No abstract

The helix 69 (H69) region of the large subunit (28S) rRNA of Homo sapiens contains five pseudouridine (C) residues out of 19 total nucleotides (26%), three of which are universally or highly conserved. In this study, the effects of this abundant modified nucleotide on the structure and stability of H69 were compared with those of uridine. The role of a loop nucleotide substitution from A in bacteria (position 1918 in Escherichia coli 23S rRNA) to G in eukaryotes (position in 3734 in H. sapiens) was also examined. The thermodynamic parameters were obtained through UV melting studies, and differences in the modified and unmodified RNA structures were examined by 1 H NMR and circular dichroism spectroscopy. In addition, a [1,3-15 N]C phosphoramidite was used to generate H69 analogs with site-specific 15 N labels. By using this approach, different C residues can be clearly distinguished from one another in 1 H NMR experiments. The effects of pseudouridine on H. sapiens H69 are consistent with previous studies on tRNA, rRNA, and snRNA models in which the nucleotide offers stabilization of duplex regions through CN1H-mediated hydrogen bonds. The overall secondary structure and base-pairing patterns of human H69 are similar to the bacterial RNA, consistent with the idea that ribosome structure and function are highly conserved. Nonetheless, pseudouridine-containing RNAs have subtle differences in their structures and stabilities compared to the corresponding uridine-containing analogs, suggesting possible roles for C such as maintaining translation fidelity.

The (1)H NMR spectra of RNAs representing E. coli 23S rRNA helix 69 with [1,3-(15)N]pseudouridine modification at specific sites reveal unique roles for pseudouridine in stabilizing base-stacking interactions in the hairpin loop region.

1585seen from Table VI, the values for the digested sera obtained by A1F MAS and the ISE show fairly good agreement, while the values for the digested sera are slightly larger than those for the nondigested sera obtained by AlF MAS. There may be a possibility that the differences of analytical results for nondigested and digested sera originate from contamination during the complicated procedures of dry ashing or from the existence of another form of fluorine; e.g., volatile organic fluorine compounds which may not be detected by AlF MAS.However, since A1F MAS has less chance of contamination because of its simplicity and rapidity, the present results may strongly support the existence of fluorine bound to protein, which was suggested by Taves (2, 3 ) .ACKNOWLEDGMENT T h e authors thank to S. Kamei for providing the blood serum samdes. We also exmess our thanks to S. Fuiiwara Taves, D. R. Nature (London) 1968, 220, 582-583. Taves, D. R. Talanta 1966, 75, 969-974.

We report the chemical synthesis and derivatization of an ortho‐functionalized tetrachlorinated azobenzene diol. A 4′,4‐dimethoxytrityl (DMT) phosphoramidite was synthesized for its site‐specific incorporation within the sense strand of an siRNA duplex to form ortho‐functionalized tetrachlorinated azobenzene‐containing siRNAs (Cl‐siRNAzos). Compared to a non‐halogenated azobenzene, ortho‐functionalized tetrachlorinated azobenzenes are capable of red‐shifting the π→π* transition from the ultraviolet (UV) portion of the electromagnetic spectrum into the visible range. Within this visible range, the azobenzene molecule can be reliably converted from trans to cis with red light (660 nm), and converted back to trans with violet wavelength light (410 nm) and/or thermal relaxation. We also report the gene‐silencing ability of these Cl‐siRNAzos in cell culture as well as their reversible control with visible light for up to 24 hours.