We employ high precision single molecule Förster Resonance Energy Transfer (sm-FRET) method in order to uncover the fundamental behavior of the helical motifs and to compare topology of RNA three way junctions (3WJ). FRET is a powerful tool for recognizing structural heterogeneities and detection of the conformational changes in the biomolecular structure. For this study we selected four RNA 3WJs with various sequences in order to understand the importance of the sequence variation on the junction formation. Overall 173 FRET pairs were measured and analyzed with Multiparameter Fluorescence Detection (MFD) toolkit. 3D structures of RNA 3WJ were resolved by FRET-restrained computational structural modeling. Additionally, errors were accurately estimated using numerous independent measurements of the different RNA constructs. The results demonstrate that each RNA 3WJ construct can be represented by one conformer and characterized by the planarity and coaxial stacking. However, the shapes of RNA 3WJ with different sequences are similar within the errors, and common cross-sequence representative structure for all RNA 3WJ can be modeled. Noteworthy, the common representative structure is not symmetric, helical positions are not equivalent and this manifests in different mutual angles when alignment via sequence is applied.

Non-coding RNAs are involved in many essential processes in the cell such as protein synthesis in the ribosome and gene regulation in the form of riboswitches. The function of these catalytic RNAs is linked to their ability to form unique tertiary structures, which is inherently dynamic and dependent on interactions with proteins or small molecule ligands such as magnesium ions. Here, we apply three-color single-molecule FRET to study the global structural dynamics of helical RNA junctions, one of the most common structural building blocks of RNA. Three-color FRET offers the possibility to monitor three distances simultaneously, allowing one to address complex coordinated motions within single molecules. The method is applied to the unrestrained RNA four-way junction of the hairpin ribozyme. By labeling three of the four helices with fluorescent dyes, we can monitor their movement and determine their relative position. Quantitative information about the three-dimensional distribution of inter-dye distances is obtained by three-color photon distribution analysis (3C-PDA), allowing us to characterize the static conformations of the RNA four-way junction at high magnesium concentrations. To study the dynamics of the junction at lower concentrations of magnesium, we extended the toolbox of single-molecule FRET experiments, consisting of qualitative indicators of dynamics and the quantitative methods of filtered fluorescence correlation spectroscopy (fFCS) and dynamic PDA, for three-color FRET experiments. Specifically, in three-color pulsed interleaved excitation experiments (PIE) with multiparameter fluorescence detection (MFD), the multidimensional information of color, lifetime and anisotropy offers superior contrast to separate the different conformational states. This increases the robustness of the analysis and the sensitivity to minor conformational states, which are otherwise hidden in two-color FRET experiments. The experimental results are supported by enhanced sampling molecular dynamics simulations that probe the conformational space of the junction.

Förster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling is the powerful tool for detection conformational changes in the biomolecular structure. In this study we combine single-molecule multi-parameter fluorescence detection (MFD) and ensemble Time-Correlated Single Photon Counting measurements (eTCSPC) to investigate structure and dynamics of RNA three-way-junctions (3WJs) which are derived from the hairpin ribozyme. Bulge and sequence variations were considered as dominant factors influencing junction conformations of RNAs. To investigate the influence of these factors on RNA 3WJ conformations a set of six different molecules with different sequences were generated, two of which have two and five unpaired nucleotides in the junction region. Overall 258 FRET pairs were measured and investigated with the analysis framework [1] that includes probability distribution analysis (PDA) [2] for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation. We also compare numerous measurements from different constructs in order to calibrate error estimation accurately. The results demonstrate that each RNA 3WJ construct has only one predominant conformer. Furthermore bulges in the junction region determine orientation and rotation of helices, inducing coaxial stacking. The stacked helices are different for the 3WJs with different bulges whereas structures without bulges do not exhibit coaxial stacking. Noteworthy Mg concentration in solution modulates structural stabilization, preventing conformational changes. Our results show that small changes in the sequence make dramatic changes in RNA 3WJ tertiary structures which impacts the biological function. [1] Kalinin, S. et al, Nature Methods, 9, 1218-1225 (2012) [2] Antonik, M. et al, J.Phys.Chem, 110,6970-6978 (2006)

