Site-directed modification of DNA duplexes by chemical ligation

Abstract

The efficiency of chemical ligation method have been demonstrated by assembling a number of DNA duplexes with modified sugar phosphate backbone. Condensation on a tetradecanucleotide template of hexa(penta)- and undecanucleotides differing only in the terminal nucleoside residue have been performed using water-soluble carbodiimide as a condensing agent. As was shown by comparing the efficiency of chemical ligation of single-strand breaks in those duplexes, the reaction rate rises 70 or 45 times if the 3'-OH group is substituted with an amino or phosphate group (the yield of products with a phosphoramidate or pyrophosphate bond is 96-100% in 6 d). Changes in the conformation of reacting groups caused by mismatched base pairs (A.A, A.C) as well as the hybrid rU.dA pair or an unpaired base make the template-directed condensation less effective. The thermal stability of DNA duplexes was assayed before and after the chemical ligation. Among all of the modified duplexes, only the duplex containing 3'-rU in the nick was found to be a substrate of T4 DNA ligase.

Preparation of modified long-mer RNAs and analysis of FMN binding to theypaAaptamer fromB. subtilis

“…In addition to enzymatic protocols for ligation of chemically synthesized fragments, chemical protocols were developed, [40][41][42][43] whereby the cupper-catalyzed azide-alkyne coupling (Click ligation) 26,29,34,44 stands out as most promising. A drawback of Click ligation is the need for terminal functionalization of fragments to be ligated (5′-azide and 3′-alkyne or vice versa), and the generation of a triazole linkage in the nucleic acid backbone.…”

New, stronger nucleophiles for nucleic acid-templated chemistry: Synthesis and application in fluorescence detection of cellular RNA

“…Addition of 3-mercaptopropionitrile and DBU resulted in complete conversion to a new product in less than one minute, which was confirmed as the cyanoethyl-protected phosphorotrithioate by mass spectrometry. Treatment of this intermediate with concentrated NH 4 OH at 60 °C resulted in the formation of a new product with an HPLC retention time consistent with the phosphorotrithioate. Mass spectral analysis revealed that the desired dT-PS3 product had been formed.…”

“…Treatment with the base DBU in the presence of 3-hydroxypropionitrile or 3-mercaptopropionitrile should provide the 3′-terminal cyanoethyl protected PS2 or PS3 groups, respectively. Normal cleavage and deprotection conditions using NH 4 OH should then yield the 3′-PS2 or PS3 modified oligonucleotides.…”

Origin of Biological Nucleic Acids and the First Genetic System (The Progene Hypothesis)

“…The complementary interaction influences positively for formation of phosphodiester bond between the dinucleotide and the aminoacyl nucleotide. Activation of the 3'hydroxyl substantially increases the probability of the phosphodiester bond formation in comparison with 5' hydroxyl activation [19,20]. The important role of a template for phosphodiester bond formation was shown by Lesli Orgel and his coworkers [21][22][23][24].…”