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Volume 3
Journal of Pharmacology and Medicinal Chemistry
Nanomedicine 2019
Biotechnology 2019
May 20-21, 2019
May 20-21, 2019 London, UK
4
th
World Biotechnology CONGRESS
Nanomedicine and Nanotechnology
4
th
International Conference on
&
Artificial RNA editing in mutated BFP (derivative of GFP) by using AID deaminase for
restoration of genetic code
Sonali Bhakta
Japan Advanced Institute of Science and Technology (JAIST), Japan
E
diting of mutated gene can be a possible means of treatment for genetic diseases caused by mutations in the normal codons.
Here, I have tried to engineer deaminase domain of AID (Activated Induced Deaminase, a family member of APOBEC)
and MS2 system to target specific Cytosine (C) to restore Thymine (T) that has been caused by the T to C mutations. For this
catalytic domain of AID deaminase has been fused with RNA binding protein MS2, which binds to MS2-RNA. Guide RNA was
designed complementary to target RNAs. Thus AID deaminase domain was carried out to desired editing site to convert C to
U. As a target, Blue Fluorescence Protein (BFP) gene was prepared by mutating at 199 nucleotide of GFP. MS2 system has the
ability to Convert Mutated Codon (CCA) to normal codon (CTA) in cellular system (e.g., HEK 293). The system converted CCA
to CTA (conversion of cytidine to uridine transforms the BFP gene into the GFP gene) and turned on green fluorescence. cDNA
was synthesized from positive cells followed by RNA extraction and PCR-RFLP was done by using BtgI restriction enzyme. The
unedited BFP gave the fluorescence in the microscopic observation, remained uncut and edited were cut, also provide the green
fluorescence expression by microscopic observation. Final confirmation was done by the Sanger’s sequencing analysis where the
restored one also gave the peak as the wild type “CTACGG” which was “CCACGG” in case of the mutated one (BFP).
Successful artificial editing of RNA in vivo by MS2 system can pioneer genetic code restoration therapy including stop-codon read
through therapy for various genetic diseases.
Key words: RNA editing. AID, BFP, Guide RNA, Genetic code.
sonali.dvm@gmail.comJ Pharmacol Med Cheml, Volume 3