Study on the inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides

Because of the high efficiency and specificity of oligonucleotides (oligos) in inhibiting gene expression, antisense and antigene oligonucleotides have great promise as therapeutic agent. Antigene oligos have an advantage over antisense oligos in that inhibition of gene expression is more efficient and in long term. This study investigates the inhibition of the E. coli lac operon gene expression by antigene oligonucleotides both theoretically and experimentally.; Based on the molecular mechanism of lac operon transcription, a steady-state genetically structured model has been developed for the theoretical analysis of the effects of antigene oligonucletides on the lac operon gene expression. The potential inhibitory effect of antigene oligonucleotides targeted to different sites on the lac operon was evaluated. The steady-state model predicts that the operator will be the best target for antigene oligos. Antigene oligonucleotide targeted to the operator will have significant inhibitory effect on the lac operon gene expression, e.g. β-galactosidase production.; A 21-mer oligo was designed to target to the operator region. This 21-mer oligo, which is supposed to form a triplex in parallel orientation with the purine-rich strand of the operator, can specifically inhibit the β-galactosidase production in a dose-dependent manner without nonspecific inhibitory effect on cell growth. In contrast to the 21-mer oligo, several control oligos do not have any inhibitory effect. The ineffectiveness of the various control oligos, along with the fact that the 21-mer oligo has no homology sequence with the lacZYA, and no mRNA is transcribed from the operator, suggests that the 21-mer oligo inhibit target gene expression by an antigene mechanism.; The inhibitory effect of oligo can be observed within 10–15 minutes, showing that the diffusion of oligo into the cell and its hybridization to the target sequence occurs relatively fast. However, if oligo is introduced into the culture after induction, its inhibitory effect can be hardly observed. This suggests the oligo must occupy the operator site on the lac operon before transcription initiation to be able to inhibit target gene expression.; A comprehensive genetically structured kinetic model was developed to simulate the transient expression of the lac operon in the presence of antigene oligonucleotides. Predictions of the kinetic model can fit the experimental data quite well by adjusting the value of oligonucleotide transport rate (k_{in_oligo}) and binding affinity constant (K _F1) to 9.0 × 10−3 (min−1) and 1.05 × 106 (M−1) respectively, which are in the range of reported literature values.