SASDA88: RAID3 in PBS (RAID3)

Basic information

• Entry: Database: SASBDB / ID: SASDA88

Sample

RAID3 in PBS

• RAID3 (RNA)

• Citation: Journal: RNA Biol / Year: 2015; Title: RAID3--An interleukin-6 receptor-binding aptamer with post-selective modification-resistant affinity.; Authors: Florian Mittelberger / Cindy Meyer / Georg H Waetzig / Martin Zacharias / Erica Valentini / Dmitri I Svergun / Katharina Berg / Inken Lorenzen / Joachim Grötzinger / Stefan Rose-John / Ulrich Hahn / ; Abstract: Aptamers are an emerging class of highly specific targeting ligands. They can be selected in vitro for a large variety of targets, ranging from small molecules to whole cells. Most aptamers selected are nucleic acid-based, allowing chemical synthesis and easy modification. Although their properties make them interesting drug candidates for a broad spectrum of applications and an interesting alternative to antibodies or fusion proteins, they are not yet broadly used. One major drawback of aptamers is their susceptibility to abundant serum nucleases, resulting in their fast degradation in biological fluids. Using modified nucleic acids has become a common strategy to overcome these disadvantages, greatly increasing their half-life under cell culture conditions or even in vivo. Whereas pre-selective modifications of the initial library for aptamer selection are relatively easy to obtain, post-selective modifications of already selected aptamers are still generally very labor-intensive and often compromise the aptamers ability to bind its target molecule. Here we report the selection, characterization and post-selective modification of a 34 nucleotide (nt) RNA aptamer for a non-dominant, novel target site (domain 3) of the interleukin-6 receptor (IL-6R). We performed structural analyses and investigated the affinity of the aptamer to the membrane-bound and soluble forms (sIL-6R) of the IL-6R. Further, we performed structural analyses of the aptamer in solution using small-angle X-ray scattering and determined its overall shape and oligomeric state. Post-selective exchange of all pyrimidines against their 2'-fluoro analogs increased the aptamers stability significantly without compromising its affinity for the target protein. The resulting modified aptamer could be shortened to its minimal binding motif without loss of affinity.

Contact author

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

Models

• Model #314: ; Type: dummy / Software: DAMMIN / Radius of dummy atoms: 2.40 A / Symmetry: P2 / Chi-square value: 0.906; Search similar-shape structures of this assembly by Omokage search (details)
• Model #315: ; Type: atomic / Software: SASREF CV / Radius of dummy atoms: 1.90 A / Symmetry: P2 / Comment: Constraint of max 8 A among the quadruplex imposed / Chi-square value: 1.04 / P-value: 0.056437; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: RAID3 in PBS / Specimen concentration: 0.2 mg/ml
• Buffer: Name: PBS / pH: 7.4 ; Composition: 137 mM NaCl; 2,7 mM KCl; 6,5 mM Na2HPO4; 1,5 mM KH2PO4

Entity #181

Type: RNA / Description: RAID3 / Formula weight: 11.267 / Num. of mol.: 2
Sequence:

GGGAGAACUG UGGGAGUGGA GGGUGGAUGG UUCU

Experimental information

• Beam: Instrument name: Diamond Light Source B21 / City: Oxfordshire / 国: UK / Shape: 1 x 5 mm / Type of source: X-ray synchrotron / Dist. spec. to detc.: 3.9 mm
• Detector: Name: Pilatus 2M

Scan

Title: RAID3 / Measurement date: Oct 31, 2013 / Unit: 1/nm /

	Min	Max
Q	0.1659	4.0114

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 671 /

	Min	Max
Q	0.0380298	0.222194
P(R) point	1	671
R	0	100

Result

Type of curve: single_conc /

	Experimental	Porod
MW	26 kDa	26 kDa
Volume	-	26 nm3

	Guinier	P(R)
Forward scattering, I0	0.002	-
Radius of gyration, Rg	2.7 nm	2.7 nm

	Min	Max
D	-	10
Guinier point	21	92