Endo Ribose Research Articles

Solid-state NMR chemical shift analysis for determining the conformation of ATP bound to Na,K-ATPase in its native membrane.

Structures of membrane proteins determined by X-ray crystallography and, increasingly, by cryo-electron microscopy often fail to resolve the structural details of unstable or reactive small molecular ligands in their physiological sites. This work demonstrates that 13C chemical shifts measured by magic-angle spinning (MAS) solid-state NMR (SSNMR) provide unique information on the conformation of a labile ligand in the physiological site of a functional protein in its native membrane, by exploiting freeze-trapping to stabilise the complex. We examine the ribose conformation of ATP in a high affinity complex with Na,K-ATPase (NKA), an enzyme that rapidly hydrolyses ATP to ADP and inorganic phosphate under physiological conditions. The 13C SSNMR spectrum of the frozen complex exhibits peaks from all ATP ribose carbon sites and some adenine base carbons. Comparison of experimental chemical shifts with density functional theory (DFT) calculations of ATP in different conformations and protein environments reveals that the ATP ribose ring adopts an C3'-endo (N) conformation when bound with high affinity to NKA in the E1Na state, in contrast to the C2'-endo (S) ribose conformations of ATP bound to the E2P state and AMPPCP in the E1 complex. Additional dipolar coupling-mediated measurements of H-C-C-H torsional angles are used to eliminate possible relative orientations of the ribose and adenine rings. The utilization of chemical shifts to determine membrane protein ligand conformations has been underexploited to date and here we demonstrate this approach to be a powerful tool for resolving the fine details of ligand-protein interactions.

RNA approaches the B-form in stacked single strand dinucleotide contexts.

Duplex RNA adopts an A-form structure, while duplex DNA interconverts between the A- and B-forms depending on the environment. The C2'-endo sugar pucker seen in B-form DNA can occur infrequently in ribose sugars as well, but RNA is not understood to assume B-form conformations. Through analysis of over 45,000 stacked single strand dinucleotide (SSD) crystal structure conformations, this study demonstrates that RNA is capable of adopting a wide conformational range between the canonical A- and B-forms at the localized SSD level, including many B-form-like conformations. It does so through C2'-endo ribose conformations in one or both nucleotides, and B-form-like neighboring base stacking patterns. As chemical reactions on nucleic acids involve localized changes in chemical bonds, the understanding of how enzymes distinguish between DNA and RNA nucleotides is altered by the energetic accessibility of these rare B-form-like RNA SSD conformations. The existence of these conformations also has direct implications in parametrization of molecular mechanics energy functions used extensively to model nucleic acid behavior., 2016. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 65-82, 2016.

The conformational structure of adenosine molecules, isolated in low-temperature Ar matrices

FTIR spectra of adenosine (Ado) molecules, isolated in low-temperature matrices, are obtained in the infrared range of 3800–400 cm−1, with a resolution of 0.3. MP2 and DT/B3LYP quantum-mechanical methods are used to calculate the population of major structural Ado isomers. It is shown that Ado can withstand prolonged evaporation at a temperature of 465 K without thermal decomposition. The structure five major conformers that are fixed in Ar matrices at 6 K, are established. Ado anti-conformers with intramolecular hydrogen bonds O2′H-N3, are found. The population of these anti-conformers is more than one and a half times less than the syn-conformer population having the intramolecular hydrogen bond O5′H-N3. In low-temperature matrices, the majority of the dominant conformers are fixed with a C2′-endo ribose ring structure. Similar to 2′-deoxyadenosine (dA), Ado conformers with intramolecular hydrogen bonds O3′H-O5 or O5′H-O3 were not found.

Surprising base pairing and structural properties of 2'-trifluoromethylthio-modified ribonucleic acids.