RNAs play multiple roles in the storage, transmission, and regulation of genetic information. They commonly exhibit helical junctions as main architectural building blocks of RNA tertiary arrangements. However, the knowledge about equilibrium structures and tertiary conformational changes of large RNAs, not restrained by external or tertiary interactions, is rather limited. A typical example is the hairpin ribozyme where helical four-way junction (RNA4WJ) is an essential structural motif. In order to rationalize the fundamentals of ribozyme architecture, we performed high-precision Förster resonance energy transfer (hpFRET) measurements at the single-molecule level and small angle X-ray scattering (SAXS) in connection with all-atom and coarse-grained molecular simulations to map the conformational space and determine structural models of a large RNA4WJ (≈ 45 kD) as a prototypic system. We resolved the structures of three coexisting conformers of a fully Watson-Crick base paired RNA4WJ. 51 donor-acceptor-pairs were measured using single-molecule multi-parameter fluorescence detection (smMFD). For each dataset, the single-molecule approach allowed for the simultaneous extraction of three distances (and their corresponding errors) belonging to one major FRET state and two minor states. Distinct Mg2+-affinities of the two minor states were used for the FRET peak assignment. Rigid body models for the major and both minor conformers were obtained by docking rigid ds A-RNA helices explicitly taking into account dye position distributions. The three rigid body models were refined by all-atom MD simulations and coarse-grained RNA folding using FRET restraints. Our results demonstrated that FRET-restrained molecular modeling can distinguish different conformational states of a complex RNA junction and determine their respective structures with a resolution of a few Ångström. Our broadly applicable method provides a powerful tool to probe the conformational ensembles of functional RNAs.

Förster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling is a powerful tool for analyzing biomolecular structures. Here we apply multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting measurements to perform FRET study on RNA three- and four-way-junctions (4WJs and 3WJs) with a high level of precision in distance better than 1% of the Förster radius [1]. We have generated a database of RNA 4WJs and six different RNA 3WJs with different bulges and sequences to study the influence of these factors on the junction conformations of RNA 3WJs. Overall 260 FRET pairs were measured with single-molecule MFD at 20 mM MgCl2 concentration and analyzed with the analysis toolkit [2] that includes probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation. Monte Carlo simulations showed that sterically allowed conformational space for RNA junctions is large. However, FRET measurements detect the existence of three different static conformers for RNA 4WJ, whereas RNA 3WJs have only one predominant static conformation. terically allowed conformational space for RNA is large. Their junction geometry was described in terms of mutual and Euler angles between helices. The FRET-derived structures suggest that the sequence dictates a junction specific conformation within the large topology space. Furthermore we see that bulges in the junction region determine orientation and rotation of helices and induce coaxial stacking between two of them. [1] Antonik, M., et al., J.Phys.Chem.B, 110, 6970-6978 (2006) [2] Kalinin, S. et al, Nat. Meth., 9, 1218-1225 (2012)

Non-protein coding RNAs perform essential functions in living organisms. They commonly exist as dynamic ensembles of conformational states. While many structurally known RNAs are trapped in one or a few conformations by interactions with proteins or tertiary contacts between stems, bulges, or loops the knowledge of equilibrium structures, conformational space, and tertiary conformational changes of large RNAs not being restrained by external or tertiary interactions is still very limited. Helical four-way and three-way junctions (4WJs and 3WJs) are an essential structural motif of the for functional RNA structures. Here we explore the topology of a set of a 4WJ and related 3WJs related to the hairpin ribozyme by measuring more than 250 different FRET-pairs using single-molecule multi-parameter fluorescence detection [1]. Using FRET restrained high-precision structural modeling combined with full atom MD simulations as a hybrid tool [2,3], we resolve the structures of three coexisting conformers of a fully Watson-Crick base paired RNA4WJ. By a suitable choice of the number of bases in the bulges the helices arrangements of the corresponding 3WJs can span a huge conformational space which is necessary for the stem communication in functional RNAs. [1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010). [2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011). [3] Kalinin, S. et al. Nat. Methods 9, 1218-1225 (2012)

and (ii) develop a qualitative picture for the folding landscape. These results are compared and contrasted to the results of a nearly identical RNA construct with a 2 nt substitutionmutation associated with the genetic disorder dyskeratosis congentia (DKC). As expected, the WT RNA construct (DG WT = 4.2 5 0.2 kcal/mol) is substantially more stable than the DKC construct (DG DKC = 0.265 0.05 kcal/mol]. The kinetic origin of this differential stability is the result of a substantially increased folding rate constant (~400 times faster) for the WT and a subtle reduction of the unfolding rate constant (~5 times slower).

