Crystal structures of two plasmid copy control related RNA duplexes: An 18 base pair duplex at 1.20 A resolution and a 19 base pair duplex at 1.55 A resolution.

The structures of two RNA duplexes, whose sequences correspond to portions of the ColE1 plasmid copy control RNA I and RNA II, have been determined. Crystals containing the 18mers 5'-CA CCGUUGGUAGCGGUGC-3' and 5'-CACCGCUACCAACGGUGC-3' diffract to 1.20 A resolution while those containing the 19mers 5'-GCACCGUUGGUAGCGGUGC-3' and 5'-GCACCGCUACCAACGGUGC-3' diffract to 1.55 A resolution. Both duplexes are standard A form, with Watson-Crick base pairing throughout. Use of anisotropic atomic displacement factors in refinement of the 1.20 A structure dramatically improved refinement statistics, resulting in a final R(free) of 15.0% and a crystallographic R-factor of 11.6%. Perhaps surprisingly, these crystals of the 18 base pair RNA exhibit a 36-fold static disorder, resulting in a structure with a single sugar-phosphate backbone conformation and an averaged base composition at each residue. Since the sugar-phosphate backbone structure is identical in the 36 different nucleotides that are superimposed, there can be no sequence-dependent variation in the structure. The average ribose pucker amplitude is 45.8 degrees for the 18 base pair structure and 46.4 degrees for the 19 base pair structure; these values are respectively 19% and 20% larger than the average pucker amplitude reported from nucleoside crystal structures. A standard RNA water structure, based on analysis of the hydration of these crystal structures and that of the TAR RNA stem [Ippolito, J. A., and Steitz, T. A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9819-9824], has been derived, which has allowed us to predict water positions in lower resolution RNA crystal structures. We report a new RNA packing motif, in which three pro-S(p) phosphate oxygens interact with an ammonium ion.     

Solution structure of a DNA duplex containing a formamide-adenine base pair

The N-(2-deoxy-beta3-D-erythro-pentofuranosyl) formamide residue results from a ring fragmentation product of thymine or cytosine. The presence of a formamide-adenine base pair in the sequence 5'd(AGGAACCACG).d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. There are two possible isomers for the formamide side chain, either cis or trans. For each isomer, we observed an equilibrium in solution between two forms. First, a species where the formamide is intrahelical and paired with the facing adenine. For the cis isomer, the formamide is in a syn conformation and two hydrogen bonds with adenine are formed. The trans isomer is in an anti conformation and a single hydrogen bond is observed. In the second form, whatever the isomer, the formamide is rejected outside the helix, whereas the adenine remains inside.     

Synthesis and duplex stability of oligonucleotides containing cytosine-thymine analogues.

The synthesis of the deoxynucleoside derived from the base P, 6H,8H-3,4-dihydro-pyrimido[4,5-c] [4,5-c] [1,2]oxazin-7-one, 2, and its introduction by established phosphoramidite and H-phosphonate chemistry into oligonucleotides is described. The melting transition temperatures (Tm) of a range of heptadecamer duplexes containing P/A and P/G base-pairs are compared with corresponding ones having N4-methoxycytosine (M) 1 and mismatched normal bases. P/A and P/G pairs allow closely similar duplex stabilities and have the potential to reduce the multiplicity of probes and primers based on amino acid sequences by removing the T/C degeneracy.     

Solution structure of an RNA duplex including a C-U base pair

The formation of the C-U base pair in a duplex was observed in solution by means of the temperature profile of (15)N chemical shifts, and the precise geometry of the C-U base pair was also determined by NOE-based structure calculation. From the solution structure of the RNA oligomer, r[CGACUCAGG].r[CCUGCGUCG], it was found that a single C-U mismatch preferred being stacked in the duplex rather than being flipped-out even in solution. Moreover, it adopts an irregular geometry, where the amino nitrogen (N4) of the cytidine and keto-oxygen (O4) of the uridine are within hydrogen-bonding distance, as seen in crystals. To further prove the presence of a hydrogen bond in the C-U pair, we employed a point-labeled cytidine at the exocyclic amino nitrogen of the cytidine in the C-U pair. The temperature profile of its (15)N chemical shift showed a sigmoidal transition curve, indicating the presence of a hydrogen bond in the C-U pair in the duplex.     