The chemical synthesis of ribonucleic acids (RNA) with novel chemical modifications is largely driven by the motivation to identify eligible functional probes for the various applications in life sciences. To this end, we have a strong focus on the development of novel fluorinated RNA derivatives that are powerful in NMR spectroscopic analysis of RNA folding and RNA ligand interactions. Here, we report on the synthesis of 2′-SCF3 pyrimidine nucleoside containing oligoribonucleotides and the comprehensive investigation of their structure and base pairing properties. While this modification has a modest impact on thermodynamic stability when it resides in single-stranded regions, it was found to be destabilizing to a surprisingly high extent when located in double helical regions. Our NMR spectroscopic investigations on short single-stranded RNA revealed a strong preference for C2′-endo conformation of the 2′-SCF3 ribose unit. Together with a recent computational study (L. Li, J. W. Szostak, J. Am. Chem. Soc. 2014, 136, 2858–2865) that estimated the extent of destabilization caused by a single C2′-endo nucleotide within a native RNA duplex to amount to 6 kcal mol−1 because of disruption of the planar base pair structure, these findings support the notion that the intrinsic preference for C2′-endo conformation of 2′-SCF3 nucleosides is most likely responsible for the pronounced destabilization of double helices. Importantly, we were able to crystallize 2′-SCF3 modified RNAs and solved their X-ray structures at atomic resolution. Interestingly, the 2′-SCF3 containing nucleosides that were engaged in distinct mismatch arrangements, but also in a standard Watson–Crick base pair, adopted the same C3′-endo ribose conformations as observed in the structure of the unmodified RNA. Likely, strong crystal packing interactions account for this observation. In all structures, the fluorine atoms made surprisingly close contacts to the oxygen atoms of the corresponding pyrimidine nucleobase (O2), and the 2′-SCF3 moieties participated in defined water-bridged hydrogen-bonding networks in the minor groove. All these features allow a rationalization of the structural determinants of the 2′-SCF3 nucleoside modification and correlate them to base pairing properties.

Fingerprinting noncanonical and tertiary RNA structures by differential SHAPE reactivity.

Many RNA structures are composed of simple secondary structure elements linked by a few critical tertiary interactions. SHAPE chemistry has made interrogation of RNA dynamics at single-nucleotide resolution straightforward. However, de novo identification of nucleotides involved in tertiary interactions remains a challenge. Here we show that nucleotides that form noncanonical or tertiary contacts can be detected by comparing information obtained using two SHAPE reagents, N-methylisatoic anhydride (NMIA) and 1-methyl-6-nitroisatoic anhydride (1M6). Nucleotides that react preferentially with NMIA exhibit slow local nucleotide dynamics and usually adopt the less common C2'-endo ribose conformation. Experiments and first-principles calculations show that 1M6 reacts preferentially with nucleotides in which one face of the nucleobase allows an unhindered stacking interaction with the reagent. Differential SHAPE reactivities were used to detect noncanonical and tertiary interactions in four RNAs with diverse structures and to identify preformed noncanonical interactions in partially folded RNAs. Differential SHAPE reactivity analysis will enable experimentally concise, large-scale identification of tertiary structure elements and ligand binding sites in complex RNAs and in diverse biological environments.

2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2′-azido cytidine and 2′-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2′-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2′-azido uridine and 2′-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2′-azido group supports the C3′-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2′-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2′-azido modifications are compatible with 2′-fluoro and/or 2′-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2′-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2′-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification.

Structure of an RNA/DNA dodecamer corresponding to the HIV-1 polypurine tract at 1.6 Å resolution

