PMC

Denaturing PAGE analysis of direct strand scission upon photolysis of (A) 5’-³²P-3, (B) 5’-³²P-4, (C) 5’-³²P-7 under anaerobic conditions.

Formation of the C2’-radical (10) was expected to give rise to direct strand scission via elimination of the β-phosphate analogous to what was first proposed by von Sonntag for DNA cleavage from the respective C4’-radical., HPLC analysis of dinucleotide 11 () that was photolyzed under anaerobic conditions was consistent with this mechanism. The dinucleotide produced the independently synthesized, anticipated elimination product, 12, in 14% yield. How the respective cation radical in RNA () is ultimately converted into the fragment containing a 3’-phosphate terminus requires further investigation.

These observations and examination of molecular models (Spartan ’02) led us to propose that the major pathway for direct strand scission from 5,6-dihydrouridin-6-yl radical (1) in duplex RNA under anaerobic conditions involves C2’-hydrogen atom abstraction from the 5’-adjacent uridine (). This hypothesis was supported by photolysis of 5’-³²P-7 in which the 5’-adjacent uridine was deuterated at C2’. Formation of 1 in 5’-³²P-7 resulted in more than an 8-fold reduction in the absolute amount of direct strand scission at the 5’-adjacent C2’-deuterated uridine compared to what is produced in 5’-³²P-4. Although the level of strand scission at the nucleotide where 1 is generated increased in the deuterated substrate, the increase did not fully compensate for the diminution in strand scission at the 5’-adjacent nucleotide. Hence, we cannot rule out an increased contribution to the radical’s overall reactivity at a position within 1 or an adjacent nucleotide that does not result in direct strand scission (e.g. C1’) when the C2’-hydrogen of the 5’-adjacent uridine is deuterated.

Hydroxyl radical and hydrogen atom preferentially add to the C5-position of pyrimidines (). We independently generated the monomeric form of the C5-hydrogen atom adduct of uridine, 5,6-dihydrouridin-6-yl radical (1) via Norrish Type I photocleavage of the nucleoside 2. Oligonucleotides containing 2 were prepared via automated solid phase oligonucleotide synthesis. Direct strand break formation following photolysis (350 nm, 4 h) of 2 was examined in single and double stranded RNA under aerobic and anaerobic conditions. Direct strand scission under aerobic conditions was less than 1/7th the amount when 1 was produced in a duplex (4) in the absence of O₂. The photocleavage of monomeric 2 is independent of O₂, suggesting that the observed trend is indicative of the effect of O₂ on one or more steps in the mechanism for strand scission from 1.