Single Molecule Analysis Research Tool (SMART): an integrated approach for analyzing single molecule data

Single molecule studies have expanded rapidly over the past decade and have the ability to provide an unprecedented level of understanding of biological systems. A common challenge upon introduction of novel, data-rich approaches is the management, processing, and analysis of the complex data sets that are generated. We provide a standardized approach for analyzing these data in the freely available software package SMART: Single Molecule Analysis Research Tool. SMART provides a format for organizing and easily accessing single molecule data, a general hidden Markov modeling algorithm for fitting an array of possible models specified by the user, a standardized data structure and graphical user interfaces to streamline the analysis and visualization of data. This approach guides experimental design, facilitating acquisition of the maximal information from single molecule experiments. SMART also provides a standardized format to allow dissemination of single molecule data and transparency in the analysis of reported data.     

荧光单分子检测技术在生命科学中的应用

荧光单分子检测技术是用荧光标记来显示和追踪单个分子的构象变化、动力学,单分子之间的相互作用以及单分子操纵的研究。过去对于生命科学分子机制的研究,都是对分子群体进行研究,然后平均化来进行单分子估测。因此,单个分子的动态性和独立性也被平均化掉而无法表现出来。荧光单分子检测技术真正实现了对单个分子的实时观测,将过去被平均化并隐藏在群体测量中不能获得的信息显示出来。近几年来,荧光单分子检测技术的飞速发展,为生命科学的发展,开辟了全新的研究领域。现就荧光单分子检测技术在研究动力蛋白、DNA转录、酶反应、蛋白质动态性和细胞信号转导方面的应用进展作一综述。     

单分子荧光共振能量转移技术

单分子荧光共振能量转移技术（single molecule fluorescence resonance energy transfer,smFRET）通过检测单个分子内的荧光供体及受体间荧光能量转移的效率,来研究分子构象的变化。在单分子探测技术发展之前,大多数的分子实验是探测分子的综合平均效应（ensemble averages）,这一平均效应掩盖了许多特殊的信息。单分子探测可以对体系中的单个分子进行研究,得到某一分子特性的分布状况,也可研究生物分子的动力学反应。介绍了近来单分子荧光共振能量转移技术的进展。     

Targeting,resolving and quantifying cellular structures by single‐molecule localization microscopy

The second ‘Single Molecule Localization Microscopy’ symposium was held in August 2012 at the École Polytechnique Fédérale de Lausanne in Switzerland. During two and a half days, around 100 researchers from across the globe, and in disciplines spanning physics, biology, chemistry and computer science, gathered to discuss the developments in single‐molecule super‐resolution imaging and its applications to address important biological questions.     

第三代测序基本原理

文章阐述了以单分子实时测序和纳米孔技术为标志第三代测序的基本原理,介绍了Helicos的Heliscope单分子测序仪、Pacific Bioscience的SMRT技术和Oxford Nanopore Technologies公司正在研究的纳米孔单分子测序技术。与其他测序技术进行了简单的对比以并提出一些单分子测序仍需面对的问题以及对未来单分子测序的展望。     

Heterogeneity of single molecule FRET signals reveals multiple active ribosome subpopulations

Single molecule methods have revealed that heterogeneity is common in biological systems. However, interpretations of the complex signals are challenging. By tracking the fluorescence resonance energy transfer (FRET) signals between the A‐site tRNA and L27 protein in single ribosomes, we attempt to develop a qualitative method to subtract the inherent patterns of the heterogeneous single molecule FRET data. Seven ribosome subpopulations are identified using this method and spontaneous exchanges among these subpopulations are observed. All of the pretranslocation subpopulations are competent in real‐time translocation, but via distinguished pathways. These observations suggest that the ribosome may function through multiple reaction pathways. Proteins 2014; 82:1–9. © 2013 Wiley Periodicals, Inc.     

