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User applications with fluorescently labeled oligos

farFRET – Single-molecule FRET Experiments beyond 10 nm

Multiply-labeled DNA custom synthesized by IBA

Single-molecule Förster resonance energy transfer (smFRET) is a powerful nanoscopic tool in studies of biomolecular structures and nanoscale objects; however, conventional smFRET measurements are typically limited to distances up to 10 nm. KRAINER, HARTMANN and SCHLIERF developed farFRET, a technique that extends the range in smFRET measurements beyond the 10 nm limit by enhanced energy transfer using multiple acceptors. To demonstrate this enhancement effect, double-stranded DNA rulers with defined lengths (dsDNA, 20 and 32 bp), custom manufactured by IBA, were labeled with a single donor (ATTO532) and increasing numbers of acceptors (ATTO647N) via click chemistry and amine reactions, respectively. Up to four acceptors were placed in close proximity on adjacent and opposite sides of the dsDNA.The authors demonstrated that with an increasing number of acceptors a pronounced shift in the FRET efficiency histograms resulted from the enhancement in energy transfer (Fig. 1). In conclusion, farFRET allows quantitative measurements of large biomolecular complexes and nanostructures thus bridging the remaining gap between FRET spectroscopy and superresolution microscopy.

Figure 1: Concept of farFRET and proof-of-principle experiments with DNA rulers. Compared with a single donor−acceptor FRET pair, the use of multiple acceptors enhances the transfer efficiency (E) for distances of 20 bp around the Förster radius (a) as well as for distances beyond 10 nm of 32 bp (b). The FRET efficiency histograms illustrate that farFRET readily extends the accessible range beyond the often experienced 10 nm limit.


Reprinted (adapted) with permission from Krainer, G.; Hartmann, A.; Schlierf, M. Nano Lett. 2015, 15 (9), 5826-5829. Copyright 2015 American Chemical Society.

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Measurements of DNA-loop formation via Cre-mediated recombination

Shoura et al. describes a novel quantitative method, in which they have used IBA’s fluorescently-labeled deoxynucleotides for FRET to measure the kinetics of Cre-recombination, in order to analyze DNA-loop formation.

 (A) Novel-plasmid DNA constructs were engineered in which pairs of tandem BbvCI and BsmI restriction sites flank respective Cre  Recombinase binding sites; loxP sites. The minor domain between centers of the loxP sites is 870bp.  Fluorophore labels (ATTO 594 in green as the donor dye; ATTO 647N in red as the acceptor dye) were incorporated by displacing the single-stranded fragments released by the tandem nicks with mutant endonucleases Nb.BbvCI and Nb.BsmI. The nicks were subsequently sealed with T4 DNA ligase resulting in a doubly-labeled covalently-closed plasmid DNA.  (B) The labeled plasmid can be linearized in the minor  or the major domain. Upon adding the Cre protein (pink circles), we monitored the kinetics of Cre-mediated loop formation of two different loop sizes using FRET. 

fluorescently-labeled deoxynucleotides for FRET
Multicolor Single-Molecule Spectroscopy

Person et al. prepared three-way and four-way junctions of DNA with oligonucleotides, manufactured by IBA, by slowly cooling the DNA strands down from 95°C. In the three-way junction, all branches were labeled with different fluorophores and the base pairs of the junction formed a bindig site for cholic acid. Three branches of the four-way Holliday junction were also labeled with different fluorophores, while biotin was bound to the fourth branch for immobilization to a glass cover slide (Fig. 1A). Three-color FRET was then used with alternating laser excitation, allowing the conformational changes between the two stable folds of the Holliday junction for immobilized molecules to be observed. Furthermore integrity of the Holliday junctions could be detected by fluorescence imaging (Fig. 1B).

Figure 1: A) Scheme of the labeled four-way junction (HJ, Holliday junction) and its conformational change.
False color representations of confocal images showing immobilized Holliday junctions. The colors blue, green, and red encode for the overall fluorescence intensity after 495 nm, 568 nm, and 650 nm excitation, respectively. This reveals singly labeled populations (blue, green and red spots), doubly labeled populations (cyan, purple and yellow spots) and intact Holliday junctions (white spots, circled).


Britta Person, Ingo H. Stein, Christian Steinhauer, Jan Vogelsang, and Philip Tinnefeld (2009)
Correlated Movement and Bending of Nucleic Acid Structures Visualized by Multicolor Single-Molecule Spectroscopy
ChemPhysChem 10: 1455 – 1460. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

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Single-Molecule STED Microscopy

A 60 bp double-stranded DNA, custom manufactured by IBA, was labeled with ATTO647N fluorescent dye by classical NHS-ester chemistry. This labeled oligonucleotide was immobilised on a streptavidin coated cover slide by a BSA-Biotin-linker, so that the fluorescent dye was bound by a DNA-“tether” of about 20 nm (Fig. 1). Using this setup for STED (Stimulated Emission Depletion) microscopy showed Kasper et al. that a reducing and oxidizing buffer system (ROXS) leads to an improvement in photostability of the fluorophores and fluorescence brightness decreases less during a series of scans (Fig. 2).

