Abstract

Non-coding RNAs or ncRNAs are RNA molecules that are not translated but play functional roles within cells. Some of these ncRNAs possess enzymatic properties. These molecules are termed as ribozymes. Ribozymes mainly catalyze nucleic acid strand scission reactions with or without the help of protein molecules. Ribozymes such as hammerhead ribozymes (HHRs) are known to mediate gene silencing and RNA processing. Single stranded RNA/DNA (ssRNA/DNA) inducible HHRs or tetracycline inducible aptazymes exist. Using these HHRs, different types of logic gates can be designed, activated by one or more inputs including ssDNA and ssRNA. Evaluating HHR kinetics of cleavage is essential to understand their mechanism, characterize HHR mutants and to properly estimate several parameters important to design RNA-based logic circuits.
Firstly, we developed a novel methodology to detect HHR kinetics using toehold mediated strand displacement reaction (TMSDR). A probe composed of a fluorophore and a quencher was designed to measure the kinetics of HHR cleavage reactions without labelling RNA molecules, regular sampling or the utilization of polyacrylamide gels. This probe consists of two DNA strands; one strand labelled with a fluorophore at its 5′ end, while the other strand labelled with a quencher at its 3′ end. These two DNA strands are complementary, but the fluorophore strand is longer than the quencher strand at its 3′. The unpaired extra nucleotides act as toehold, which is utilized by a detached cleaved fragment, coming from a self-cleaving hammerhead ribozyme, as the starting point for the strand displacement reaction. This reaction will cause the separation of the fluorophore strand from the quencher strand, culminating in fluorescence detectable in a plate reader. This fluorescence is proportional to the amount of detached cleaved-off RNA strand displacing the DNA quencher strand. This method can be used to replace radio-hazardous unstable 32P as a means of measurement of the kinetics of ribozyme cleavage reactions; it also eliminates the need for use of polyacrylamide gels for the same purpose. Critically, this method allows experimenters to distinguish between the amount of cleaved ribozyme and the amount of detached cleaved-off fragments, resulting from the cleavage.
Secondly, we developed doubler HHRs that cleave twice upon induction with a single input strand (ssDNA/ssRNA). Outputs can be heterogeneous (Hetero doubler) or identical (Homo doubler). Homo doublers were designed to work as amplifying components in RNA amplifiers. We showed two potential doubler HHRs from two different designs (First doubler and D1 doubler). In conclusion, we found that the concentration of detached cleaved-off fragments is relatively low and hence we developed homo-doublers to increase the concentration of cleaved-off fragments.