Characterization of a fluorescence-signaling and RNA-cleaving deoxyribozyme

DNA enzymes (deoxyribozymes or DNAzymes) are single-stranded DNA molecules with catalytic function. Previously, we isolated several RNA-cleaving deoxyribozymes with fluorescence-signaling properties. These special DNA molecules are capable of cleaving an RNA linkage embedded within a DNA sequence and flanked by a pair of deoxyribothymidines modified with a fluorophore (fluorescein) and a quencher (dabcyl). Here we report on the sequence truncation and secondary structure characterization of one such deoxyribozyme known as pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicates that pH6DZ1 has a four-way junction-like secondary structure comprising four short duplexes, three hairpin loops, and three inter-helical unpaired elements.