Figure 1: Scheme of the model used to estimate the influence of the linker length on resolution.

Figure 2: Fluorescence images of single ATTO647N-labeled 60-bp oligonucleotides immobilized under different aqueous buffer conditions in the absence (confocal) and presence of the STED beam (750 nm) in three successive scans (1-3). To visualize the effect of STED-beam-induced
photobleaching, the image of the first scan is color encoded red and overlaid with the image of the third scan ("1 + 3"). Measurements were performed in PBS in the absence (a) and presence (b) of the ROXS. In (b) there is almost no difference between the first and third scan.


Robert Kasper, Benjamin Harke, Carsten Forthmann, Philip Tinnefeld, Stefan W. Hell, and Markus Sauer (2010)
Single-Molecule STED Microscopy with Photostable Organic Fluorophores.
Small 6(13): 1379–1384. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

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DNA hairpin folding

Short DNA sequences with 3–9 loop nucleotides (thymines, dT), enclosed on both ends by 0–2 complementary nucleotides (cytosine, dC, and guanosine, dG) that can form a stem, were custom synthesized by IBA GmbH. Kim et al. labeled these oligos at their 5’ end with MR121 via an aliphatic amino modifier C3 using classical Nhydroxysuccinimidylester (NHS-ester) chemistry. MR121 is a fluorophore which can form nonfluorescent complexes with dG. Upon hairpinloop formation, a change in fluorescence is detectable and the loop formation can be investigated and related to the introduction of complementary stem nucleotides.

Figure: Schematic representation of conformational fluctuations of F-labeled DNA hairpin structures. MR121 (F = fluorophore) is attached at the 5’ end and connected via a loop of polythymines (dT) to the intrinsic quencher guanosine (dG). The stem consists of 1 or 2 cytosine (dC, incorporated at the 5’ end) and dG (incorporated at the 3’ end) pairs. kcl, kop: constants for closing and opening of the hairpinloop (Kindly provided by Prof. Dr. Markus Sauer)


Jiho Kim, Sören Doose, Hannes Neuweiler and Markus Sauer (2006)
The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy.
Nucleic Acids Research 34(9): 2516–2527

"We were always fully satisfied with the exceptional quality, purity and reliability of the amount of unlabeled and labeled oligonucleotides as well as with the customer support provided by 
Prof. Dr. Markus Sauer, Biotechnology & Biophysics, Julius-Maximilians-University Würzburg, Germany

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FRET measures bends and kinks in DNA

The Nanoanalytic group led by Dr. Filipp Oesterhelt, analysed three dimensional structures of the DNA double strands containing unpaired adenosines, so called A bulges (Fig.) via single molecule Fluorescence Resonance Energy Transfer (FRET).
To measure a set of donor-acceptor distances within each double-stranded DNA, three DNA oligonucleotides labeled with the donor fluorophore Alexa Fluor 488 at different positions and four complementary strands, labeled with the acceptor Cy5 at different positions, were combined.
The series of 12 FRET-distances, each with two basepairs difference, made it possible to determine the tree dimensional structure of the whole DNA constructs and also revealed a sequence-dependent bending of the double-stranded regions.
All oligonucleotides were synthesized and fluorescently labeled via 5-C6-aminoallyldeoxythymidines by IBA.

Figure: 3D structures of the doublestranded DNA sequences with bends resulting from A bulges. One, three or five unpaired adenosines (A) were inserted. Positions of the fluorescent dyes are marked by circles: gray for donor Alexa fluor 488 and black for acceptor Cy5.


Anna K. Wozniak, Gunnar F. Schröder, Helmut Grubmüller, Claus A.M. Seidel, and Filipp Oesterhelt(2008)
Singlemolecule FRET measures bends and kinks in DNA.
PNAS 105(47): 18337–18342. Copyright (2008) National Academy of Sciences, U.S.A.

"IBA has always been open for special requests. We have been able to professionally discuss all technical inquiries and IBA’s specialists have offered a very helpful customer service." 
Prof. Dr. Filip Oesterhelt, Institute for molecular physical chemistry, Heinrich-Heine-University Düsseldorf, Germany

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Investigation of Fl1- and TMR2-dyes used for FRET

A stem-loop oligonucleotide complementary to the transactivation region of the HIV-1 genome (cTAR) was doubly labeled with a pair of dyes that are frequently used for FRET, Fl1 and TMR2. Labeling of the 5’ terminus with TMR was performed via an amino linker with a ten-carbon spacer arm. The 3’ terminus of the oligonucleotides was labeled with Fl using a special solid support with the dye already attached. Synthesis and labeling of the oligonucleotides was performed by IBA GmbH. Because both dyes are bound at the stem of the hairpin-loop, Bernacchi et al. demonstrated that these dyes form a ground state heterodimer that shows a unique optical signature instead of the spectral properties of the individual dyes.