Triple helical DNA in a duplex context and base pair opening

It is fundamental to explore in atomic detail the behavior of DNA triple helices as a means to understand the role they might play in vivo and to better engineer their use in genetic technologies, such as antigene therapy. To this aim we have performed atomistic simulations of a purine-rich antiparallel triple helix stretch of 10 base triplets flanked by canonical Watson–Crick double helices. At the same time we have explored the thermodynamic behavior of a flipping Watson–Crick base pair in the context of the triple and double helix. The third strand can be accommodated in a B-like duplex conformation. Upon binding, the double helix changes shape, and becomes more rigid. The triple-helical region increases its major groove width mainly by oversliding in the negative direction. The resulting conformations are somewhere between the A and B conformations with base pairs remaining almost perpendicular to the helical axis. The neighboring duplex regions maintain a B DNA conformation. Base pair opening in the duplex regions is more probable than in the triplex and binding of the Hoogsteen strand does not influence base pair breathing in the neighboring duplex region.     

Azole substituted oligonucleotides promote antiparallel triplex formation at non-homopurine duplex targets.

The ability of certain azole substituted oligodeoxy-ribonucleotides to promote antiparallel triple helix formation with duplex targets having CG or TA interruptions in the otherwise homopurine sequence was examined. 2'-Deoxyribonucleosides of the azoles, which include pyrazole, imidazole, 1,2,4-triazole and 1,2,3,4-tetrazole were synthesized using the stereo-specific sodium salt glycosylation procedure. These nucleosides were successfully incorporated using solid-support, phosphoramidite chemistry, into oligonucleotides designed to interact with the non-homopurine duplex targets. The interaction of these modified oligonucleotides with all four possible base pairs was evaluated and compared to similar data for a series of natural oligonucleotides. The oligonucleotides containing simple azoles enhanced the triplex forming ability considerably at non-homopurine targets. Binding of these modified oligonucleotides to duplex targets containing TA inversion sites was particularly noteworthy, and compare favorably to unmodified oligonucleotides for binding to duplex targets containing CG as well as TA base pairs. The selectivity exhibited by certain azoles is suggestive of base pair specific interactions. Thus, the azoles evaluated during this study show considerable promise for efforts to develop generalized triplex formation at non-homopurine duplex sequences.     

Mu insertion duplicates a 5 base pair sequence at the host inserted site.

Nucleotide sequences were analyzed across the two ends of lysogenic Mu DNA. These ends were cloned separately in lambdapMu hybrid particles that derived from a single Mu lysogen in the lac Z part of lambdaplac5. The obtained data imply that Mu lysogenization was associated with the duplication of 5 base pairs present in lac DNA at the Mu insertion site. As a result of this duplication, Mu DNA is flanked by two copies of five identical base pairs oriented as direct repeats. A similar conclusion has been obtained independently by other investigators with the use of a different Mu lysogen (D. Kamp and R. Kahmann, personal communication). Thus Mu insertion seems to have a striking similarity to typical IS-mediated insertions that were found to be associated with a short DNA duplication at the target site.     

Mammalian telomeres end in a large duplex loop.

Mammalian telomeres contain a duplex array of telomeric repeats bound to the telomeric repeat-binding factors TRF1 and TRF2. Inhibition of TRF2 results in immediate deprotection of chromosome ends, manifested by loss of the telomeric 3' overhang, activation of p53, and end-to-end chromosome fusions. Electron microscopy reported here demonstrated that TRF2 can remodel linear telomeric DNA into large duplex loops (t loops) in vitro. Electron microscopy analysis of psoralen cross-linked telomeric DNA purified from human and mouse cells revealed abundant large t loops with a size distribution consistent with their telomeric origin. Binding of TRF1 and single strand binding protein suggested that t loops are formed by invasion of the 3' telomeric overhang into the duplex telomeric repeat array. T loops may provide a general mechanism for the protection and replication of telomeres.     

