Detection of Naja atra Cardiotoxin Using Adenosine-Based Molecular Beacon.

This study presents an adenosine (A)-based molecular beacon (MB) for selective detection of Naja atra cardiotoxin (CTX) that functions by utilizing the competitive binding between CTX and the poly(A) stem of MB to coralyne. The 5'- and 3'-end of MB were labeled with a reporter fluorophore and a non-fluorescent quencher, respectively. Coralyne induced formation of the stem-loop MB structure through A2-coralyne-A2 coordination, causing fluorescence signal turn-off due to fluorescence resonance energy transfer between the fluorophore and quencher. CTX3 could bind to coralyne. Moreover, CTX3 alone induced the folding of MB structure and quenching of MB fluorescence. Unlike that of snake venom α-neurotoxins, the fluorescence signal of coralyne-MB complexes produced a bell-shaped concentration-dependent curve in the presence of CTX3 and CTX isotoxins; a turn-on fluorescence signal was noted when CTX concentration was ≤80 nM, while a turn-off fluorescence signal was noted with a further increase in toxin concentrations. The fluorescence signal of coralyne-MB complexes yielded a bell-shaped curve in response to varying concentrations of N. atra crude venom but not those of Bungarus multicinctus and Protobothrops mucrosquamatus venoms. Moreover, N. nigricollis venom also functioned as N. atra venom to yield a bell-shaped concentration-dependent curve of MB fluorescence signal, again supporting that the hairpin-shaped MB could detect crude venoms containing CTXs. Taken together, our data validate that a platform composed of coralyne-induced stem-loop MB structure selectively detects CTXs.

Molecular recognition of nucleic acids: coralyne binds strongly to poly(A).

The small molecule coralyne was found to bind preferentially and strongly to single-stranded poly(A) with an apparent association constant (Ka) of (1.8+/-0.3) x 10(6)M(-1). Binding of coralyne to poly(A) is predominantly enthalpically driven with a stoichiometry of one coralyne per four adenine bases. Poly(A) forms a coralyne dependent secondary structure with a melting temperature of 60 degrees C, for the conditions of our study.

Dimerization of coralyne and its propyl analogue and their association with DNA.

Dimerization of coralyne, Ia, and its propyl analogue Ic has been analyzed at neutral pH as a function of ionic strength. Even at low ionic strength (I = 0.02) dimerization constants (molar units) for both compounds are 10(5) or greater, which is considerably larger than values obtained for similar intercalating molecules. Coralyne seems to undergo association to higher aggregates somewhat easier than Ic, which could be due to the fact that the propyl group on Ic provides some steric hinderance in forming higher aggregates. Both compounds readily associate with DNA. At high ratios of ligand to DNA, the spectra for Ia or Ic are similar to that of a highly aggregated complex. At low ratios of ligand to DNA, the spectra for both compounds approach a limit, which is relatively independent of ionic strength and concentration, and can be identified with the intercalated species. The slight differences between Ia and Ic in dimerization or in association with DNA do not seem capable of explaining the significant differences in antileukemic activity of these compounds.

Formation of protoberberine-type alkaloids by the tubers of somatic embryo-derived plants of Corydalis yanhusuo.

The effects of 0.5 - 5 mg/l abscisic acid [ABA], 0.5 - 10 mg/l (2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol [paclobutrazol] and 0.5 - 2 mg/l alpha-cyclopropyl-alpha-(4-methoxyphenyl)-5-pyrimidinemethanol [ancymidol], 0.5 - 5 mg/l gibberellic acid [GA(3)] and 15 - 100 mg/l polyethylene glycol [PEG] 4000 supplemented in half-strength Murashige and Skoog's (MS) medium on the production of the two major protoberberine-type alkaloids (D,L-tetrahydropalmatine and D-corydaline) by the tubers of somatic embryo-derived plants of Corydalis yanhusuo were examined. Somatic embryo derived plants were also maintained for 6 months on half-strength MS medium containing 0.1 mg/l GA(3) or 0.5 mg/l paclobutrazol. The alkaloid contents were determined by high performance liquid chromatography (HPLC). The analysis revealed that the contents of D,L-tetrahydropalmatine and D-corydaline in the tubers of somatic embryo-derived plants were higher than the marketed crude drug and varied with growth regulator/PEG-4000 treatment and the age of the plant.