The crystal structure of an RNA/DNA hybrid dodecamer, r(5'-uaaaagaaaagg):d(5'-CCTTTTCTTTTA), which contains three-quarters of the polypurine tract (PPT) sequence of theHIV RNA genome is reported. The hybrid structure was determined at 1.6 Å resolution and was found to have the A-form conformation. However, the presence of alternate conformations along the RNA template strand indicated increased flexibility of the PPT sequence. Two segments (atnucleotides 1-2 and 6-8) of the RNA chain have two conformations exhibiting differences in torsion and pseudorotation angles. For conformation I((1-2), (6-8)), 25% of the RNA sugars have the C2'-exo pucker and the rest have the expected C3'-endo pucker. The II(1-2) and II(6-8) conformations of the RNA strand have one sugar with the C2'-exo pucker. None of theribose rings exist in the C2'-endo form, in contrast to a previous report which postulated a C2'-endo ribose as a key structural element of the PPT. The widths of the minor groove for conformations I((1-2), (6-8)) and II((1-2), (6-8)) of the RNA strand are 9.2-10.5 and 9.4-10.7 Å, respectively. Both ranges are very close to the intervals accepted for A-form RNA duplexes. On the opposing DNA primer strand most of the sugars are C3'-endo, except for the 3'-terminal sugars, which are C2'-endo (T22) or O4'-endo (T23 and A24). The duplex includes a noncanonical u1(anti)·A24(syn) base interaction with only one hydrogen bond between the bases. This noncanonical base interaction at the 5'-end of the template distorts the values of the helical parameters of the adjacent base pair.

Conformational Variation Revealed by the Crystal Structure of Rnase U2A Complexed with Ca Ion and 2´-Adenylic Acid at 1.03 Å Resolution

Asparagine can be non-enzymatically deamidated and isomerized via succinimide to isoaspartate. This post-translational modification can potentially alter the physical properties or the function of the parent protein. Asn32 of ribonuclease U2A from Ustilago sphaerogena is known to rapidly deamidate and isomerize in alkaline conditions. The crystal structure of ribonuclease U2A complexed with 2'-adenylic acid and calcium ions was determined at 1.03 Å resolution. In this structure, the region from Asp29 to Asp37 winds around a calcium ion, and the main-chain of Asn32-Gly33 adopts an extended conformation. Rotation of the side-chain of Asn32 could bring Asn32C(γ) into close proximity to Gly33N, in a conformation suitable for succinimide formation. The structure suggests that in solution the region around Asn32-Gly33 is likely to be in equilibrium between multiple conformers, with the deamidation of Asn32 proceeding when the region adopts an extended conformation.

Conformations of ribonucleoside uridine in the low temperature Ar matrices

The conformational equilibrium of the ribonucleoside uridine molecules in low-temperature Ar matrices was investigated by the FTIR matrix isolation spectroscopy and quantum-chemical calculations at the DFT and MP2 levels of theory. It was established that conformers with a carbonyl group C2O, which takes part in the intramolecular hydrogen bonds, dominate in the low temperature matrices. Populations of syn- and anti- conformers with intramolecular hydrogen bonds O5′H–O2 and O2′–O2, respectively, are equal practically. Unlike 2′-deoxyuridine, the syn- conformers of Ur with C3′-endo structure of the ribose ring (South) have been found in Ar matrices. At the same time the conformations of C2′-endo (North) ribose ring prevail both in anti- and syn- conformers.

Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester

We analyzed cleavage of precursor tRNAs with an LNA, 2′-OCH3, 2′-H or 2′-F modification at the canonical (c0) site by bacterial RNase P. We infer that the major function of the 2′-substituent at nt −1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c0 site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3′ -endo ribose and without the H-bond donor function of the 2′-substituent. LNA and 2′-OCH3 suppressed processing at the major aberrant m−1 site; instead, the m+1 (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c0 and m+1 sites had different pH profiles, with a higher Mg2+ requirement for c0 versus m+1 cleavage. The strong catalytic defect for LNA and 2′-OCH3 supports a model where the extra methylene (LNA) or methyl group (2′-OCH3) causes a steric interference with a nearby bound catalytic Mg2+ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects.

An ab initio study of a nucleoside: Uridine

Ab initio SCF molecular orbital computations have been performed for a nucleoside, uridine, in a specific conformational arrangement (3'-endo conformation of the ribose, gg orientation of the extracyclic CH2OH group, and anti orientation of the base with respect to the sugar) and the results compared with some of those pertaining to the isolated component fragments, uracil and 3'-endo ribose. While the modifications of the atomic charges or of the energies of the molecular orbitals upon the formation of the nucleoside are small, the perturbations produced in some portions of the corresponding electrostatic molecular potential are more profound and more significant. Those occurring in the vicinity of the CO4 carbonyl group account for the probable increase in the proton affinity of uridine with respect to uracil. Those relevant to the O1′ oxygen indicate that the proton affinity of this atom in the free ribose is appreciably shielded in the nucleoside. The oxygens O2′ and O3′ represent non-negligible sites of attraction for positive ions in the free ribose and in the nucleoside.

