Comparative Analysis of Inhibition Efficiency of Chimeric Antisense Oligonucleotides with Various Chemical Modifications and Their Application for Exogenous Control of Gene Expression in Escherichia coli

Background: Antisense oligonucleotides (ASOs) are synthetic nucleic acid analogs that can suppress gene expression through sequence-specific hybridization with target RNA. Enzymatic degradation, low bioavailability, and limited cellular uptake constrain their applications. Chemical modifications to the backbone and sugar moieties can enhance nuclease resistance, improve hybridization affinity, and extend in vivo half-life. This study compared the effects of structural and chemical modifications on the inhibitory activity of cell-penetrating peptide-conjugated ASOs in a bacterial reporter system. Methods: Seven ASO variants were evaluated, encompassing nucleotide deletions and additions, locked nucleic acid residues, 2′-O-methyl modifications, thiophosphoramidate morpholino (TMO) chemistry, and phosphorothioate (PS) linkages. We assessed inhibitory activity in Escherichia coli using a lacZ reporter assay. We quantified their effects by measuring residual β-galactosidase activity, providing a functional readout of reporter inhibition across the seven designs. Results: The TMO-modified ASO produced the lowest residual reporter activity among the tested designs, corresponding to 97% inhibition at 120 μg/mL. The PS-modified ASO achieved 92% inhibition at the same concentration. Both designs substantially reduced reporter activity under the tested experimental conditions, with the TMO-modified variant showing the greatest observed inhibition. Conclusions: Structural and chemical modifications influenced the inhibitory activity of peptide-conjugated ASOs in the E. coli lacZ reporter system under the conditions examined. The results support further evaluation of TMO-containing designs for bacterial reporter-gene suppression. Chemical optimization may help develop ASO-based tools for external control of gene expression, with potential applications in synthetic biology, biotechnology, and metabolic engineering.