'Cyclicons' as hybridization-based fluorescent primer-probes: Synthesis, properties and application in real-time PCR

We have studied the use of 'pseudocyclic oligonucleotides' (PCOs) (Jiang et al. Bioorg. Med. Chem. 1999, 7, 2727) as hybridization-based fluorescent p robes. The resulting fluorescent tag-attached PCOs are called 'cyclicons'. Cyclicons consist of two oligonucleotides linked to each other through 3'-3 ' or 5'-5' ends. One of the oligos is the probe or primer-probe sequence th at is complementary to a target nucleic acid (mRNA/DNA), and the other is a modifier oligo that is complementary to one of the ends of the probe oligo . A fluorescence molecule and a quancher molecule are attached at an approp riate position in the cyclicons. In the absence of the target nucleic acid, the fluorophore and the quencher are brought in close proximity to each ot her because of the formation of an intramolecular cyclic structure, resulti ng in fluorescence quenching. When the cyclicon hybridizes to the complemen tary target nucleic acid strand, the intramolecular cyclic structure of the cyclicon is destabilized and opened up, separating the fluorophore and que ncher groups, resulting in spontaneous fluorescence emission. Fluorescent s tudies in the presence and absence of a target nucleic acid suggest that cy clicons exist in intramolecular cyclic structure form in the absence of the target and form the duplex with the target sequence when present. Both the cyclicons are useful for nucleic acid detection. The studies with DNA poly merase on 5'-5'-attached cyclicons suggest that the presence of quencher mo iety in the probe sequence does not inhibit chain elongation by polymerase. The experiments with a 5'-5'-attached cyclicon suggest the new design serv es as an efficient unimolecular primer-probe in real-time PCR experiments. (C) 2000 Elsevier Science Ltd. All rights reserved.