Facile synthesis of nucleoside 5'-(α-P-seleno)-triphosphates and phosphoroselenoate RNA transcription

Abstract

Phosphoroselenoate RNA (PSe-RNA) is nuclease resistant and has great potentials in X-ray crystal structure and function studies of noncoding RNAs and protein-RNA interactions. In order to conveniently synthesize PSe-RNA via transcription, we have developed a one-pot synthetic method for the nucleoside 5'-(α-P-seleno)-triphosphates (NTPαSe) analogs without protecting any functionality of the ribonucleosides. The NTPαSe diastereomers have been purified, fully characterized, and incorporated into RNAs by T7 RNA polymerase. The transcribed RNAs are diastereomerically pure, and the Se-derivatized ribozymes are generally active. Furthermore, we have established an affinity purification strategy by using immobilized boronate to conveniently purify NTPαSe analogs. Though the affinity-purified NTPαSe analogs are diastereomeric mixtures, they can be directly used in transcription without a significant impact on the transcription efficiency. Moreover, we found that the PSe-nucleotide is stable during polyacrylamide gel purification, indicating that the PSe-RNAs can be purified straightforwardly for crystal structural and functional studies.

FIGURE 1.

Chemical structures of the Se-derivatized nucleotides. (A) Nucleoside 5′-(α-P-seleno)triphosphates (NTPαSe, Sp and Rp). (B) Phosphoroselenoate RNA (PSe-RNA, Rp).

FIGURE 2.

Facile synthesis of the NTPαSe analogs. (DMF) Dimethylformamide; (TBA) tributylamine.

FIGURE 3.

Enzymatic incorporation of NTPαSe analogs purified by HPLC to (hammerhead mutant) HHM RNA. Minor cleavage (the small faster-moving bands) was observed with the native, Se-C and Se-U ribozymes. NTP (all native nucleoside triphosphates), A₁ (ATPαSe I), A₂ (ATPαSe II), A_1&2 (I and II; I vs II = 1:1); C₁ (CTPαSe I), C₂ (CTPαSe II), C_1&2 (I and II; I vs II = 1:1); G₁ (GTPαSe I), G₂ (GTPαSe II), G_1&2 (I and II; I vs II = 1:1); U₁ (UTPαSe I), U₂ (UTPαSe II), U_1&2 (I and II; I vs II = 1:1).

FIGURE 4.

HHM RNAs transcribed with the NTPαSe analogs (diastereomer mixture) purified by boronate column. The shorter fragments are self-cleaved products.

FIGURE 5.

MALDI-TOF MS analysis of transcribed RNAs. (A) Native RNA42.1 (22 nt), molecular formula: C₂₀₉H₂₆₁N₈₂O₁₆₅P₂₄; observed [M+H]⁺ = 7305 (calcd. [M+H]⁺ = 7303). PSe-U RNA42.1, molecular formula: C₂₀₉H₂₆₁N₈₂O₁₅₉P₂₄Se₆; observed [M+H]⁺ = 7687 (calcd. [M+H]⁺ = 7686). Their mass difference of 382 (7687 − 7305 = 382) reflects incorporation of six Se-modified U units (6Se-6O = 6 × 80 − 6 × 16 = 384). (B) PSe-U HHM, molecular formula: C₆₅₇H₈₁₄N₂₆₄O₄₆₅P₆₈Se₁₅ (calcd [M] = 23,144); the matrix (2′,4′,6′-trihydroxyacetophenone, THAP), molecular formula: C₈H₈O₄, molecular weight: 168.0; observed mass of [M + THAP]: 23,314 (calcd. 23,312).

FIGURE 6.

Secondary structure of wild-type hammerhead ribozyme (HHN) derived from the satellite RNA of tobacco ringspot virus. Conserved bases are labeled in gray. The mutant hammerhead ribozyme (HHM) has a G→A mutation.

FIGURE 7.

Transcription of the hammerhead ribozymes. (M) Mutant HHM; (N) wild-type HHN; (NTP) all native NTPs; (A) ATPαSe I and other natives; (C) CTPαSe I and other natives; (G) GTPαSe I and other natives; (U) UTPαSe I and other natives. One band from each cleavage is shown here, since the other band (12 nt) ran off the gel.