S-DNA is oversupercoiled DNA with 1.94-2.19 A rise per base pair along duplex axis

Supercoiled pGEMEX DNA with length of 3993 nucleotides was immobilized on four substrates (freshly cleaved mica, standard amino mica, modified amino mica with increased and decreased surface charge density compared with standard amino mica) and it was visualized by atomic force microscopy (AFM) in air. Plectonomically supercoiled DNA molecules as well as single molecules with extremely high level of compaction (i.e. molecules with significantly higher superhelix density values on comparison with previously experimentally measured and theoretically investigated ones) were visualized on modified amino mica which was characterized by increased surface charge density. Distance between base pairs along duplex axis was determined by measurements of contour length of single oversupercoiled DNA molecules. Determined rise per base pair was varied from 1.94 to 2.19 A. These compressed supercoiled DNA molecules like a spring with decreased rise/base pair on comparison with well-known DNA forms were called new DNA form--S-DNA. A model of S-DNA was built. Formation of the S-DNA molecules was suggested to be an intermediate stage on the compaction of the single supercoiled DNA molecules up to the spheroids and minitoroids. Oversupercoiling and further compression of the supercoiled DNA molecules was shown to cause by high surface charge density of amino mica on which DNA molecules were immobilized.     

Altered structural fluctuations in duplex RNA versus DNA: a conformational switch involving base pair opening

DNA and RNA are known to have different structural properties. In the present study, molecular dynamics (MD) simulations on a series of RNA and DNA duplexes indicate differential structural flexibility for the two classes of oligonucleotides. In duplex RNA, multiple base pairs experienced local opening events into the major groove on the nanosecond time scale, while such events were not observed in the DNA simulations. Three factors are indicated to be responsible for the base opening events in RNA: solvent-base interactions, 2'OH(n)-O4'(n+1) intra-strand hydrogen bonding, and enhanced rigid body motion of RNA at the nucleoside level. Water molecules in the major groove of RNA contribute to initiation of base pair opening. Stabilization of the base pair open state is due to a 'conformational switch' comprised of 2'OH(n)-O4'(n+1) hydrogen bonding and a rigid body motion of the nucleoside moiety in RNA. This rigid body motion is associated with decreased flexibility of the glycosyl linkage and sugar moieties in A-form structures. The observed opening rates in RNA are consistent with the imino proton exchange experiments for AU base pairs, although not for GC base pairs, while structural and flexibility changes associated with the proposed conformational switch are consistent with survey data of RNA and DNA crystal structures. The possible relevance of base pair opening events in RNA to its many biological functions is discussed.     

Inosine.adenine base pairs in a B-DNA duplex.

The structure of the synthetic deoxydodecamer d(C-G-C-I-A-A-T-T-A-G-C-G) has been determined by single crystal X-ray diffraction techniques at 2.5A resolution. The refinement converged with a crystallographic residual, R = 0.19 and the location of 64 solvent molecules. The sequence crystallises as a B-DNA helix with 10 Watson-Crick base-pairs (4 A.T. and 6 G.C) and 2 inosine.adenine (I.A) pairs. The present work shows that in the purine.purine base-pairs the adenine adopts syn orientation with respect to the furanose moiety while the inosine is in the trans (anti) orientation. Two hydrogen bonds link the I.A. base-pair, one between N-1(I) and N-7(A), the other between O-6(I) and N-6(A). This bulky purine.purine base-pair is incorporated in the double helix at two positions with little distortion of either local or global conformation. The pairing observed in this study is presented as a model for I.A base-pairs in RNA codon-anticodon interactions and may help explain the thermodynamic stability of inosine containing base-pairs. Conformational parameters and base stacking interactions are presented and where appropriate compared with those of the native compound, d(C-G-C-G-A-A-T-T-C-G-C-G) and with other studies of oligonucleotides containing purine.purine base-pairs.     

Thermodynamics of RNA melting,one base pair at a time

The melting of base pairs is a ubiquitous feature of RNA structural transitions, which are widely used to sense and respond to cellular stimuli. A recent study employing solution nuclear magnetic resonance (NMR) imino proton exchange spectroscopy provides a rare base-pair-specific view of duplex melting in the Salmonella FourU RNA thermosensor, which regulates gene expression in response to changes in temperature at the translational level by undergoing a melting transition. The authors observe “microscopic” enthalpy–entropy compensation—often seen “macroscopically” across a series of related molecular species—across base pairs within the same RNA. This yields variations in base-pair stabilities that are an order of magnitude smaller than corresponding variations in enthalpy and entropy. A surprising yet convincing link is established between the slopes of enthalpy–entropy correlations and RNA melting points determined by circular dichroism (CD), which argues that unfolding occurs when base-pair stabilities are equalized. A single AG-to-CG mutation, which enhances the macroscopic hairpin thermostability and folding cooperativity and renders the RNA thermometer inactive in vivo, spreads its effect microscopically throughout all base pairs in the RNA, including ones far removed from the site of mutation. The authors suggest that an extended network of hydration underlies this long-range communication. This study suggests that the deconstruction of macroscopic RNA unfolding in terms of microscopic unfolding events will require careful consideration of water interactions.     

