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Budweis Single-Molecule Group

Our research group is taking the advantage of living on the border in between biology and physics. We are mostly focused on single-molecule research and we are currently looking for motivated students of all degrees!


Research Interests Contact Information

Structure and dynamics of nucleic acids (e.g. those induced by epigenetic modifications)

Address:
Building C (room 01037) 
Faculty of Science
University of South Bohemia in České Budějovice
Branišovská 1760
370 05 České Budějovice
Phone: 00420387776237
Email: fessl at prf.jcu.cz
          lbujak at prf.jcu.cz
GPS: 48.9772847N, 14.4451364E
Epigenetic modification of DNA can affect transcription via several mechanisms. (a) Change in flexibility of nucleosome DNA can be induced by epigenetic modification. This can regulate transcription via chromatin condensation or decondensation. (b) Occurrence and stability of secondary structures in promoter region as (a) 4-way junction, (b) G-quadruplex, (c) i-motif or (d) hairpin can also regulate transcription. Lab Members

Structure and dynamics of IRESs

Researchers:
Tomáš Fessl, Ph.D.
Dr Łukasz Bujak
Radek Litvín, Ph.D.
Prof. František Vácha, Ph.D.

Students:
Eva Sýkorová
Jiří Štangl
Jakub Strejc

Three-way junction of the HCV IRES element. (A) Schematic of the secondary structure of the IRES. The three-way helical junction studied here is Circled.Exploration of the physically reasonable range of axial extension and winding of the junction, statistically evaluated using Pearson’s χ2 test. There are two minima within the range of core extension from approximately −3 Å to 3 Å and winding from −180° to 180°. Expanded cross sections of the critical region of the global minimum are shown to demonstrate the level of confidence. This work was done in laboratory of Prof. David Lilley (Dudnee, UK)

Collaborators:

Impact of physiological (in-cell) environment on biological machinery

Alena Bruce-Krejčí's group
Martin Vácha's group
Radek Litvín's group
Roman Tůma's group
Inside a living cell, nucleic acids (NA) are exposed to a very complex environment and their structures are, depending on their sequence, modulated by non-specific factors such as viscosity, molecular crowding or by specific interactions with ions and small molecular weight compounds. While the complexity of the intracellular environment as a general property of every living organism is generally appreciated, there is a lack of appropriate tools to analyse NA structures in a cellular context. Thus we introduced in-cell smFRET on NA as a non-invasive technique capable of precise cellular localization, offering temporal and distance information.

Plasmon resonance phenomena in single-molecule methodology

Phenomena of plasmon resonance can be used to alter spectroscopic properties of fluorescent probes such as fluorescence intensity. It can also increase efficiency of FRET and thus enlarge useful range of this method. To achieve enhancement of fluorescence intensity (b) or FRET, labelled molecule should be placed into suitable distance from metallic nanoparticle (a). Conjugation of labelled molecules to nanoparticles can be checked using FCS via increase in diffusion times (~ hydrated radius) after labelling (c).

Single-mol photosynthesis and light harvesting ...

Maps of fluorescence intensity and lifetimes of Light Harvesting Complex 2 (LH2) from purple bacteria, excited with 405nm laser P=2,3uW, repetition rate 40MHz. Intensity map is shown on the left (acquisition time 10ms). Intensity time trace for molecule in white circle is shown on the upper right part of figure. Corresponding lifetimes of its different states are shown at the right bottom.

Single-Molecule Facility

Currently, we have two optical tables with flexible single-molecule set-ups capable of:
  1. TIRF & epi-fluorescent widefield
  2. Multicolor Confocal microscopy
  3. Multicolor Fluorescence lifetime imaging (FLIM)
  4. Fluorescence correlation spectroscopy (FCS
  5. Burst  intensity fluorescence analysis (BIFL)
  6. Multicolor Fluorescence lifetimes & anisotropy
  7. Fluorescence Intensity Distribution Analysis (FIDA
We are always keen to collaborate. If you want to implement single-molecule techniques into your research, do not hesitate to contact us.

Selected publications

Bacteriochlorophyll Aggregates Self-Assembled on Functionalized Gold Nanorod Cores as Mimics of Photosynthetic Chlorosomal Antennae: A Single Molecule Study. 
S. Furumaki, F. Vacha, S. Hirata, M. Vacha (2014)
ACS Nano, 8(3), 2176-2182

Measurement of the Change in Twist at a Helical Junction in RNA Using the Orientation Dependence of FRET 
Fessl, DMJ Lilley (2013)
Biophysical journal 105 (9), 2175-2181

Single-Molecule Observation of the Induction of k-Turn RNA Structure on Binding L7Ae Protein
J Wang, T Fessl, KT Schroeder, J Ouellet, Y Liu, ADJ Freeman, DMJ Lilley (2012)
Biophysical Journal 103 (12), 2541-2548

Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET
T Fessl, F Adamec, Tomáš Fessl, František Adamec, Tomáš Polívka, Silvie Foldynová-Trantírková, František Vácha, Lukáš Trantírek (2012)
Nucleic acids research 40 (16), e121-e121

A comparative study of the binding of QSY 21 and Rhodamine 6G fluorescence probes to DNA: structure and dynamics
M Kabeláč, F Zimandl, T Fessl, Z Chval, F Lankaš (2010)
Physical Chemistry Chemical Physics 12 (33), 9677-9684

Depth of focus extended microscope configuration for imaging of incorporated groups of molecules, DNA constructs and clusters inside bacterial cells
T Fessl, S Ben-Yaish, F Vacha, F Adamec, Z Zalevsky (2009)
Optics Communications 282 (13), 2495-2501

Bacteriochlorophyll Aggregates Self-Assembled on Functionalized Gold Nanorod Cores as Mimics of Photosynthetic Chlorosomal Antennae: A Single Molecule Study
S. Furumaki, F. Vacha, S. Hirata, M. Vacha (2014)
ACS Nano, 8(3), 2176-2182

Absorption linear dichroism measured directly on a single light-harvesting system: the role of disorder in chlorosomes of green photosynthetic bacteria
S. Furumaki, F. Vacha, S. Habuchi, Y. Tsukatani, D.A. Bryant, M. Vacha (2011)
JACS 133(17), 6703-6710