference among the mRNA groups (p?<?0.01), both in fluorescent population percentage and in florescence intensity. We showed here the necessity of the polyadenylation step in order to achieve cell expression of the eGFP observable under fluorescence microscopy. The presence of the Kozak sequence, as a 5′ element, seems to augment significantly the level of protein produced upon mRNA transfection. We presented here an easy protocol to allow production of functioning mRNAs from any DNA construct. The molecules produced may aid in the standardisation and controlling most of the RNA-related experiments as well as it gives proper guidance for researchers performing expression of other proteins through mRNA transfection.     

RNA templating of molecular assembly and covalent modification patterning in early molecular evolution and modern biosystems

The Direct RNA Template (DRT) hypothesis proposes that an early stage of genetic code evolution involved RNA molecules acting as stereochemical recognition templates for assembly of specific amino acids in sequence-ordered arrays, providing a framework for directed covalent peptide bond formation. It is hypothesized here that modern biological precedents may exist for RNA-based structural templating with functional analogies to hypothetical DRT systems. Beyond covalent molecular assembly, an extension of the DRT concept can include RNA molecules acting as dynamic structural template guides for the specific non-covalent assembly of multi-subunit complexes, equivalent to structural assembly chaperones. However, despite numerous precedents for RNA molecules acting as scaffolds for protein complexes, true RNA-mediated assembly chaperoning appears to be absent in modern biosystems. Another level of function with parallels to a DRT system is possible if RNA structural motifs dynamically guided specific patterns of catalytic modifications within multiple target sites in a pre-formed polymer or macromolecular complex. It is suggested that this type of structural RNA templating could logically play a functional role in certain areas of biology, one of which is the glycome of complex organisms. If any such RNA templating processes are shown to exist, they would share no necessary evolutionary relationships with events during early molecular evolution, but may promote understanding of the practical limits of biological RNA functions now and in the ancient RNA World. Awareness of these formal possibilities may also assist in the current search for functions of extensive non-coding RNAs in complex organisms, or for efforts towards artificial rendering of DRT systems.     

Targeted messenger RNA profiling of transfected breast cancer gene in a living cell

Selective messenger RNA (mRNA) profiling of transfected breast cancer gene expression in a living cell is demonstrated. Atomic force microscope (AFM) probe tips are structurally modified to create a dielectrophoretic force that attracts mRNA molecules within the cell nucleus. The tip end is chemically modified to hybridize only to the target mRNA from a pool of molecules within the nucleus. We successfully combined this scheme with standard assay techniques to develop an assay technology that can be used for early disease detection and basic studies in cell biology.     

Force spectroscopy with single bio-molecules

For many biological molecules, force is an important functional and structural parameter. With the rapidly growing knowledge about the relation between structure, function, and force, single-molecule force spectroscopy has become a versatile analytical tool for the structural and functional investigation of single bio-molecules in their native environments. Within the past year, detailed insights into binding potentials of receptor ligand pairs, protein folding pathways, molecular motors, DNA mechanics and the functioning of DNA-binding agents (such as proteins and drugs), as well as the function of molecular motors, have been obtained.     

Five challenges to bringing single-molecule force spectroscopy into living cells

In recent years, single-molecule force spectroscopy techniques have been used to study how inter- and intramolecular interactions control the assembly and functional state of biomolecular machinery in vitro. Here we discuss the problems and challenges that need to be addressed to bring these technologies into living cells and to learn how cellular machinery is controlled in vivo.     

How Do We Know when Single-Molecule Force Spectroscopy Really Tests Single Bonds?

Single-molecule force spectroscopy makes it possible to measure the mechanical strength of single noncovalent receptor-ligand-type bonds. A major challenge in this technique is to ensure that measurements reflect bonds between single biomolecules because the molecules cannot be directly observed. This perspective evaluates different methodologies for identifying and reducing the contribution of multiple molecule interactions to single-molecule measurements to help the reader design experiments or assess publications in the single-molecule force spectroscopy field. We apply our analysis to the large body of literature that purports to measure the strength of single bonds between biotin and streptavidin as a demonstration that measurements are only reproducible when the most reliable methods for ensuring single molecules are used.