A completely randomized RNA pool as well as a degenerate pool comprised of an RNA sequence which binds citrulline with a dissociation constant of 0 muM were used to select for tight binding arginine specific RNA aptamers. A modified in vitro selection scheme, based on affinity chromatography was applied to allow the enrichment of high affinity solution binders. The selection scheme included a negative selection with the non-cognate ligand citrulline, and a heat denaturation step prior to affinity elution with an excess of the cognate ligand arginine. After 20 cycles the majority of the pools bound specifically to the arginine matrix even after denaturation/renaturation in the presence of 20 mM of a non-cognate amino acid. When denatured and eluted in the presence of 20 mM arginine, the selected RNAs quantitatively washed off the column. These RNA aptamers were cloned and sequenced. Equilibrium dialysis performed with the most abundant clone among the selected sequence revealed Kd values of 330 nM for the RNA/arginine affinity, which is nearly a 200-fold improvement over the tightest binding arginine binding RNAs known to date. Arginine recognition by this RNA is highly enantioselectice: L- arginine is bound 12 000-fold better than D-arginine. Chemical modification analysis revealed that the secondary structure of the aptamer might contain a pseudoknot motif. Our tight binding arginine aptamers join a number of natural and in vitro selected RNAs which recognize arginine. The RNAs described here compare in their binding affinity with the tightest binding RNA aptamers for low molecular weight molecules isolated in other in vitro selection experiments.

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Die gemahlene Droge von Scrophuluriu nodosu L. (Scrophulariaceae) wurde mit einer 5prO.z. Losung von Na,C03 . 1 OH,O extrahiert (um gleichzeitig eventuell vorliegende Ester der Iridoidglykoside zu verseifen). Der an Tierkohle adsorbierte und an einer A1,03-Saule vorgereinigte Extrakt enthielt nach der Dunnschichtchromatographie mindestens 4 Iridoidglykoside, von denen die 3 Hauptprodukte rein isoliert werden konnten. Mit Hilfe analytischer und spektroskopischer Methoden wurden fur diese Verbindungen die folgenden Konstitutionen ermittelt: Aucubin (1; RF-Wert = 0.33), Harpagid (2; R,-Wert = 0.25), 6-cc-~-Rhamnopyranosylcatalpol (3; R,-Wert = 0.15). Die Ausbeuten betrugen 2.6% 1, 1.5% 2 und 8% 3, bezogen auf den vorgereinigten Extrakt. Natural Products from Medicinal Plants, XXIII'). -Iridoid Glycosides from Scrophularia nodosa L.The ground drug of Scrophuluriu nodosu L. (Scrophulariacease) is extracted with a 5% solution of Na2C03 . 10H20 to saponify esters of iridoid glycosides which might be present. The extract prepurified by adsorption on charcoal and Al,03 chromatography shows at least four iridoid glycosides in its thin layer chromatogram. The three main components have been isolated in the pure state. With the aid of analytical and spectroscopic methods the following constitutions of these compounds are elucidated: R , value 0.33 = aucubin (l), R , value 0.25 = harpagide (2), R, value 0.1 5 = 6-a-~-rhamnopyranosylcatalpol (3). The yields referred to prepurified extract amount to: 2.6% 1, 1.5% 2, and 8% 3.

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The chemical syntheses of novel digoxigenin-derivatized compounds are described which are modified substrates for enzymatically or photochemically non-radioactive digoxigenin labeling of nucleic acids. Various activated digoxigenin-haptens are coupled to 5-aminoallyl-substituted 2'-deoxyuridine-5'-triphosphate. This results in digoxigeninmodified nucleoside triphosphates of variable spacer lengths (Dig-[4]-dUTP/Dig-[ll]-dUTP/Dig-[16]-dUTP) which can be used as substrates for enzymatic labeling of DNA with digoxigenin-haptens by Klenow enzyme-catalysed random-primed synthesis. In addition the synthesis of N-, a photoactivatable analog of digoxigenin, is described which can be applied for photolabeling of DNA and RNA with digoxigenin-haptens leaving the nucleic acid molecules intact. Nicht-radioaktiveMarkierung und Detektion von Nucleinsäuren: IV. Synthese und Eigenschaften von Digoxigenin-modifizierten 2'-Desoxyuridin-5'-triphosphaten und eines photoaktivierbaren Analogons (Photodigoxigenin) Zusammenfassung: Es werden die chemischen Synthesen von solchen neuen, Digoxigenin-derivatisierten Komponenten beschrieben, die als modifizierte Substrate zur enzymatischen oder photochemischen nicht-radioaktiven Digoxigenin-Markierung von Nucleinsäuren eingesetzt werden können. Verschiedene aktivierte Digoxigenin-Haptene werden an 5-Aminoallyl-substituierte 2'-Desoxyuridin-5'-triphosphate gekoppelt. Es resultieren Digoxigenin-modifizierte Nucleosidtriphosphate mit unterschiedlichen Spacerlängen (Dig-[4]-dUTP/Dig-[ll]-dUTP/Dig-[16]-dUTP), die als Substrate zur enzymatischen Markierung von DNA mit Digoxigenin-Haptenen über die Klenow-Enzym-katalysierte Random-primed-Synthese eingesetzt werden können. Zusätzlich wird die Synthese von yV-[4]-Azidobenzoyl]-;V-[3-O-digoxigenininyl)methylcarbonyl]-l,8-diamino-3,6-dioxaoctan (Photodigoxigenin), einem photoaktivierbaren Analogon von Digoxigenin, beschrieben. Dieses Substrat kann zur Photomarkierung von DNA und RNA mit Digoxigenin-Haptenen angewendet werden; bei dieser Markierungsreaktion bleiben die Nucleinsäu-remoleküle intakt.