RNA three-way junctions are important ribosomal structural motifs. They are also widely used as building blocks and functional components in nanotechnology applications. Förster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling in combination with molecular dynamics simulations was used to determine the structure of RNA three-way junction (3WJ) without bulges, RNA three-way junction with a small (two unpaired nucleotides) bulge and RNA three way junction with bigger (5 unpaired nucleotides) bulge. In total 81 Donor-Acceptor pairs were measured using single-molecule multi-parameter fluorescence detection (smMFD). This allows us to observe structural changes of the molecule induced by addition of bulge to the initial structure. Rigid body models for the major conformers were obtained by docking rigid double-stranded A-RNA helices explicitly taking into account dye position distributions. This is done many times (1000 iterations) with random starting conformations, yielding all local minima. Obtained models were then refined by all-atom MD simulations [1,2]. First results indicate the presence of two coaxially stacked helices for 3WJs with additional bulges at the junction, and absence of such stacking in case with no bulge. noteworthy the stacked helices are different for the 3WJs with two and five nucleotides in the bulge. Our studies showed that high precision FRET measurements are a valuable tool to complement the structural information obtained by X-ray crystallography or NMR spectroscopy as these techniques are limited in detecting minority conformers. References 1. Sindbert S, et al. (2011) Accur ate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity. J Am Chem Soc 133(8):2463-2480. 2. Kalinin S,et al. (2012) A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Methods in revision.

Like for many other non-coding RNAs, helical four-way and three-way junctions (4WJs and 3WJs) are an essential structural motif of the for functional RNA structures. Using FRET restrained high-precision structural modeling as a hybrid tool we resolve the structures of three coexisting conformers of a fully Watson-Crick base paired RNA4WJ based on the hairpin ribozyme. 51 different FRET-pairs were measured using single-molecule multi-parameter fluorescence detection (smMFD). For each dataset, the single-molecule approach allowed for the simultaneous extraction of three distances (and their corresponding errors) belonging to one major FRET state and two minor states. Distinct Mg2+-affinities were used for the assignment of the two minor states to the corresponding conformers. Rigid body models for the major and both minor conformers were obtained by docking rigid ds A-RNA helices explicitly taking into account dye position distributions. The three rigid body models were refined by all-atom MD simulations and coarse-grained RNA folding using FRET-restraints. A cluster analysis gives confidence levels for the proposed ensemble of models, and the precision was assessed via bootstrapping. The achieved precisions are significantly better than the uncertainty of the dye position with respect to the macromolecule. The structure of the 4WJ was compared with FRET restrained structures of related RNA 3WJs, where one stem was removed. The types of 3WJs were studied: (I) without bulges, (II) with a small bulge (two unpaired nucleotides) and (III) with larger bulge (5 unpaired nucleotides). In conclusion the overall geometry of the RNA helices depends drastically on the junction type. [1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010) [2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011) [3] Kalinin, S. et al. Nat. Methods in press

