Rational design for cooperative recognition of specific nucleobases using β-cyclodextrin-modified DNAs and fluorescent ligands on DNA and RNA scaffolds

We propose a binary fluorimetric method for DNA and RNA analysis by the combined use of two probes rationally designed to work cooperatively. One probe is an oligonucleotide (ODN) conjugate bearing a β-cyclodextrin (β-CyD). The other probe is a small reporter ligand, which comprises linked molecules of a nucleobase-specific heterocycle and an environment-sensitive fluorophore. The heterocycle of the reporter ligand recognizes a single nucleobase displayed in a gap on the target labeled with the conjugate and, at the same time, the fluorophore moiety forms a luminous inclusion complex with nearby β-CyD. Three reporter ligands, MNDS (naphthyridine-dansyl linked ligand), MNDB (naphthyridine-DBD), and DPDB (pyridine-DBD), were used for DNA and RNA probing with 3′-end or 5′-end modified β-CyD-ODN conjugates. For the DNA target, the β-CyD tethered to the 3′-end of the ODN facing into the gap interacted with the fluorophore sticking out into the major groove of the gap site (MNDS and DPDB). Meanwhile the β-CyD on the 5′-end of the ODN interacted with the fluorophore in the minor groove (MNDB and DPDB). The results obtained by this study could be a guideline for the design of binary DNA/RNA probe systems based on controlling the proximity of functional molecules.