Enzymatic Synthesis Of Modified Oligonucleotides By PEAR And Primary Study Of Reading Frame Restoration Of Frameshift Mutation In E.Coli

Synthetic oligonucleotides have been widely used as an important tool for gene regulating, and have been developed into therapeutic drugs, such as antisense oligonucleotides (ASONs), CpG-containing oligonucleotides (CpG-ONs) and nucleic acid aptamer. Large-scale production of oligonucleotides is extremely difficult and costly, because it requires expensive equipment and tedious purification process, and the products is usually contaminated with highly homologous failure sequences. We previously reported polymerase-endonuclease amplification reaction (PEAR) for amplification of natural and 5’-O-(1-thiotriphosphate) (S)-modified oligonucleotides. Here, we extended the PEAR technique for enzymatic preparation of 2’-Deoxy-2’-fluoro-(2’-F) and 2’-F/S double modified oligonucleotides. The result showed that KOD and Phusion DNA polymerase could synthesize oligonucleotides with one or two modified nucleotides, and KOD DNA polymerase is more suitable than Phusion DNA polymerase for PEAR amplification of 2’-F and 2’-F/S double modified oligonucleotides. The composition of PEAR products were analyzed by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection and showed that the sequence of the PEAR products are maintained at an extremely high accuracy, and after digestion the area percent of full-length modified oligonucleotides reaches 89.2%. PEAR is suitable for synthesis of modified oligonucleotides efficiently and with high purity.As a common phenomenon in vivo, homologous recombination not only play an important role in biological evolution, but has the potential to transform specific gene. It is reported that microorganisms have the ability to take up foreign single-stranded DNA and insert them into homologous region of genome resulting site-specific mutation. Based on this, we anticipate that double-stranded oligonucleotides or modified double-stranded oligonucleotieds may have the ability to incerase recombinant frequency. First of all, we took the plasmid pBR322 as research object, designed a point deletion in β-lactamase gene of this plasmid. Under the effect of synthetic wild-type single-stranded oligonucleotides, this frameshift mutation site was repaired, but double-stranded oligonucleotides does not have the same effect. We also found that, the reading frame of the frameshift mutation had the ability to restore spontaneously without the treatment of synthetic oligonucleotides. Sub-culturing of the revertants showed that the growth rate increases with the increase of as generations, and the reading frame restoration is a gradualgradual process. Sanger sequencing showed that, under the pressure of antibiotic, one nucleotide is inserted into DNA duplex at some random sites near the deletion site.