Synthesis of 4-Thiopseudoisocytidine and 4-Thiopseudouridine as Components of Triplex-forming Oligonucleotides

Abstract In this paper, we report convenient methods for the synthesis of 4-thiopseudoisocytidine (s4ψiC) and 4-thiopseudouridine (s4Ψ). 1H NMR spectral analysis of these modified nucleosides showed that both s4Ψ and s4ψiC prefer C3′-endo ribose puckering. These conformational properties are favorable for the stabilization of triplex formation.

Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions

The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm5s2U by largely fixing the C3′-endo ribose puckering, ensuring stable and accurate codon–anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm5s2U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.

NMR conformational analysis of p-tolyl furanopyrimidine 2′-deoxyribonucleoside and crystal structure of its 3′,5′-di- O-acetyl derivative

The conformation of a representative molecule of a new, potent class of antiviral-active modified nucleosides is determined. A bicyclic nucleoside, 3-(2′-deoxy-β- d-ribofuranosyl)-6-(4-methylphenyl)-2,3-dihydrofuro[2,3- d]pyrimidin-2-one, shows C2′- endo and C3′- endo ribose conformations in solution (63:37, 37 °C; DMSO- d 6), as determined by 1H NMR studies. The crystal structure of a 3′,5′-di- O-acetyl-protected derivative (monoclinic, P2 1, a/ b/ c = 6.666(1)/12.225(1)/24.676(2) Å, β = 90.24(1)°, Z = 4) shows exclusively C2′- endo deoxyribose puckering. The base is found in the anti position both in solution and in crystalline form.

Structural investigation of the binding of nucleotide to phosphoenolpyruvate carboxykinase by NMR.

The enzyme phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the reversible conversion of oxalacetate and GTP to phosphoenolpyruvate (PEP), GDP, and CO2. PEPCK from higher organisms is a monomer, specifically requires GTP or ITP, and uses Mn2+ as the activating cation. Currently, there is no crystal structure of GTP-utilizing PEPCKs. The conformation of the bound nucleotide was determined from transferred nuclear Overhauser effects (trnOe) experiments to determine internuclear proton distances. At 600 MHz in the presence of PEPCK, nOe effects were observed between nucleotide protons. Internuclear distances were calculated from the initial rate of the nOe buildup. These distance constraints were used in energy minimization calculations to determine the conformation of PEPCK-bound GTP. The bound nucleotide has the base oriented anti to the C2'-endo(2E) ribose ring conformation. Relaxation rate studies indicate that there is an additional relaxation effect on the C1' proton upon nucleotide binding to PEPCK. Nucleotide binding to PEPCK-Mn2+ was studied by 1H relaxation rate studies, but results were complicated by long dipole-dipole distances and the presence of competing complexes. Modification of PEPCK by iodoacetamido-TEMPO leads to an inactive enzyme that is spin-labeled at cys273. The interaction of TEMPO-PEPCK with GTP allows for the measurement of nuclear distances between GTP and the spin label. The results suggest that cys273 lies near the ribose ring of the bound nucleotide, but it is too far to be implicated in direct hydrogen bonding interactions consistent with previous results [Makinen, A. L., and Nowak, T. J. Biol. Chem. (1989) 264, 12148], suggesting that cys273 does not actively participate in catalysis. Modification of PEPCK with several cysteine specific modifying agents causes no change in the ability of the enzyme to bind nucleotide as monitored by fluorescence quenching. A correlation between the size of the modifying agent and the maximal observed quenching upon saturation of the enzyme with nucleotide is observed. This suggests a mechanism for inactivation of PEPCK by cysteine modification due to inhibition of a dynamic motion that may occur upon nucleotide binding.

