SASDA88: RAID3 in PBS (RAID3)

基本情報

• 登録情報: データベース: SASBDB / ID: SASDA88

試料

RAID3 in PBS

• RAID3 (RNA)

• 引用: ジャーナル: RNA Biol / 年: 2015; タイトル: RAID3--An interleukin-6 receptor-binding aptamer with post-selective modification-resistant affinity.; 著者: 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 / ; 要旨: 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.

登録者

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

モデル

• モデル #314: ; タイプ: dummy / ソフトウェア: DAMMIN / ダミー原子の半径: 2.40 A / 対称性: P2 / カイ2乗値: 0.906; Omokage検索でこの集合体の類似形状データを探す (詳細)
• モデル #315: ; タイプ: atomic / ソフトウェア: SASREF CV / ダミー原子の半径: 1.90 A / 対称性: P2; コメント: Constraint of max 8 A among the quadruplex imposed; カイ2乗値: 1.04 / P-value: 0.056437; Omokage検索でこの集合体の類似形状データを探す (詳細)

試料

• 試料: 名称: RAID3 in PBS / 試料濃度: 0.2 mg/ml
• バッファ: 名称: PBS / pH: 7.4 ; 組成: 137 mM NaCl; 2,7 mM KCl; 6,5 mM Na2HPO4; 1,5 mM KH2PO4

要素 #181

タイプ: RNA / 記述: RAID3 / 分子量: 11.267 / 分子数: 2
配列:

GGGAGAACUG UGGGAGUGGA GGGUGGAUGG UUCU

実験情報

• ビーム: 設備名称: Diamond Light Source B21 / 地域: Oxfordshire / 国: UK / 形状: 1 x 5 mm / 線源: X-ray synchrotron / スペクトロメータ・検出器間距離: 3.9 mm
• 検出器: 名称: Pilatus 2M

スキャン

タイトル: RAID3 / 測定日: 2013年10月31日 / 単位: 1/nm /

	Min	Max
Q	0.1659	4.0114

距離分布関数 P(R)

ソフトウェア P(R): GNOM 5.0 / ポイント数: 671 /

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

結果

カーブのタイプ: single_conc /

	実験値	Porod
分子量	26 kDa	26 kDa
体積	-	26 nm3

	Guinier	P(R)
前方散乱 I0	0.002	-
慣性半径, Rg	2.7 nm	2.7 nm

	Min	Max
D	-	10
Guinier point	21	92