Characterization of TectoRNA Assembly with Cationic Conjugated Polymers

Association between RNAs with preprogrammed molecular recognition units can be quantified by using cationic, water-soluble conjugated polymers. The method uses a fluorophore-labeled probe RNA (RNA-F*), which is treated with a target structure (RNA-T). Heterodimer formation, (RNA-T/RNA-F*), increases the total negative charge on the F*-bearing macromolecule and reduces the number of negatively charged molecules (relative to unbound RNA-T+ RNA-F*). On the basis of electrostatic interactions, we anticipated more effective binding between CCP and (RNA_T/RNA-F*), a reduction of the average CCP- - -F* distance, and more effective FRET upon excitation of the conjugated polymer. The resulting signals benefit from the optical amplification characteristic of emissive conjugated polymers. Solution dissociation constants can be determined by monitoring F* intensity changes as a function of [RNA-F*] and the ratio:  [I(T) − I(NB)]/I(NB), where I(T) and I(NB) are the F* intensities in the presence of the target RNA (RNA-T) and a nonbinding RNA (RNA-NB), respectively, while keeping the concentration of the conjugated polymer constant. By focusing on [I(T) − I(NB)]/I(NB) as a function of RNA concentration, one can detect the concentration range wherein increased fluorescence is the result of dimerization.