1349-Pos Board B241 Internal Friction of a Migrating Holliday Junction Hergen Brutzer, Alexander Huhle, Daniel Klaue, Ralf Seidel. TU Dresden, Dresden, Germany. Friction within biomolecules has recently gained increasing interest. Here we present a method that allows to study the friction that occurs during fast, few nm-sized refolding processes of nucleic acids. Branch migration of a homologous Holliday junction serves as an ideal system where such friction can be investigated. In this four-arm DNA junction the opposing arms possess identical sequences with respect to the junction center. In the absence of external constraints the junction is mobile such that one pair of homologous arms can expand at the expense of the other in single base pair diffusive steps.Wemeasure the dynamics of the branch migration process by stretching a torsionally constrained Holliday junction using magnetic tweezers and measuring the length fluctuations of the arms with high-speed videomicroscopy at ~3 kHz. Since DNA has a helical structure, branch migration causes twisting of the arms with one turn per helical pitchmoved. This constrains themovement of the junction within the tweezers to ~10 bp. Single base pair diffusive steps are expected to occur on a sub-millisecond time scale and to be much smaller than the overall DNA length fluctuations. Thus they cannot be directly resolved. However, power-spectral-density analysis of the length fluctuations is able to clearly resolve the overall dynamics of the branch migration process. Theoretical modeling considering the elastic coupling of DNA bending fluctuations and the junction movement allows to quantitatively determine the stepping rate and thus the friction of the branch migration process. We expect that our method iswidely applicable to study local-scalemolecular friction in biological systems.

Measuring in solution and utilizing the single-molecule advantage of fluorescence detection we established a toolbox to generate FRET-constrained structure models of biomolecules which can also show their heterogeneity and flexibility. Our approach comprises seven steps: (1) Quantitative measurement of FRET by multiparameter fluorescence detection of single molecules [1]; (2) Rigorous analysis and error determination of FRET derived donor-acceptor distances by analyzing the photon distributions and time resolved anisotropies of the dyes; (3) Appropriate description for the spatial distribution of the fluorophore by fast accessible volume (AV) simulations [2] to determine the dye positions relative to the biomolecule; (4) Search for possible structures via a FRET positioning system using a spring-network algorithm. Possible structures are generated either by a model-based approach with rigid body docking or model free by selecting suitable models from a huge structure library; (5) Docking is repeated many times to find all possible arrangements and assure the completeness of generated structural ensemble; (6) The obtained models are ranked according to their violation of FRET constraints and steric clashes. Then they are assigned to clusters of related structural organization in order to judge the uniqueness of structural models; (7) The precision (RMSD) of the structure models is determined using a bootstrapping procedure. We demonstrate the accuracy of high-precision (hp) FRET in two experiments - determination of the DNA position in HIV-1 reverse transcriptase:primer/template complexes and arrangement of a primer/template DNA bound by HIV-1 reverse transcriptase and analysis of the internal structural heterogeneity of human guanylate binding protein 1 (hGBP1). These studies show that hpFRET studies are valuable tool to complement the structure information obtained by classical methods. [1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010). [2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011).

collection of simulation tools and workflow for single-molecule Förster resonance energy transfer (smFRET) allows highly quantitative structural modeling. This hybrid approach yields a model of reverse-transcriptase binding to DNA at sub-angstrom accuracy when benchmarked against a crystal structure and can resolve a flexible single-stranded template overhang.

So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. We used multi-parameter fluorescence detection (MFD) to perform smFRET studies of free diffusing biomolecules. We demonstrate that the simultaneous acquisition of most fluorescent parameters by MFD allows for a robust assessment of all possible artefacts involved in single-molecules FRET and offers unsurpassed capabilities regarding the identification and analysis of individual species present in population of molecules [1]. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility and position of fluorophores and conformational dynamics [2] have to be taken into account when determining FRET related distances with high accuracy. Although static structures are known for many biomolecules, the functions of biomolecules are governed ultimately by their dynamic character. In this view we give various examples of smFRET experiments of DNA- and RNA-junctions. Their Mg-dependent structural dynamics is studied in detail. These studies show that smFRET studies are valuable tool to complement the structural and dynamic information obtained by X-ray crystallography or NMR spectroscopy. [1] Sisamakis, E., Valeri, A., Kalinin, S., Rothwell, P. J., Seidel, C. A. M.; Accurate single-molecule FRET studies using multiparameter fluorescence detection. Methods in Enzymology 475 (Single Molecule Methods, Part B: Multiparameter, super-resolution, tethering, and force based methods, Ed. Nils Walter) Chapter 18, 455-514 (2010). [2] Kalinin, S., Valeri A., Antonik M., Felekyan, S., Seidel, C. A. M.; Detection of structural dynamics by FRET: A photon distribution and fluorescence lifetime analysis of systems with multiple states. J. Phys. Chem. B. 114, 7983-7995 (2010).