Structural and functional accommodation of nucleotide variations at a conserved tRNA tertiary base pair.

The U8:A14 tertiary base pair of transfer RNAs (tRNAs) stabilizes the sharp turn from the acceptor stem to the dihydrouridine stem. This tertiary base pair is important for the overall L-shaped tRNA structure. Inspection of tRNA sequences shows that U8:A14 is highly conserved. However, variations of U8:A14 are found in natural sequences. This raises the question of whether all 16 permutations of U8:A14 can be accommodated by a single tRNA sequence framework and by the bacterial translational apparatus. Here we expressed the wild type and 15 variants of U8:A14 of an alanine tRNA amber suppressor in Escherichia coli and tested the ability of each to suppress an amber mutation. We showed that 12 of the 15 variants are functional suppressors (sup+) and 3 are nonfunctional (sup-). Of the 12 functional suppressors, the G8:G14 variant is the most efficient suppressor, whose suppression efficiency is indistinguishable from that of the wild type. Analysis of tRNA structure with chemical probes and the lead-cleavage reaction, however, showed a distinct difference between the G8:G14 variant and the wild type. Thus, two different structures of E. coli tRNAAla/CUA share an identical functional phenotype in protein synthesis. The remaining 11 sup+ variants with reduced suppression efficiencies are likely to have other structural variations. We suggest that the variations of these sup+ mutants are structurally and functionally accommodated by the bacterial translational apparatus. In contrast, the three sup- mutants harbor variations that alter the backbone structure in the corner of the L. These variations are likely to reduce the stability of the tRNA inside the cell or, among others, to interfere with the ability of the tRNA to functionally interact with elongation factor Tu and with the ribosome.     

Inhibition of polyoma DNA synthesis by base pair substitutions at the replication origin.

The effect of base pair substitutions on the function of the polyoma virus origin of DNA replication was studied. The mutations were all C-G to T-A transitions, induced by bisulfite treatment of recombinant DNA molecules. The mutagenesis was directed to short single-stranded gaps in duplex DNA, or to loops in heteroduplex molecules. Modification of a 34 base pair sequence of dyad symmetry led to cis-acting inhibition of viral DNA synthesis, ranging from slight defects to total inactivation. One of the mutants was temperature sensitive. Mutants with base changes in an adjacent DNA segment, including an 18 base pair long purine-pyrimidine tract, had similar, but less severe, deficiences. In contrast to the effect of mutations in the homologous region of the simian virus 40 genome, there was no strict relationship between mutation of the putative large T-antigen-binding base sequence GPuGGC and defective viral DNA synthesis.     

Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution

Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein–DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein–DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.     

Homopurine and homopyrimidine strands complementary in parallel orientation form an antiparallel duplex at neutral pH with A-C,G-T,and T-C mismatched base pairs

DNA sequences d-TGAGGAAAGAAGGT (a 14-mer) and d-CTCCTTTCTTCC (a 12-mer) are complementary in parallel orientation forming either Donahue (reverse Watson-Crick) base pairing at neutral pH or Hoogsteen base pairing at slightly acidic pH. The structure of the complex formed by dissolving the two strands in equimolar ratio in water has been investigated by nmr. At neutral pH, the system forms an ordered antiparallel duplex with five A : T and four G : C Watson-Crick base pairs and three mismatches, namely G-T, A-C, and T-C. The nuclear Overhauser effect cross-peak pattern suggests an overall B-DNA conformation with major structural perturbations near the mismatches. The duplex has a low melting point and dissociates directly into single strands with a broad melting profile. The hydrogen-bonding schemes in the mismatched base pairs have been investigated. It has been shown earlier that in acidic pH, the system prefers a triple-stranded structure with two pyrimidine strands and one purine strand. One of the pyrimidine strands has protonated cytosines, forms Hoogsteen base pairing, and is aligned parallel to the purine strand; the other has nonprotonated cytosines and has base-pairing scheme similar to the one discussed in this paper. The parallel duplex is therefore less stable than either the antiparallel duplex or the triplex, in spite of its perfect complementarity. © 1997 John Wiley & Sons, Inc. Biopoly 41: 773–784, 1997     