Interactions of a didomain fragment of the Drosophila sex-lethal protein with single-stranded uridine-rich oligoribonucleotides derived from the transformer and Sex-lethal messenger RNA precursors: NMR with residue-selective [5-2H]uridine substitutions.

Proteins that contain two or more copies of the RNA-binding domain [ribonucleoprotein (RNP) domain or RNA recognition motif (RRM)] are considered to be involved in the recognition of single-stranded RNA, but the mechanisms of this recognition are poorly understood at the molecular level. For an NMR analysis of a single-stranded RNA complexed with a multi-RBD protein, residue-selective stable-isotope labeling techniques are necessary, rather than common assignment methods based on the secondary structure of RNA. In the present study, we analyzed the interaction of a Drosophila Sex-lethal (Sx1) protein fragment, consisting of two RBDs (RBD1-RBD2), with two distinct target RNAs derived from the tra and Sxl mRNA precursors with guanosine and adenosine, respectively, in a position near the 5'-terminus of a uridine stretch. First, we prepared a [5-2H]uridine phosphoramidite, and synthesized a series of 2H-labeled RNAs, in which all of the uridine residues except one were replaced by [5-2H]uridine in the target sequence, GU8C. By observing the H5-H6 TOCSY cross peaks of the series of 2H-labeled RNAs complexed with the Sx1 RBDI-RBD2, all of the base H5-H6 proton resonances of the target RNA were unambiguously assigned. Then, the H5-H6 cross peaks of other target RNAs, GU2GU8, AU8, and UAU8, were assigned by comparison with those of GU8C. We found that the uridine residue prior to the G or A residue is essential for proper interaction with the protein, and that the interaction is tighter for A than for G. Moreover, the H1' resonance assignments were achieved from the H5-H6 assignments. The results revealed that all of the protein-bound nucleotide residues, except for only two, are in the unusual C2'-endo ribose conformation in the complex.

A UV resonance Raman investigation of poly(rI): Evidence for cation-dependent structural perturbations

Poly(rI) stabilized by either Na+ or K+ was investigated using uv resonance Raman (UVRR) spectroscopy. Raman excitation profiles of inosine 5′-monophosphate demonstrated the 250 nm excitation selectively enhances base stacking interactions, while ribose and carbonyl stretching vibrations are preferentially enhanced with 210 nm excitation. These wavelengths were used to examine the structure of poly(rI) in the presence of either K+ or Na+ as a function of temperature. UVRR studies revealed that the K+ stabilized form is more thermally stable, yielding a Tm of ∼ 47°C compared to a Tm of ∼ 30°C for the Na+ stabilized form. We observed that both the ribosyl conformation and the coordination of the carbonyl groups depend on the nature of the cation. The C6O stretching frequency indicates that Na+ coordinates much more strongly to the carbonyl groups than K+ (1672 cm−1 Na+ vs 1684 cm−1 K+ at 4°C). Conformationally sensitive modes of the phosphate backbone and the ribosyl ring indicate that Na+ stabilized poly(rI) predominantly exists in a C3′-endo ribose conformation, whereas K+ stabilized poly(rI) adopts a C2′-endo conformation possibly as a consequence of the larger ionic radius of the K+ ion. © 1998 John Wiley & Sons, Inc. Biopoly 46: 475–487, 1998

Kinetic, Binding, and NMR Studies of Perdeuterated Yeast Phosphoglycerate Kinase and Its Interactions with Substrates

Perdeuterated yeast phosphoglycerate kinase (2HPGK) was prepared from yeast cells grown in 99.9% 2H2O and an acid hydrolysate from deuterated algal cells. Kinetic and binding studies suggested that perdeuterated enzyme was similar to the isotopically normal PGK. The use of 2HPGK not only eliminated the spectral overlap between the enzyme and substrate nuclear Overhauser effect (NOE) cross-peaks, but also permitted observation of weak transfer NOE cross-peaks between the substrate protons that are greater than 4 Å apart. Intensity of NOE cross-peaks was used to determine the interproton distances of enzyme bound Mg(II)dATP. These distances were then used in model building studies to determine the conformation of Mg(II)dATP. The average conformation of enzyme bound dATP is anti with O4′/C2′ endo ribose pucker and trans, gauche about the C4′-C5′ bond. Although many spin diffusion pathways were eliminated by protein deuteration, spin diffusion was still observed between the protons of the substrate at mixing times longer than 25 ms.

