The nonamer r(GCUUCGGC)dBrU, where dBrU is 5-bromo-2'-deoxyuridine, contains the tetraloop sequence UUCG. It crystallizes in the presence of Rh(NH3)6CI3. In solution the oligomer is expected to form a hairpin loop but the x-ray structure analysis, to a resolution of 1.6 A, indicates an eight-base-pair A-RNA duplex containing a central block of two GNU and two C U pairs. Self-pairs which approximate to Watson-Crick geometry are also formed in the extended crystal structure between symmetry-related BrU residues and are part of infinite double-helical stacks. The G U pair is a wobble base pair analogous to the GOT pair found in DNA fragments. The CU mismatch involves one hydrogen-bonded contact between the bases and a bridging water molecule which ensures a good fit ofthe base pair in the RNA helix. The BrU BrU pair is held by two hydrogen bonds in an orientation which is compatible with duplex geometry. The structure observed within the crystal has some parallels with the structure of globular RNAs, and the presence of stable, noncanonical base pairs has implications for the prediction of RNA secondary structure.Tertiary structure is essential to the biological function of many single-stranded RNAs. Base pairing between complementary segments in a sequence generates a series of doublehelical stems that connect unpaired regions or loops. These can fold further into compact assemblies stabilized by additional hydrogen bonds and by hydrophobic and electrostatic interactions (1). The various levels of structural order are most completely characterized in tRNAs, for which a number of crystal structures have been determined (2-4). In the case of larger and more complex RNA species, including ribosomal components and RNase P, present understanding of secondary structure relies heavily on assumptions about the relative stabilities of possible base-pairing arrangements (5). These follow the specificities of adenine for uracil and guanine for cytosine observed in both ribonucleotides and deoxyribonucleotides during replication and transcription and are also mirrored in the experimentally measured stabilities of short oligonucleotide duplexes. Although the double helix is a central feature of these systems, none is a conspicuously direct model of the environment within a globular RNA structure.The present analysis of an RNA double helix is the second to be reported for an oligonucleotide containing a "tetraloop" sequence. Tetraloops are common in natural singlestranded RNA and, although the stems of such structural elements are variable, the unpaired regions are found to be tetranucleotides of mainly two types: a group with the general sequence r(GNRA) and the specific r(UUCG). Examples of both classes have been examined extensively in solution, where they exist as monomeric, thermally stable, looped species which parallel the behavior of the parent sequences in RNA of higher molecular weight (6,7).In crystals of both the present sequence and a dodecamer studied by Holbrook et al. (8), where r(UUCG) is embedded in se...

Influenza virus causes seasonal epidemics and dangerous pandemic outbreaks. It is a single stranded (−)RNA virus with a segmented genome. Eight segments of genomic viral RNA (vRNA) form the virion, which are then transcribed and replicated in host cells. The secondary structure of vRNA is an important regulator of virus biology and can be a target for finding new therapeutics. In this paper, the secondary structure of segment 5 vRNA is determined based on chemical mapping data, free energy minimization and structure-sequence conservation analysis for type A influenza. The revealed secondary structure has circular folding with a previously reported panhandle motif and distinct novel domains. Conservations of base pairs is 87% on average with many structural motifs that are highly conserved. Isoenergetic microarray mapping was used to additionally validate secondary structure and to discover regions that easy bind short oligonucleotides. Antisense oligonucleotides, which were designed based on modeled secondary structure and microarray mapping, inhibit influenza A virus proliferation in MDCK cells. The most potent oligonucleotides lowered virus titer by ~90%. These results define universal for type A structured regions that could be important for virus function, as well as new targets for antisense therapeutics.

Structures of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 have been determined by NMR spectroscopy under low salt conditions. All protons and phosphorus nuclei resonances have been assigned. Signals of H5'/5" have been assigned stereospecifically. All 3JH,H and 3JP,H coupling constants have been measured. The structures were determined and refined using an iterative relaxation matrix procedure (IRMA) and the restrained MD simulation. Both duplexes form half-turn, right-handed helices with several conformational features which deviate significantly from a canonical A-RNA structure. Duplexes are characterised as having C3'-endo sugar pucker, very low base-pair rise and high helical twist and inclination angles. Helices are overwound with <10 bp per turn. There is limited inter-strand guanine stacking for CG steps. Within CG steps of both duplexes, the planes of the inter-strand cytosines are not parallel while guanines are almost parallel. For the GC steps this pattern is reversed. The 2'-O-methyl groups are spatially close to the 5'-hydrogens of neighbouring residues from the 3'-side and are directed towards the minor groove of 2'-O-Me(CGCGCG)2 forming a hydrophobic layer. Solution structures of both duplexes are similar; the effect of 2'-O-methylation on the parent RNA structure is small. This suggests that intrinsic properties imposed by alternating CG base pairs govern the overall conformation of both duplexes.

Highly amphiphilic fragments of hydrolytically stable 3′‐peptidyl transfer RNA analogues are stepwise assembled on solid support. Their secondary structure and thermal denaturation are studied by using CD and UV spectroscopy, their supramolecular assemblage by using AFM and DLS (see picture).

No abstract