Bridge PCR,An Easy Way for Concatemerizing DNA Tags / 中国生物工程杂志

In MAST (mRNA accessible site tagging),the DNA tags from synthesized library were employed for identifying mRNA accessible sites. A large number of tags were amplified and subcloned for sequencing to verify mRNA binding profiles. A PCR was designed by using one primer which bridges over the tag terminal sequences. In PCR reaction DNA tag fragments were concatemerized by a bridge primer in reaction cycles. The concatemerized tag fragments were subcloned and sequenced. Dozens of the concatemerized sequences contained thousands tags. The PCR was a simple,effective way which for sequencing tags in a high through put manner.

Design and Identification of Antisense Oligonucleotide Targeting to 16S rRNA of E.coli / 中国生物工程杂志

Targeting rRNA of bacteria is a new strategy for antibiotic agent development. The rRNA such as mRNA are naturally self-folded molecules which expose only limited accessible target-sites for binding. These accessible sites are pivotal for designing the effective antisense oligonucleotides, ribozymes, and DNAzymes. MAST, an RNA accessible site screening method, illustrated 6 accessible sites on 16S rRNA by immobilizing 16S rRNA and hybridizing with oligonucleotide library. 5 of the accessible sites were identified valid, and the antisense oligonucleotides targeted to which showed inhibition effectiveness on the proliferation. Among the 5 target sites, one showed the priority of accessibility. Ribozyme designed to this site showed obvious inhibition to the growth when induced expressing in the transfection E.coli.

Antisense Sites Screening of Fas Gene mRNA and Its Validation in vitro / 中国生物工程杂志

Three candidate antisense target sites of mouse Fas gene were screened by PARASS (poly-A anchored RNA accessible sites screening) technology. They were target at Fas gene 297nt-317nt, 618nt- 638nt and 662nt-682nt. Antisense oligos (A1, A2 and A3) and DNAzymes (D1, D2, and D3) for every target site were designed and synthesized. In vitro, the validation of the sites were judged by antisense oligos included RNase H splicing and the DNAzyme degradation. The results indicated that A1, A2 and A3 introduced RNase H degradation. DNAzymes D1, D2 and D3 cleaved Fas mRNA effectively. Neither degradation observed in antisense oligo RNase H group in non-target site (1211-1231nt) and 2 bases mismatched of A3, nor splicing occurred in DNzyme group in non-target site ( 1211-1231nt) and 2 bases mismatched of D3. Site 2 and 3 were at the same positions with those of ISIS Pharmaceuticals. The effective antisense oligos and DNAzymes for Fas gene could be used for the research subsequently.