1 5(and 6)-carboxyfluorescein
2 5(and 6)-carboxytetramethylrhodamine

Figure: The oligonucleotide sequence of cTAR.
(Kindly provided by Prof. Dr. Yves Mély)


Serena Bernacchi, Etienne Piémont, Noelle Potier, Alain van Dorsselaer, and Yves Mély (2003)
Excitonic Heterodimer Formation in an HIV-1 Oligonucleotide Labeled with a Donor-Acceptor Pair Used for Fluorescence Resonance Energy Transfer.
Biophysical Journal 84: 643–654

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2-aminopurine substitution to analyze oligonucleotide-NC-peptide interaction

In this study hexanucleotides were substituted with 2-aminopurine (2Ap), a fluorescent adenine analogue, at positions 2 and 5 (custom manufactured by IBA GmbH). 2Ap is an environmentally sensitive fluorescent probe which can be used to detect oligonucleotide dynamics. Fluorescence of 2Ap within oligonucleotides is quenched due to interactions with its neighbour bases. During interaction of the oligonucleotide with NC-peptide (the HIV-1 nucleocapsid protein) quenching is reduced because of reduced oligonucleotide flexibility and 2Ap local mobility. This restriction in the oligonucleotide dynamics appears as an important mechanistic component of the nucleic acid chaperone properties of NC.


S. V. Avilov, E. Piemont, V. Shvadchak, H. de Rocquigny and Y. Mély (2008)
Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine.
Nucleic Acids Research 36: 885-896

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A two-color fluorescence dye as alternative to FRET

Several oligonucleotides (e.g. SL2 RNA), custom produced by IBA GmbH, were used as interaction partners for the NC-peptide (the HIV -1 nucleocapsid protein), which is thought to be critically involved in the viral life cycle, mainly through interaction with nucleic acids. The NC-peptide was labeled with a fluorescent dye (3 - hydroxychromone, 3HC), which has dual fluorescence emission. Upon interaction with nucleic acids, the ratio of the dye’s two emission bands clearly changed (Fig. 1). The described method is an alternative to FRET in interaction studies and has the advantage of requiring only single and not double labeling.

Figure: Changes in the fluorescence emission spectra of the labeled peptide 3HC-NC on binding to SL2 RNA. The spectra of 0.4 μM 3HC-NC was recorded in the absence (black) and in the presence of 0.2 μM (red), 0.4 μM (blue) and 0.6 mM (green) SL2 RNA. Excitation wavelength was 340 nm.
(Kindly provided by Prof. Dr. Yves Mély)


Volodymyr V. Shvadchak, Andrey S. Klymchenko, Hugues de Rocquigny and Yves Mély (2009)
Sensing peptide–oligonucleotide interactions by a two-color fluorescence label: application to the HIV-1 nucleocapsid protein.
Nucleic Acids Research 37(3): e25

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Time-resolved and FCS studies of the Hepatitis C virus core protein

The complementary oligonucleotides cTAR and dTAR form stem-loop structures and were synthesized and fluorescently labeled for timeresolved and FCS studies by IBA GmbH. In the case of the doubly labeled ODNs, the 5’ terminus was labeled with TMR1 or Rh6G2 via an amino-linker with a six carbon spacer arm, while the 3’ terminus was labeled with either Dabcyl3 or Fl4 using a special solid support with the dye already attached. The studies revealed that peptides from the Hepatitis C virus core protein chaperone the annealing of HIV-1 cTAR and dTAR, taken as models, and increase their annealing kinetics by at least three orders of magnitude. Two kinetic pathways were identified with a fast pre-equilibrium intermediate that then slowly converts into the final extended duplex. The nucleic acid chaperone properties of the core protein are mainly supported by its basic clusters.

1 carboxytetramethylrhodamine
2 Rhodamin 6G
3 4-(40-dimethylaminophenylazo) benzoic acid
4 5(and 6)-carboxyfluorescein


Kamal kant Sharma, Pascal Didier, Jean Luc Darlix, Hugues de Rocquigny, Hayet Bensikkadour, Jean-Pierre Lavergne, François Pénin, Jean-Marc Lessinger and Yves Mély (2010)
Kinetic analysis of the nucleic acid chaperone activity of the Hepatitis C virus core protein.
Nucleic Acids Research 38(11): 3632-42