In Förster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A detailed analysis of possible orientation effects (κ(2) problem) indicates that, for short linkers and unknown local environments, additional κ(2)-related uncertainties are clearly outweighed by better defined dye positions.

An overview of recent advances in high resolution fluorescence microscopy will be given. In structured illumination the sample is illuminated with a number of different patterns of light. In our case this is a series of sinusoidal grids at different grid positions and orientations generated by a programmable spatial light modulator or a physical phase grating. Experimental datasets acquired under these conditions and reconstructed results from these data, demonstrating a resolution improvement of up to a factor of two over standard widefield microscopy are presented. The non-linear approach of saturating optical transitions (for structured illumination as well as beam-scanning approaches) has a great potential especially in combination with photo-switchable dyes such as the recently described IrisFP protein from Ulrich Nienhaus’group or the Cy3-Alexa647 system used in Xiaowei Zhuang's group. An interesting approach is to push molecules into dark states in a patterned way shortly before imaging and exploiting the saturation of this transition. Finally a method will be presented in which the emitted fluorescence of a confocal microscope passes through two separate paths. These paths are interferometrically recombined in such a way that the images undergo a mutual rotation of 180 degrees. The self-interference of the fluorescent light is only constructive, if it originated from the optical axis of the scanning laser beam, thus leading to an efficient detection of a high resolution fluorescence images. K. Wicker, S. Sindbert, R. Heintzmann, Characterisation of resolution enhancing image inversion interferometers, Optics Express 17, 15491-15501, 2009 L. Hirvonen, K. Wicker., O. Mandula and R. Heintzmann, Structured illumination microscopy of a living cell, Europ. Biophys. J. 38, 807-812, 2009

Using a confocal fluorescence microscope multiparameter fluorescence detection (MFD) enables us to simultaneously collect all fluorescence information such as intensity, lifetime, anisotropy in several spectral ranges) from picoseconds to seconds. MFD and fluorescence correlation spectroscopy is applied to perform single-molecule FRET studies with an ultimate level of precision in determining separations with FRET of 1% of the Förster radius [J.Phys.Chem.B 110, 6970 (2006), J.Phys.Chem.B 112, 8361 (2008)]. In addition we can unambiguously distinguish between stochastic processes and broadening due to static or dynamic heterogeneity. In this way we measured bends and kinks in dsDNA. The high accuracy allowed us the detection of sequence-dependent DNA bending by 16° [PNAS 105, 18773 (2008)]. Moreover we studied the Mg-dependent structural dynamics of a four-way DNA (Holliday-) junction in order to find out whether the postulated extended square structure accumulates indeed as an intermediate or whether it should be considered more as a very short lived transition state. We found a complex Mg dependent kinetics, which must be described by a four species model with only two distinct FRET and two kinetic levels. The species with the same FRET value differ in their conformational flexibility: one is quasi-static, the other is dynamic. Our FRET data are clearly inconsistent with an accumulation of a single extended square junction structure at very low Mg concentrations. Finally we compare the structure and dynamics of DNA- and RNA four-way junctions. Thereby the structure of RNA four-way junction was characterized by 24 FRET distances, which allowed us to prove the existence of 3 of the 4 possible stacking conformers. These studies show that sm FRET studies are valuable tool to complement the structural and dynamic information obtained by X-ray crystallography or NMR spectroscopy.

Image inversion interferometers have the potential to significantly enhance the lateral resolution and light efficiency of scanning fluorescence microscopes. Self-interference of a point source's coherent point spread function with its inverted copy leads to a reduction in the integrated signal for off-axis sources compared to sources on the inversion axis. This can be used to enhance the resolution in a confocal laser scanning microscope. We present a simple image inversion interferometer relying solely on reflections off planar surfaces. Measurements of the detection point spread function for several types of light sources confirm the predicted performance and suggest its usability for scanning confocal fluorescence microscopy.

We present experimental results on image inversion interferometric setups with significant potential to increase efficiency and resolution in fluorescence confocal microscopy. Results agree with theory in terms of a reduction of FWHM.