Making connections—strategies for single molecule fluorescence biophysics

Fluorescence spectroscopy and fluorescence microscopy carried out on the single molecule level are elegant methods to decipher complex biological systems; it can provide a wealth of information that frequently is obscured in the averaging of ensemble measurements. Fluorescence can be used to localise a molecule, study its binding with interaction partners and ligands, or to follow conformational changes in large multicomponent systems. Efficient labelling of proteins and nucleic acids is very important for any fluorescence method, and equally the development of novel fluorophores has been crucial in making biomolecules amenable to single molecule fluorescence methods. In this paper we review novel coupling strategies that permit site-specific and efficient labelling of proteins. Furthermore, we will discuss progressive single molecule approaches that allow the detection of individual molecules and biomolecular complexes even directly isolated from cellular extracts at much higher and much lower concentrations than has been possible so far

Distance dependence of single-molecule energy transfer to graphene measured with DNA origami nanopositioners

Despite the thorough investigation of graphene since 2004, altering its surface chemistry and reproducible functionalization remain challenging. This hinders fabrication of more complex hybrid materials with controlled architectures, and as a consequence the development of sensitive and reliable sensors and biological assays. In this contribution, we introduce DNA origami structures as nanopositioners for placing single dye molecules at controlled distances from graphene. The measurements of fluorescence intensity and lifetime of single emitters carried out for distances ranging from 3 to 58 nm confirmed the d–4 dependence of the excitation energy transfer to graphene. Moreover, we determined the characteristic distance for 50% efficiency of the energy transfer from single dyes to graphene to be 17.7 nm. Using pyrene molecules as a glue to immobilize DNA origami nanostructures of various shape on graphene opens new possibilities to develop graphene-based biophysics and biosensing

DNA origami-based single-molecule force spectroscopy elucidates RNA Polymerase III pre-initiation complex stability

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP II

Strong plasmonic enhancement of single molecule photostability in silver dimer optical antennas

Photobleaching is an effect terminating the photon output of fluorophores, limiting the duration of fluorescence-based experiments. Plasmonic nanoparticles (NPs) can increase the overall fluorophore photostability through an enhancement of the radiative rate. In this work, we use the DNA origami technique to arrange a single fluorophore in the 12-nm gap of a silver NP dimer and study the number of emitted photons at the single molecule level. Our findings yielded a 30× enhancement in the average number of photons emitted before photobleaching. Numerical simulations are employed to rationalize our results. They reveal the effect of silver oxidation on decreasing the radiative rate enhancement.We acknowledge funding by a starting grant (SiMBA, EU 261162) of the European Research Council (ERC) and the Deutsche Forschungsgesellschaft (AC 279/2-1 and TI 329/9-1). IK is grateful for the support by the Mobility Plus grant 1269/MOB/IV/2015/0 from the Polish Ministry of Science and Higher Education (MNiSW). CV thanks a scholarship of the Studienstiftung des deutschen Volkes. AIF-D and AC-G acknowledge funding from the Spanish MINECO under Contracts FIS2015- 64951-R and MDM-2014-0377-16-4, respectively. AIF-D also acknowledges funding from EU Seventh Framework Programme under Grant Agreement FP7-PEOPLE- 2013-CIG-630996. GA and PT acknowledge funding of the state ministry for research of lower saxony in the frame of the “Quantum- and Nanometrology” (QUANOMET) strategic research area. Quanomet is part of the LUH-TUBS research allianc

Directing single-molecule emission with dna origami-assembled optical antennas

We demonstrate the capability of DNA self-assembled optical antennas to direct the emission of an individual fluorophore, which is free to rotate. DNA origami is used to fabricate optical antennas composed of two colloidal gold nanoparticles separated by a predefined gap and to place a single Cy5 fluorophore near the gap center. Although the fluorophore is able to rotate, its excitation and far-field emission is mediated by the antenna, with the emission directionality following a dipolar pattern according to the antenna main resonant mode. This work is intended to set out the basis for manipulating the emission pattern of single molecules with self-assembled optical antennas based on colloidal nanoparticles

Controlled reduction of photobleaching in DNA origami gold nanoparticle hybrids

The amount of information obtainable from a fluorescence-based measurement is limited by photobleaching: Irreversible photochemical reactions either render the molecules nonfluorescent or shift their absorption and/or emission spectra outside the working range. Photobleaching is evidenced as a decrease of fluorescence intensity with time, or in the case of single molecule measurements, as an abrupt, single-step interruption of the fluorescence emission that determines the end of the experiment. Reducing photobleaching is central for improving fluorescence (functional) imaging, single molecule tracking, and fluorescence-based biosensors and assays. In this single molecule study, we use DNA self-assembly to produce hybrid nanostructures containing individual fluorophores and gold nanoparticles at a controlled separation distance of 8.5 nm. By changing the nanoparticles? size we are able to systematically increase the mean number of photons emitted by the fluorophores before photobleaching.Fil: Pellegrotti, Jesica Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Acuña, Guillermo. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; AlemaniaFil: Puchkova, Anastasiya. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; AlemaniaFil: Holzmeister, Phil. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; AlemaniaFil: Gietl, Andreas. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; AlemaniaFil: Lalkens, Birka. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; AlemaniaFil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Tinnefeld, Philip. Technische Universität Braunschweig. Institute for Physical and Theoretical Chemistry. NanoBioSciences Group; Alemani

Advanced Fluorescence Microscopy Techniques-FRAP, FLIP, FLAP, FRET and FLIM

Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research