Characterization of a fluorophore binding RNA aptamer by fluorescence correlation spectroscopy and small angle X-ray scattering

Werner, Ansgar; Konarev, Petr V.; Svergun, Dmitri I.; Hahn, Ulrich

doi:10.1016/j.ab.2009.03.018

Cited by 20 publications

(13 citation statements)

References 51 publications

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“…FCS is a correlation analysis of fluctuations of fluorescence radiation. The method enables the direct measurement of the interaction of antigen molecules with specific monoclonal antibodies [7,8,9,10,11,12,13,14,15]. Specific antibodies are used for the detection of the GAD 65 antigen.…”

Section: Methodsmentioning

confidence: 99%

The Diabetic Antigen Glutamic Acid Decarboxylase (GAD 65) in the Human Peripheral Blood

Tilz¹,

Dausset²,

Wiltgen

2009

Int Arch Allergy Immunol

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Background: Glutamic acid decarboxylase (GAD 65) is a diabetes-associated antigen which is generally considered to be strictly intracellular. In order to better understand autoimmunity, this study demonstrates the appearance of GAD 65 in the peripheral human blood and presents implications for the diagnosis and therapy of some autoimmune diseases. Methods: The GAD 65 molecules are detected by their interaction with monoclonal antibodies labeled with dyes in an experimental setup with fluorescence correlation spectroscopy (FCS). These interactions result in changes in Brownian motion measured as fluorescence fluctuations. Sera from 153 patients with diabetes mellitus type 1 and controls were investigated. To enable the representation of the molecule as a model for further discussions, we present structural visualizations of its hydrophobic properties, leading to possible interactions with the cell membrane lipids and epitope locations. Results: The GAD65 antigen could be measured with a sensitivity of 2.65 µg/ml in ‘clean systems’ resulting from spiking experiments and human sera. The GAD 65 antigen could be identified in 8 patient sera: 4 children with diabetes mellitus type 1 and 4 adults initially taken as controls but who retrospectively showed signs of autoimmunity. Conclusion: We conclude that these findings are of significance for the concept of autoimmunity, i.e. in an initial step the immune system is primed by its accessibility to GAD 65. Our experimental results may also be important for the therapy of diabetes mellitus type 1 and other autoimmune diseases by the passive administration of GAD 65 antibodies.

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Section: Methodsmentioning

confidence: 99%

The Diabetic Antigen Glutamic Acid Decarboxylase (GAD 65) in the Human Peripheral Blood

Tilz¹,

Dausset²,

Wiltgen

2009

Int Arch Allergy Immunol

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“…This buffer composition was used for SAXS experiments of fluorophore binding RNA aptamer in ref. [1]. Sufficient amount (>10 mL) of matching buffer(s) must be brought to the SAXS station to make the buffer collections and to dilute the samples if necessary.…”

Section: Methodsmentioning

confidence: 99%

“…Depending on the binding mode between target and aptamer, the aptamer can interact as monomer or dimer for instance. This oligomerization state might change dependent on the bound molecule [1]. To monitor the dynamics of free and/or complexed aptamers one can use fluorescence correlation spectroscopy (FCS) and small angle X-ray scattering (SAXS).…”

Section: Introductionmentioning

confidence: 99%

Exploring RNA Oligomerization and Ligand Binding by Fluorescence Correlation Spectroscopy and Small Angle X-Ray Scattering

Magbanua

Konarev²,

Svergun³

et al. 2013

Methods in Molecular Biology

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RNA forms defined structures and binds specifically to target molecules. The combination of data which results from fluorescence correlation spectroscopy (FCS) and small angle X-ray scattering (SAXS) measurements can be used to determine intermolecular interactions between RNA and its binding partners. To define oligomerization states of free RNA and its complexes with bound target molecules, hydrodynamic radii, radii of gyration as well as the maximum sizes of the components have to be determined and compared. Furthermore, the program OLIGOMER allows calculating the portions of monomeric and dimeric RNA, for instance, within a mixture.

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“…The optimum concentration range for FCS is nano- to micromolar, mimicking physiological concentrations and minimizing sample amounts. These methods have been applied on the characterization of ribonucleic acids in vitro (9,10). …”

Section: Introductionmentioning

confidence: 99%

Predicting translational diffusion of evolutionary conserved RNA structures by the nucleotide number

Werner

2010

Nucleic Acids Research

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Ribonucleic acids are highly conserved essential parts of cellular life. RNA function is determined to a large extent by its hydrodynamic behaviour. The presented study proposes a strategy to predict the hydrodynamic behaviour of RNA single strands on the basis of the polymer size. By atom-level shell-modelling of high-resolution structures, hydrodynamic radius and diffusion coefficient of evolutionary conserved RNA single strands (ssRNA) were calculated. The diffusion coefficients D of 17–174 nucleotides (nt) containing ssRNA depended on the number of nucleotides N with D = 4.56 × 10−10 N−0.39 m2 s−1. The hydrodynamic radius RH depended on N with RH = 5.00 × 10−10 N0.38 m. An average ratio of the radius of gyration and the hydrodynamic radius of 0.98 ± 0.08 was calculated in solution. The empirical law was tested by in solution measured hydrodynamic radii and radii of gyration and was found to be highly consistent with experimental data of evolutionary conserved ssRNA. Furthermore, the hydrodynamic behaviour of several evolutionary unevolved ribonucleic acids could be predicted. Based on atom-level shell-modelling of high-resolution structures and experimental hydrodynamic data, empirical models are proposed, which enable to predict the translational diffusion coefficient and molecular size of short RNA single strands solely on the basis of the polymer size.

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Characterization of a fluorophore binding RNA aptamer by fluorescence correlation spectroscopy and small angle X-ray scattering

Cited by 20 publications

References 51 publications

The Diabetic Antigen Glutamic Acid Decarboxylase (GAD 65) in the Human Peripheral Blood

The Diabetic Antigen Glutamic Acid Decarboxylase (GAD 65) in the Human Peripheral Blood

Exploring RNA Oligomerization and Ligand Binding by Fluorescence Correlation Spectroscopy and Small Angle X-Ray Scattering

Predicting translational diffusion of evolutionary conserved RNA structures by the nucleotide number

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