Fluorescent proteins for single‐molecule fluorescence applications

We present single‐molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single‐molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single‐molecule applications, demonstrated by high photostability and rare fluorescence‐intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single‐molecule fluorescence experiments. Due to its spectral characteristics and short fluorescence lifetime of 1.46 ns, mCherry complements other existing fluorescent proteins and is recommended for tracking and localization of target molecules with high accuracy, fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), or multicolor applications. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Microfabricated arrays of cylindrical wells facilitate single‐molecule enzymology of α‐chymotrypsin

Single‐molecule enzymology allows scientists to examine the distributions of kinetic rates among members of a population. We describe a simple method for the analysis of single‐molecule enzymatic kinetics and provide comparisons to ensemble‐averaged kinetics. To isolate our model enzyme, α‐chymotrypsin, into single molecules, we use an array of cylindrical poly(dimethylsiloxane) wells 2 μm in diameter and 1.35 μm in height. Inside the wells, a protease assay with a profluorescent substrate detects α‐chymotrypsin activity. We hold the concentration of α‐chymotrypsin at 0.39 nM in a given well with an enzyme‐to‐substrate ratio of 1:6,666 molecules. Fluorescence emitted by the substrate is proportional to enzyme activity and detectable by a charge‐coupled device. This method allows for the simultaneous real‐time characterization of hundreds of individual enzymes. We analyze single‐molecule kinetics by recording and observing their intensity trajectories over time. By testing our method with our current instruments, we confirm that our methodology is useful for the analysis of single enzymes for extracting static inhomogeneity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Meeting Report of the 2nd International Workshop on 2‐D DIGE Applications in Proteomics

The second international workshop on “2‐D DIGE Applications in Proteomics” took place at the Medizinisches Proteom‐Center, Ruhr‐Universität Bochum, from February 27^th to March 2^nd, 2007. The four day “hands‐on” course was addressed to all interested scientists from the field of Proteomics, inter alia to members of HUPO and DGPF, with a greater focus on image analysis and statistical analysis of 2‐D DIGE experiments.     

生物过程化学机制的单分子荧光探测

单分子荧光检测越来越广泛地被应用于生命科学领域。这项技术可以对生物过程的化学机制进行定量、仔细的探究,与传统系综实验形成很好的互补。本文简介近几年单分子荧光检测研究的若干典型实例,以此展示这项技术的特点、优势及其可能的应用。它们涉及从简单的生化反应到复杂的蛋白表达调控等重要的生物过程。     

A cell type annotation Jamboree—Revival of а communal science forum

Cell Atlases are currently being constructed for human tissues as well as several model organisms. New technologies make creation of vast datasets in many species possible, but the value of such data crucially depends on the quality of annotation. The tools of annotating single cell data and creating knowledge representations comparable across organisms have been lagging. We argue that successfully creating Cell Atlases will require a revival of a boot‐camp style forum for communal annotation combined with an intensive learning workshop, dubbed a “Jamboree”. We report on our experience of successfully developing a structure and curriculum and running such a Jamboree for Xenopus Embryonic Cell Types at the Janelia Farms campus of the Howard Hughes Medical Institute.     

小波变换及滚球算法在单分子荧光能量共振转移图像分析中的应用

单分子荧光共振能量转移技术是通过检测单个分子内的荧光供体及受体间荧光能量转移的效率来研究分子构象的变化．要得到这些生物大分子的信息就需要对大量的单分子信号进行统计分析,人工分析这些信息,既费时费力又不具备客观性和可重复性,因此本文将小波变换及滚球算法应用到单分子荧光能量共振转移图像中对单分子信号进行统计分析．在保证准确检测到单分子信号的前提下,文章对滚球算法和小波变换算法处理图像后的线性进行了分析,结果表明,滚球算法和小波变换算法不但能够很好地去除单分子FRET图像的背景噪声,同时还能很好地保持单分子荧光信号的线性．最后本文还利用滚球算法处理单分子FRET图像及统计15 bp DNA的FRET效率的直方图,通过计算得到了15 bp DNA的FRET效率值．     

Mechanical stability and differentially conserved physical–chemical properties of titin Ig‐domains

