ABSTRACT
The most widely used technique for the production of DNA aptamers/oligonucleotides is chemical synthesis. Despite its effectiveness, this technique cannot be performed "in house", making the user fully dependent on a supplier. In this work, we present a simplified method by which it is possible to enzymatically produce DNA aptamers "in house". This new method uses the rolling circle replication followed by a unique cleavage step using the SchI endonuclease. Potentially, any oligonucleotide can be produced by the enzymatic method proposed in this study. To illustrate, we present the production of three variations of the 31-TBA aptamer, a single stranded DNA which has anticoagulant action.
Subject(s)
Aptamers, Nucleotide/biosynthesis , DNA, Single-Stranded/biosynthesis , Nucleic Acid Amplification Techniques , Oligodeoxyribonucleotides/biosynthesis , Anticoagulants/chemical synthesis , Anticoagulants/metabolism , Aptamers, Nucleotide/genetics , Base Sequence , DNA Restriction Enzymes/metabolism , DNA, Single-Stranded/genetics , G-Quadruplexes , Humans , Oligodeoxyribonucleotides/geneticsABSTRACT
Growth of fastidious adenovirus serotype 40 (Ad40) in several cell lines was investigated. Ad40 was able to readily propagate in human intestinal cell line, HRT 18. Coinfection assays were made in non-permissive and permissive cells between Ad40 and Ad5dl312 or dl1520, mutants deleted in E1A and E1B regions, respectively, to test the ability of Ad40 to complement these mutants and vice versa. Ad40 could enhance Ad5dl312 DNA synthesis in HRT18 and HeLa cells, although its own DNA disappeared in the presence of this mutant in HRT18 cells. In coinfection with dl1520, Ad40 DNA synthesis was inhibited by dl1520 in HRT18 cells and dl1520 DNA synthesis was inhibited by Ad40 in 293 cells. This might reflect the presence of unusual products encoded by Ad40 E1B region.