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 S_P 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 (S_P) with the oligonucleotide acceptor, ApApA, was shown to have the R_P 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.