The dog-bone probe is a 100 nt strand of circular DNA that has a secondary structure containing two 36 single-stranded nucleotide loops separated by a 14 nucleotide double-stranded stem. It is very stable in its “locked” form (-G<10 kcal/mol) (NUPACK).
When the miRNA is in proximity of the probe it will bind to a 9 nucleotide complementary “toehold” section of the dog bone.. The stem and the unbound miRNA part are complementary. Once the miRNA binds to the toehold section of the dog bone, the rest of the miRNA will break open the stem through a strand displacement mechanism [1], turning the dog bone into an open single-stranded loop with one section of double stranded DNA-RNA hybrid.
One benefit of a dog-bone probe is that it does not need a ligation step during the assay compared to other RCA methods for detecting oligonucleotides. Thus, our detection system does not have to deal with ligation inefficiencies which will affect the sensitivity of our system. In addition, the toe-hold initiated attachment of miRNA to DNA and subsequent strand displacement are highly specific to the miRNA, making this probe robust and resistant to false positives [2].
Sources:
1. Toehold-initiated rolling circle amplification for visualizing individual microRNAs in situ in single cells.
2. A new class of homogeneous nucleic acid probes based on specific displacement hybridization.