Chemical ligation of DNA: the first non-enzymatic assembly of a biologically active gene

Abstract

An artificial gene comprising 183 base pairs has been assembled by template-directed condensation of 35- to 53-membered oligodeoxyribo nucleotides with cyanogen bromide as a condensing agent. The reaction is complete within several minutes at 0 degrees C in buffer. The resulting mini-gene was cloned and expressed in vivo and in vitro. We have also found that the polymerase inhibition technique (toe-printing) is a good way to ascertain that translation initiation complexes form in the case of single-stranded DNAs as well. Thus, along with the fully chemical assembly of synthetic genes, a rapid and sufficiently reliable method for determining their ribosome-binding properties was developed.

Chemistry on Nucleic Acid Templates

“…Several chemistries more distantly related to the NA backbone have been explored as potential ligation/polymerization reactions, including Nielsen and Orgels RNA-templated coupling of peptide nucleic acids (PNAs) [46], DNAtemplated peptide conjugation of Joyce and co-workers [45], DNA-templated phosphoramidate formation of von Kiedrowski and co-workers [44], and reductive aminations of Lynn and co-workers [9] [51]. Shabarova et al used cyanogen bromide in the presence of metal ions as condensating agent to synthesize a t-RNA gene by chemical ligation of mutually annealed fragments [20]. Lynn and co-workers optimized this ligation method by covalently linking imidazole and other amines to the template strand opposite the ligation site [21].…”

“…The quest for suited NA-detection chemistries has led to the emergence of numerous templated reactions, contributing significantly to todays knowledge NA-templated chemistry (Tables 1 and 2). Orgels replication systems and Shabarovas approach to synthesize a t-RNA gene, for instance, have provided first chemical means for a nonenzymatic phosphodiester formation [15] [20]. von Kiedrowskis studies on prebiotic replication pioneered catalytic systems performing multiple turnover [19], sometimes supported by thermal cycling [44].…”

Mapping of RNA–protein interactions

“…In the past, "toeprinting" has proven effective for studying the characterization of translation sites and mechanisms of prokaryotic and eukaryotic systems [41,42]. Several other studies were carried out with primer extension inhibition, specifically on ribosome entry site-mediated translation initiation complexes [43][44][45][46][47][48].…”

Enzyme‐Free Ligation of 5′‐Phosphorylated Oligodeoxynucleotides in a DNA Nanostructure

“…However, such chemical ligations are known to be rather slow and prone to give incomplete conversion . A condensing agent giving fast, but often incomplete reactions is cyanogen bromide (BrCN) . Unfortunately, the rapid release of HBr and the potential for side reactions limit its use in large multistrand assemblies .…”