The mechanisms that determine mechanical stabilities of protein folds remain elusive. Our understanding of these mechanisms is vital to both bioengineering efforts and to the better understanding and eventual treatment of pathogenic mutations affecting mechanically important proteins such as titin. We present a new approach to analyze data from single‐molecule force spectroscopy for different domains of the giant muscle protein titin. The region of titin found in the I‐band of a sarcomere is composed of about 40 Ig‐domains and is exposed to force under normal physiological conditions and connects the free‐hanging ends of the myosin filaments to the Z‐disc. Recent single‐molecule force spectroscopy data show a mechanical hierarchy in the I‐band domains. Domains near the C‐terminus in this region unfold at forces two to three times greater than domains near the beginning of the I‐band. Though all of these Ig‐domains are thought to share a fold and topology common to members of the Ig‐like fold family, the sequences of neighboring domains vary greatly with an average sequence identity of only 25%. We examine in this study the relation of these unique mechanical stabilities of each I‐band Ig domain to specific, conserved physical–chemical properties of amino acid sequences in related Ig domains. We find that the sequences of each individual titin Ig domain are very highly conserved, with an average sequence identity of 79% across species that are divergent as humans, chickens, and zebra fish. This indicates that the mechanical properties of each domain are well conserved and tailored to its unique position in the titin molecule. We used the PCPMer software to determine the conservation of amino acid properties in titin Ig domains grouped by unfolding forces into “strong” and “weak” families. We found two motifs unique to each family that may have some role in determining the mechanical properties of these Ig domains. A detailed statistical analysis of properties of individual residues revealed several positions that displayed differentially conserved properties in strong and weak families. In contrast to previous studies, we find evidence that suggests that the mechanical stability of Ig domains is determined by several residues scattered across the β‐sandwich fold, and force sensitive residues are not only confined to the A′‐G region. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

生物分子计算机

生物分子计算机是一种用生物分子元件组装成的具有并行数据处理、三维存储器和神经网络等特征的智能化计算机。它不但运行速度快、能处理复杂性的非线性问题、具有一定的逻辑推理等认知能力 ,而且具备生物分子形成的细胞样的自行修复或自体复制能力。也许从分子计算机到蛋白质计算机、DNA计算机再到生物分子计算机是一种研究和制造生物分子计算机的路径。     

The use of optical spectroscopy in combinatorial chemistry

Infrared and Raman spectroscopy allow direct spectral analysis of the solid‐phase, thus avoiding the tedious cleavage of compounds from the solid support. With diagnostic bands in starting materials or products, infrared and Raman spectroscopy are efficient in monitoring each reaction step directly on the solid phase. Consequently, infrared and Raman spectroscopy have evolved as the premier analytical methodology for direct analysis on the solid support. While infrared transmission spectroscopy is a general analytical method for resin samples, internal reflection spectroscopy is especially suited for solid polymer substrates known as “pins” or “crowns.” Single bead analysis is done best by infrared microspectroscopy, whereas photoacoustic spectroscopy allows totally nondestructive analysis of resin samples. With an automated accessory, diffuse reflection spectroscopy provides a method for high throughput on‐bead monitoring of solid‐phase reactions. Providing identification based on molecular structure, HPLC‐FTIR is, therefore, complementary to LC‐MS. Additionally, Raman spectroscopy as a complement to infrared spectroscopy can be applied to resin samples and—using a Raman microscope—to single beads. Fluorometry as an extremely sensitive spectroscopic detection method allows rapid quantification of organic reactions directly on the resin. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:179–187, 1998/1999.     

Structural evolution and membrane interactions of Alzheimer's amyloid‐beta peptide oligomers: New knowledge from single‐molecule fluorescence studies

Amyloid‐β peptide (Aβ) oligomers may represent the proximal neurotoxin in Alzheimer's disease. Single‐molecule microscopy (SMM) techniques have recently emerged as a method for overcoming the innate difficulties of working with amyloid‐β, including the peptide's low endogenous concentrations, the dynamic nature of its oligomeric states, and its heterogeneous and complex membrane interactions. SMM techniques have revealed that small oligomers of the peptide bind to model membranes and cells at low nanomolar‐to‐picomolar concentrations and diffuse at rates dependent on the membrane characteristics. These methods have also shown that oligomers grow or dissociate based on the presence of specific inhibitors or promoters and on the ratio of Aβ40 to Aβ42. Here, we discuss several types of single‐molecule imaging that have been applied to the study of Aβ oligomers and their membrane interactions. We also summarize some of the recent insights SMM has provided into oligomer behavior in solution, on planar lipid membranes, and on living cell membranes. A brief overview of the current limitations of the technique, including the lack of sensitive assays for Aβ‐induced toxicity, is included in hopes of inspiring future development in this area of research.     

Crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å

The dibenzothiophene (DBT) monooxygenase DszC, which is the key initiating enzyme in “4S” metabolic pathway, catalyzes sequential sulphoxidation reaction of DBT to DBT sulfoxide (DBTO), then DBT sulfone (DBTO₂). Here, we report the crystal structure of DszC from Rhodococcus sp. XP at 1.79 Å. Intriguingly, two distinct conformations occur in the flexible lid loops adjacent to the active site (residue 280–295, between α9 and α10). They are named “open”' and “closed” state respectively, and might show the status of the free and ligand‐bound DszC. The molecular docking results suggest that the reduced FMN reacts with an oxygen molecule at C4a position of the isoalloxazine ring, producing the C4a‐(hydro)peroxyflavin intermediate which is stabilized by H391 and S163. H391 may contribute to the formation of the C4a‐(hydro)peroxyflavin by acting as a proton donor to the proximal peroxy oxygen, and it might also be involved in the protonation process of the C4a‐(hydro)xyflavin. Site‐directed mutagenesis study shows that mutations in the residues involved either in catalysis or in flavin or substrate‐binding result in a complete loss of enzyme activity, suggesting that the accurate positions of flavin and substrate are crucial for the enzyme activity. Proteins 2014; 82:1708–1720. © 2014 Wiley Periodicals, Inc.     

The influence of dead time related distortions on live cell fluorescence lifetime imaging (FLIM) experiments

Recent developments in the field of fluorescence lifetime imaging microscopy (FLIM) techniques allow the use of high repetition rate light sources in live cell experiments. For light sources with a repetition rate of 20–100 MHz, the time‐correlated single photon counting (TCSPC) FLIM systems suffer serious dead time related distortions, known as “inter‐pulse pile‐up”. The objective of this paper is to present a new method to quantify the level of signal distortion in TCSPC FLIM experiments, in order to determine the most efficient laser repetition rate for different FLT ranges. Optimization of the F ‐value, which is the relation between the relative standard deviation (RSD) in the measured FLT to the RSD in the measured fluorescence intensity (FI), allows quantification of the level of FI signal distortion, as well as determination of the correct FLT of the measurement. It is shown that by using a very high repetition rate (80 MHz) for samples characterized by high real FLT's (4–5 ns), virtual short FLT components are added to the FLT histogram while a F ‐value that is higher than 1 is obtained. For samples characterized with short real FLT's, virtual long FLT components are added to the FLT histogram with the lower repetition rate (20–50 MHz), while by using a higher repetition rate (80 MHz) the “inter‐pulse pile‐up” is eliminated as the F ‐value is close to 1. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Exploration of the conformational landscape in pregnane X receptor reveals a new binding pocket

Ligand‐regulated pregnane X receptor (PXR), a member of the nuclear receptor superfamily, plays a central role in xenobiotic metabolism. Despite its critical role in drug metabolism, PXR activation can lead to adverse drug‐drug interactions and early stage metabolism of drugs. Activated PXR can induce cancer drug resistance and enhance the onset of malignancy. Since promiscuity in ligand binding makes it difficult to develop competitive inhibitors targeting PXR ligand binding pocket (LBP), it is essential to identify allosteric sites for effective PXR antagonism. Here, molecular dynamics (MD) simulation studies unravelled the existence of two different conformational states, namely “expanded” and “contracted”, in apo PXR ligand binding domain (LBD). Ligand binding events shifted this conformational equilibrium and locked the LBD in a single “ligand‐adaptable” conformational state. Ensemble‐based computational solvent mapping identified a transiently open potential small molecule binding pocket between α5 and α8 helices, named “α8 pocket”, whose opening‐closing mechanism directly correlated with the conformational shift in LBD. A virtual hit identified through structure‐based virtual screening against α8 pocket locks the pocket in its open conformation. MD simulations further revealed that the presence of small molecule at allosteric site disrupts the LBD dynamics and locks the LBD in a “tightly‐contracted” conformation. The molecular details provided here could guide new structural studies to understand PXR activation and antagonism.     

Tracking bio‐molecules in live cells using quantum dots

Single particle tracking (SPT) techniques were developed to explore bio‐molecules dynamics in live cells at single molecule sensitivity and nanometer spatial resolution. Recent developments in quantum dots (Qdots) surface coating and bio‐conjugation schemes have made them most suitable probes for live cell applications. Here we review recent advancements in using quantum dots as SPT probes for live cell experiments. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)