Enzymic oligoribonucleotide synthesis with T4 RNA ligase

Abstract

The substrate specificity of T4 RNA ligase has been examined to determine whether the intermolecular reaction is sufficiently general to realize its potential in the enzymatic synthesis of oligoribonucleotides of defined sequence. Reactions between a variety of acceptor molecules with 3'- and 5'-bydroxyl groups and donor molecules with 3'- and 5'-phosphates indicate that the minimal substrates are a trinucleoside diphosphate acceptor and a nucleoside 3',5'-bisphosphate donor. Increasing the chain length of either the acceptor or donnor has little effect on the rate or extent of reaction. Although the base composition of the donor has only a small effect on the reaction rate, the presence of uridine in the acceptor greatly reduces the amount of product formed. The presence of a phosphate on the 3' terminus of the donor molecule permits a unique intermolecular product with a 5'-hydroxyl and a 3'-phosphate. By enzymatically either adding a 5'-phosphate or removing the 3'-phosphate, a new donor or acceptor is prepared so synthesis of an oligomer chain can proceed in either direction. With the simplicity of this enzymatic pathway and the rather broad substrate specificity of T4 RNA ligase, a convenient method for the synthesis of oligoribonucleotides is established.

Cleavage of tRNA with imidazole and spermine imidazole constructs: a new approach for probing RNA structure

“…All other products were of the highest quality available. (27). After is unfolded (lane 2) and that the cleavage occurs essentially non-randomly when the reaction is performed in conditions stabilising the RNA structure, i.e.…”

In vitro circularization of RNA

“…This is A > G C > U for the 3 0 -terminal nucleotide of the acceptor strand, and pC > pU > pA > pG for the 5 0 -terminal nucleotide of the donor strand. [47][48][49][50] T4 Rnl1 assisted RNA ligation has been used to produce large amounts of homogeneous and pure circRNA. 51 A key element of these syntheses was the exploitation of the RNA intrinsic secondary structure for orientation of the 5 0 -and 3 0 -termini for efficient ligation.…”

Computational analysis of fitness landscapes and evolutionary networks from in vitro evolution experiments

“…The efficiency of ligating adapters to RNA or DNA sequences depends on the nucleotides at the terminal regions of those sequences. For instance, England and Uhlenbeck [19] and Middleton et al [20], showed that sequences containing , and at the end, and , and at the end, were observed more frequently after ligation, while sequences with at the end and at the end were observed less frequently. This effect can be corrected for during computational analysis (see following section).…”