A vibrational spectroscopic study of the self‐association of polyinosinic acid and polyguanylic acid in aqueous solution

We have studied by Raman and ir spectroscopy the structure of self-associated polyinosinic acid and polyguanylic acid in aqueous solution. The results are consistent with the formation of a four-stranded complex, which melts cooperatively near 60 degrees C in the case of poly(I) in the presence of K+ ions. The conformation of the ribose in both systems is mixed C2'-endo/C3'-endo, giving a structure that is intermediate between the extremes proposed previously from x-ray diffraction studies. Characteristic Raman bands for the C2'-endo ribose conformation in polyribonucleotides are identified. The four-stranded structure of poly(I) appears to be very flexible, with approximately 15% of the tetrameric segments being disrupted and approximately 30% of the ribose units adopting a disordered conformation prior to melting. This disordering process increases to approximately 75% above the melting transition, with the remaining approximately 25% of the ribose units keeping an ordered C2'-endo or C3'-endo conformation.

Solution structure of pentameric and heptameric branched-RNA modelling the lariat structure of group II or nuclear m-RNA introns studied by one- and two-dimensional nmr spectroscopy at 500 mhz

A detailed 500 MHz 1H NMR study of the branched RNA systems 3 (pentamer) and 4 (heptamer) is reported. The conformational properties of 3 and 4 were compared with those of the previously studied smaller branched RNAs 1 (trimer) and 2 (tetramer). Compounds 1 – 3 are structural constituents of 4, which corresponds to the sequence at the branch site of Group II intron b11 from yeast mitochondria. The present data provide further substantiation to our previous preliminary conclusion that trimer 1 and pentamer 3 show very similar conformational features, which are remarkably distinct from tetramer 2 and heptamer 4. It is found that the absence of a nucleotide unit at the 5′-site of the branch point (A) leads to a conformation at the branch point which is characterized by C2′-endo (S) ribose, syn orientation of the A-base, and A2′ → 5′G base stacking. This conformation is encountered for trimer 1, and -to a larger extent- also for pentamer 3. The introduction of a mono- or dinucleotide unit at the 5′-site of the branch point introduces a marked conformational transition at the branchpoint, in such a way that the conformation becomes C3′-endo (N) ribose, anti orientation of the base, and disruption of A2′ → 5′G base stacking. This conformation is found for tetramer 2 and heptamer 4. The structural differences between 1,3 and 2,4 are highly reminiscent of the 5′-conformational transmission effect for a series of linear trimer and tetramer RNA molecules. We therefore propose that the 5′-conformational model is also of great interest with respect to our understanding of the structural preferences of branched RNAs. However, our chemical shift vs. temperature profiles and NOE data on 3 and 4 show several additional conformational features. We propose a model for the secondary structure of pentamer 3, which entails peculiar backfolding of the 3′-linked chain in such a way that stacking is achieved between the remote bases C+2 (in the 3′-linked chain), and branch point A. Also, our NOE data on heptamer 4 led to the suggestion that a stack exists between the bases U+2 (in the 2′-linked chain), and U-1 (upstream of A). All our experimental observations on 3 and 4 were integrated into preliminary structural models, shown in Figure 10. It was impossible, however, to obtain strong support for these models from NOE data, since virtually no interresidual NOE crosspeaks appeared in our 2D NOESY spectra. Only in the case of 4, we could observe four weak interresidual NOEs at mixing times of 300, 500 and 900 ms in the aromatic spectral region, provided that a sample temperature of 292 K was used; elevation of the sample temperature to 303 K rendered these NOEs invisible.