Team:REC-CHENNAI/Project-Inspiration
PROJECT INSPIRATION
The interactions with a distinguished scientist Dr.Srujan Marepally from Centre for Stem Cell Research, Vellore and Dr.P.Rajasekar, an eminent professor of our college as part of our Integrated Human Practices were a source of inspiration for us to deliver the cargo in an unconjugated form with the CPP to increase the efficiency of delivery. This non-covalent strategy of delivery has been advocated in previous studies involving the delivery of proteins [1] and nucleic acid in plant cells via the CPP BP100-(KH)9. [2, 3] The advantages of this strategy are ease of use, the versatility of cargo delivered and preservation of the biological activity of the cargo.
Chang et. al. have synthesized a novel siRNA based probe utilizing two short 21-nt RNA complementary strands as the stem and highly flexible poly(ethylene glycol) (PEG) molecule covalently linked to the RNA strands as the loop. This inspired us to design an shRNA-like siRNA.
In order to leverage RNAi towards diagnostic and monitoring applications, chang et. al. utilized molecular beacons (MB) consisting of a donor and an acceptor fluorophore. A commonly used FRET fluorophore pair, Cy3 and Cy5 fluorophores, was conjugated to the 3´terminus of the antisense and 5´ terminus of the sense strand, respectively. [4]
DNA molecular beacons are specifically designed hairpin-like structures of DNA that are used as fluorescent probes. The molecular beacons are in the form of stem-loop structure. The loop region carries the sequence complementary to the target gene. This is flanked by two short oligonucleotide sequences that are self-complementary and form the stem portion. The molecular beacon’s stem-loop structure holds the fluorescence-donor (fluorophore) and the fluorescence-acceptor (quencher) in close proximity so that no signal is passed due to fluorescence resonance energy transfer (FRET). The spatial separation of the fluorophore and the quencher due to intermolecular hybridization results in the restoration of fluorescence. [5]
Inspired by the DNA molecular beacons, we designed siRNA molecular beacons i.e. siRNA conjugated with a fluorophore and a quencher that provides a novel approach to the preliminary screening of RNAi in living cells without the need for prior separation of unbound probes.
