SASDA84: RNA Aptamer (SRB2m)

Basic information

• Entry: Database: SASBDB / ID: SASDA84

Sample

RNA Aptamer

• SRB2m (RNA), SRB2m

• Citation: Journal: Anal Biochem / Year: 2009; Title: Characterization of a fluorophore binding RNA aptamer by fluorescence correlation spectroscopy and small angle X-ray scattering.; Authors: Arne Werner / Petr V Konarev / Dmitri I Svergun / Ulrich Hahn / ; Abstract: Using fluorescence correlation spectroscopy (FCS), we have established an in vitro assay to study RNA dynamics by analyzing fluorophore binding RNA aptamers at the single molecule level. The RNA aptamer SRB2m, a minimized variant of the initially selected aptamer SRB-2, has a high affinity to the disulfonated triphenylmethane dye sulforhodamine B. A mobility shift of sulforhodamine B after binding to SRB2m was measured. In contrast, patent blue V (PBV) is visible only if complexed with SRB2m due to increased molecular brightness and minimal background. With small angle X-ray scattering (SAXS), the three-dimensional structure of the RNA aptamer was characterized at low resolution to analyze the effect of fluorophore binding. The aptamer and sulforhodamine B-aptamer complex was found to be predominantly dimeric in solution. Interaction of PBV with SRB2m led to a dissociation of SRB2m dimers into monomers. Radii of gyration and hydrodynamic radii, gained from dynamic light scattering, FCS, and fluorescence cross-correlation experiments, led to comparable conclusions. Our study demonstrates how RNA-aptamer fluorophore complexes can be simultaneously structurally and photophysically characterized by FCS. Furthermore, fluorophore binding RNA aptamers provide a tool for visualizing single RNA molecules.

Contact author

• Petr Konarev (EMBL-Hamburg, European Molecular Biology Laboratory (EMBL) - Hamburg outstation, Notkestraße 85, Geb. 25A, 22607 Hamburg, Deutschland, Germany)

Models

• Model #48: ; Type: dummy / Chi-square value: 3.265249; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: RNA Aptamer / Sample MW: 35 kDa / Specimen concentration: 0.5 mg/ml
• Buffer: Name: Hepes / pH: 7.4

Entity #46

Name: SRB2m / Type: RNA / Description: SRB2m / Formula weight: 16.55 / Num. of mol.: 2
Sequence:

GGAACCUCGC UUCGGCGAUG AUGGAGAGGC GCAAGGUUAA CCGCCUCAGG UUCC

Experimental information

• Beam: Instrument name: DORIS III X33 / City: Hamburg / 国: Germany / Type of source: X-ray synchrotronSynchrotron
• Detector: Name: MAR 345 Image Plate

Scan

Title: SRB2m with PBV / Measurement date: Oct 24, 2007 / Storage temperature: 15 °C / Cell temperature: 15 °C / Exposure time: 120 sec. / Number of frames: 2 / Unit: 1/nm /

	Min	Max
Q	0.2449	5.0717

Distance distribution function P(R)

Sofotware P(R): GNOM 4.5a / Number of points: 512 /

	Min	Max
Q	0.2483	4.98
P(R) point	1	512
R	0	8

Result

D max: 8 / Type of curve: single_conc /

	Experimental	Porod
MW	20 kDa	-
Volume	-	24 nm3

	P(R)	Guinier
Forward scattering, I0	177	180.23
Radius of gyration, Rg	2.27 nm	2.25 nm