Bioinformatics designing of 10-23 deoxyribozyme against noncoding region before start codon of beta-galactosidase gene (lacZ) in pGEM-T vector

Deoxyribozymes are oligoribodeoxynucleotides that catalyze reactions such as cutting RNA and have diagnostic and therapeutic applications. Deoxyribozyme 10-23 includes a catalytic domain dependent on a fixed 15-nucleotic (mer) cation and two variable binding arms that cause the specificity of enzymes. Lactose operon is used in the white-blue screening process. This operon includes three polycistronic genes. In this study, a deoxyribozyme against α-peptide beta-galactosidase gene in the lactose operon was designed.
pGEM-T map was obtained from addgene server and α-peptide gene sequence was determined. Then, using expasy website proper protein frame in comparison with various reading frames was determined. In this step, whole sequence was reversed and mRNA sequence was achieved. Secondary structure with the lowest free energy was gained using mfold server. Considering the fact that 10-23 deoxyribozyme has cutting capability between a unpaired purine and pairs pyrimidine; an AC was selected in ribosome binding site in the untranslated region and then 9 open bases on either side of it was used as a binding arms. Investigation of the absence of similar sequences in host bacteria was performed by NCBI server. Finally, activity and binding of deoxyribozyme was predicted by the mfold server.
The results of this study showed that the designed deoxyribozyme had a relatively high Tm with two 9-nucleotide arms, which increased its effectiveness.
The results of this study can be used to control the expression of lacZ gene as a biomarker.