An Experimental and Theoretical Study of the Inhibition of Escherichia coli lac Operon Gene Expression by Antigene Oligonucleotides Biao Cheng, Ronald L. Fournier, Patricia A. Relue, Jonathan Schisler Department of Bioengineering, University of Toledo, Toledo, Ohio 43606, USA; telephone: 419-530-8167; fax: 419-530-8076; e-mail: rfournie@eng.utoledo.edu Received 21 September 2000; accepted 18 February 2001 Abstract: Previously, we have developed a genetically structured mathematical model to describe the inhibition of Escherichia coli lac operon gene expression by anti- gene oligos. Our model predicted that antigene oligos targeted to the operator region of the lac operon would have a significant inhibitory effect on -galactosidase production. In this investigation, the E. coli lac operon gene expression in the presence of antigene oligos was studied experimentally. A 21-mer oligo, which was de- signed to form a triplex with the operator, was found to be able to specifically inhibit -galactosidase production in a dose-dependent manner. In contrast to the 21-mer triplex-forming oligonucleotide (TFO), several control oli- gos showed no inhibitory effect. The ineffectiveness of the various control oligos, along with the fact that the 21-mer oligo has no homology sequence with lacZYA, and no mRNA is transcribed from the operator, suggests that the 21-mer oligo inhibits target gene expression by an antigene mechanism. To simulate the kinetics of lac operon gene expression in the presence of antigene oli- gos, a genetically structured kinetic model, which in- cludes transport of oligo into the cell, growth of bacteria cells, and lac operon gene expression, was developed. Predictions of the kinetic model fit the experimental data quite well after adjustment of the value of the oligo- nucleotide transport rate constant (9.0 × 10 -3 min -1 ) and oligo binding affinity constant (1.05 × 10 6 M -1 ). Our val- ues for these two adjusted parameters are in the range of reported literature values. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 74: 220–229, 2001. Keywords: Escherichia coli lac operon; antigene oligo- nucleotide; triplex-forming oligonucleotide (TFO); ge- netically structured model INTRODUCTION Oligonucleotides (oligos) can bind with high affinity to spe- cific regions on DNA or RNA, and therefore inhibit gene expression (He ´le `ne, 1991; He ´le `ne and Toulme ´, 1990) at the level of transcription (antigene oligo) or translation (anti- sense oligo). The ability of oligonucleotides to inhibit target gene expression with high efficiency and high specificity is of great interest, and it has potential applications in phar- maceutical design (Cohen and Hogan, 1994; Maher, 1996; Stein and Cheng, 1993). Because many copies of mRNA can be produced from one DNA template by transcription, intervention of gene expression at the transcription level should be much more efficient than at the translation level (Uhlmann and Peyman, 1995). Therefore, the antigene oligo approach is of special interest in our study, and we have selected the Escherichia coli lac operon as the model system. We previously have developed a steady-state model (Cheng et al., 2000) to de- scribe quantitatively the mechanism of antigene oligonucle- otides. Using our steady-state model, a priori predictions of the inhibition of E. coli lac operon gene transcription by antigene oligonucleotides were made. The model predicts that antigene oligonucleotides targeted to the operator re- gion of the lac operon will have a significant inhibitory effect on the lac operon gene expression. As a follow-up to our previous theoretical study, we now report the experimental results of E. coli lac operon gene expression in the presence of antigene oligonucleotides. In addition, a comprehensive, genetically structured kinetic model has been developed to describe the transient expres- sion of the E. coli lac operon in the presence of antigene oligonucleotides. MATERIALS AND METHODS Materials and Equipment E. coli strain W3110 was kindly provided by Dr. J. Keasling (University of California, Berkeley, CA). IPTG, ONPG, and -galactosidase standard were obtained from Sigma Chemi- cal Co. (St. Louis, MO). Chemicals and accessories for DNA synthesis were obtained from PerSeptive Biosystems (Framingham, MA). Design of Oligonucleotide Sequences Because our steady-state model predicted that oligos tar- geted to the operator would have significant inhibitory ef- Correspondence to: R. L. Fournier © 2001 John Wiley & Sons, Inc.