thiouridine and phosphoramidite

5-Carboxymethylaminomethyluridine (cmnm(5)U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm(5)s(2)U) are located at the wobble position in several cytosolic and mitochondrial tRNA sequences. In this paper, we report the first site-selected incorporation of cmnm(5)U and cmnm(5)s(2)U into RNA sequences by phosphoramidite chemistry on a CPG solid support. Trifluoroacetyl and 2-(trimethylsilyl)ethyl were selected for the protection of the amine and carboxyl functions, respectively.

Topics: Base Sequence; Glycine; Molecular Structure; Organophosphorus Compounds; RNA; Thiouridine

The synthesis of phosphoramidite of 5-fluoro-4-thio-2'-O-methyluridine is described. An appropriate set of protecting groups was optimized including the 4-thio function introduced via 4-triazolyl as the 4-(2-cyanoethyl)thio derivative, and the t-butyldimethyl silyl for 2' and 3' hydroxyl protection, enabling efficient synthesis of the phosphoramidite. These protecting groups prevented unwanted side reactions during oligonucleotide synthesis. The utility of the proposed synthetic route was proven by the preparation of several oligonucleotides via automated synthesis. Photochemical experiments confirmed the utility of the synthon.

Topics: Oligodeoxyribonucleotides; Oligonucleotides; Organophosphorus Compounds; Photoaffinity Labels; Thiouridine

The hammerhead ribozyme has been intensively studied for approximately 15 years, but its cleavage mechanism is not yet understood. Crystal structures reveal a Y-shaped molecule in which the cleavage site is not ideally aligned for an S(N)2 reaction and no RNA functional groups are positioned appropriately to perform the roles of acid and base or other functions in the catalysis. If the ribozyme folds to a more compact structure in the transition state, it probably does so only transiently. We have used photocrosslinking as a tool to trap hammerhead ribozyme-substrate complexes in various stages of folding. Results suggest that the two substrate residues flanking the cleavage site approach and stack upon two guanosines (G8 and G12) in domain 2, moving 10-15 A closer to domain 2 than they appear in the crystal structure. Most crosslinks obtained with the nucleotide analogues positioned in the ribozyme core are catalytically inactive; however, one cobalt(III) hexaammine-dependent crosslink of an unmodified ribozyme retains catalytic activity and confirms the close stacking of cleavage site residue C17 with nucleotide G8 in domain 2. These findings suggest that residues involved in the chemistry of hammerhead catalysis are likely located in that region containing G8 and G12.

Topics: Animals; Binding Sites; Catalysis; Cross-Linking Reagents; Deoxyguanosine; Guanine; Guanosine; Nucleic Acid Conformation; Organophosphorus Compounds; Photochemistry; Pyrimidines; RNA, Catalytic; Schistosoma mansoni; Sequence Analysis, RNA; Substrate Specificity; Thionucleosides; Thiouridine

Oligonucleotides containing 2-thiouridine (s2U) in place of uridine form stable RNA duplexes with complementary RNAs. Particularly, this modified nucleoside has proved to recognize highly selectively adenosine, the genuine partner, without formation of a mismatched base pair with the guanosine counterpart. In this paper, we describe new methods for the synthesis of 2-thiouridine and various 2'-O-alkyl-2-thiouridine derivatives. Oligoribonucleotides having these modified nucleoside derivatives were synthesized, and their hybridization and structural properties were studied in detail by the 1H NMR analysis of these modified nucleosides and Tm experiments of RNA duplexes with their complementary RNA strands.

Topics: Magnetic Resonance Spectroscopy; Nucleic Acid Conformation; Nucleic Acid Denaturation; Nucleic Acid Heteroduplexes; Nucleic Acid Hybridization; Oligonucleotides; Organophosphorus Compounds; RNA, Complementary; Temperature; Thiouridine; Uracil

The HIV transcription initiation complex involves a putative interaction between the primer tRNA anticodon and a conserved A-rich loop in the HIV genome. Surface plasmon resonance was used to demonstrate that the hypermodified nucleosides in the tRNA anticodon stem loop (ASL) stabilize RNA-RNA interactions in a model for the anticodon/A-loop complex. tRNA ASL hairpins with the modifications of Escherchia coli tRNALys and human tRNALys,3 each form stable complexes. Partially modified tRNA ASLs bind the A-loop hairpin with lesser affinity, and it was found that the modifications of the bacterial and mammalian tRNAs make distinct contributions toward stabilizing the RNA complex. One model for the anticodon/A-loop RNA complex that is consistent with the known modification effects on tRNA structure and function is that of complementary tRNAs, as seen for the published crystal structure of tRNAAsp.

Topics: Adenosine; Anticodon; HIV-1; Kinetics; Nucleic Acid Conformation; Nucleosides; Organophosphorus Compounds; RNA, Transfer, Lys; RNA, Viral; Surface Plasmon Resonance; Thiouridine