Phosphorothioate substrates for T4 RNA ligase

Stereochemical details of the T4 RNA ligase reaction mechanism have been delineated by examining the reactivity of phosphorothioate analogues in the adenosine 5'-triphosphate (ATP) dependent and ATP-independent RNA ligase reactions. Only the SP isomer of the diastereomeric dinucleoside thiopyrophosphate, App(s)I, was active as an activated donor substrate in the ATP-independent RNA ligase reaction. The thiophosphodiester linkage in the ligation product, ApApAp(s)I, that is formed by the reaction of App(s)I (SP) with the oligonucleotide acceptor, ApApA, was shown to have the RP configuration. This indicates that phosphodiester bond formation occurs by a direct, nucleophilic displacement of AMP from App(s)I by the 3'-hydroxyl group of ApApA with inversion of configuration at phosphorus. The adenylylated intermediate, App(s)Ap, that is formed from the phosphorothioate donor, p(s)Ap, in the ATP-dependent RNA ligase reaction was shown to have the same stereochemical configuration as is required for the ATP-independent RNA ligase reaction. These results indicate that RNA ligase maintains a preferred chirality at phosphorus through the adenylylation and ligation steps of the reaction mechanism. An unusual result is the accumulation of adenosine cyclic 2',3'-phosphate 5'-phosphorothioate in the ATP-dependent RNA ligase reaction employing the donor p(s)Ap when the acceptor ApApA is present. This observation suggests that there are two distinct but reactive modes for donor molecules.