Pilot Study Of Nucleic Acid Antibiotics Targeted To Bacterial 16S RRNA

Objectives: The development of antisense technique provides a new approach to study the new generation of antibiotics. In our study, we chose E.coli 16S rRNA as the drug target gene, and used the MAST technique, a RNA accessible site screening method we had established to select accessible sites. Afterwards we verified the in vitro and in vivo effects of the selected accessible sites in order to obtain effective site sequences for the binding of antisense oligonucleotides. The designed antisense phosphorothioate oligonucleotides according to the effective site were tested to inhibit the E.coli growth for the further development of nucleic acid antibiotics.Methods: 16S rRNA was produced by in vitro transcription reactions according to the normal procedures except that all transcription reactions were supplemented with biotin-UTP, and immobilized to streptoavidin labeled magnetic beads. By this means, the immobilized 16S rRNA could be kept its natural state best. Afterwards the immobilized 16S rRNA was hybridized to the oligonucleotide library flanked with two primer sequences to screen accessible sites. This method was called the MAST technique (mRNA accessible site tagging). Six accessible sites of 16S rRNA were obtained. Then six antisense oligonucleotides and five 10-23 DNAenzymes targeted to these six sites were designed and synthesized individually. The in vitro effects of the six antisense oligonucleotides were identified by RNase H dependent analysis, and the in vitro catalytic effects of DNAenzymes were identified by interaction of DNAenzymes with 16S rRNA. The results showed that five of these six accessible sites were effective and the accessible site V (907-926nt) was the best. In order to test the in vivo effect of the...