Effects of osmolytes on stable UUCG tetraloops and their preference for a CG closing base pair

Osmolytes have the potential to affect the stability of secondary structure motifs and alter preferences for conserved nucleic acid sequences in the cell. To contribute to the understanding of the in vivo function of RNA we observed the effects of different classes of osmolytes on the UNCG tetraloop motif. UNCG tetraloops are the most common and stable of the RNA tetraloops and are nucleation sites for RNA folding. They also have a significant thermodynamic preference for a CG closing base pair. The thermal denaturation of model hairpins containing UUCG loops was monitored using UV-Vis spectroscopy in the presence of osmolytes with different chemical properties. Interestingly, all of the osmolytes tested destabilized the hairpins, but all had little effect on the thermodynamic preference for a CG base pair, except for polyethylene glycol (PEG) 200. PEG 200 destabilized the loop with the CG closing base pair relative to the loop with a GC closing base pair. The destabilization was linear with increasing concentrations of PEG 200, and the slope of this relationship was not perturbed by changes in the hairpin stem outside of the closing pair. This result suggests that in the presence of PEG 200, the UUCG loop with a GC closing base pair may retain some preferential interactions with the cosolute that are lost in the presence of the CG closing base pair. These results reveal that relatively small structural changes may influence how osmolytes tune the stability, and thus the function of a secondary structure motif in vivo.     

Recognition of a guanine-cytosine base pair by 8-oxoadenine.

Two oligodeoxyribonucleotides, d-CTTCTTTTTTATTTT, I(A), and d-ATTATTTTTTATTTT, II(A), where C is 5-methylcytosine and A is 8-oxoadenine, were prepared and their interactions with the duplex d-GAAGAAAAAAYAAAA/d-TTTTZTTTTTTCTTC, III.IV(Y.Z), were studied. Oligomers I(A) and II(A) each form triplexes with III.IV(G.C) at temperatures below 20 degrees C as shown by continuous variation experiments, melting experiments, and circular dichroism (CD) spectroscopy. The CD spectra of these triplexes are almost identical to those formed by I(C) and II(C), oligomers which contain cytosine in place of 8-oxoadenine. This suggests that the 8-oxoadenine-containing triplexes have conformations which are very similar to those of the cytosine-containing triplexes. The melting temperature (Tm) for dissociation of the third strand of triplex II.III.IV(A.G.C) is 22 degrees C at pH 7.0 and 8.0, whereas the Tm of the corresponding transition in triplex II.III.IV(C.G.C) decreases from 28 degrees C at pH 7.0 to 17 degrees C at pH 8.0. The pH dependence of the Tm in the latter triplex reflects the necessity of protonating the N-3 of cytosine in order for it to form two hydrogen bonds with G of the G.C base pair. It appears that the keto form of 8-oxoadenine can potentially form two hydrogen bonds with the N-7 and O-6 atoms of G of the G.C base pair, when the 8-oxoadenine is in the syn conformation and in contrast to cytosine does not require protonation of the base. Oligomer I(A) does not form triplexes with III.IV(Y.Z) when Y.Z is A.T or T.A.(ABSTRACT TRUNCATED AT 250 WORDS)     

IS1 insertion generates duplication of a nine base pair sequence at its target site.

Three independent integrations of the E. coli insertion sequence, IS1, into the gal operon have been analyzed. DNA sequences of portions of the wild-type galT gene which act as the target sites for these insertions, as well as the corresponding gal/IS1 junctions, are reported. Two features are particularly noteworthy. First, similar sequences appearing in inverted orientation consitute the ends of IS1: 18 of the terminal 23 base pairs at each end are identical. Second, in all three insertions, a 9 base pair segment found once in the wild-type sequence at the site of insertion is duplicated and appears in the same orientation at each end of the inserted element. The sequence of this 9 base pair repeat is different for each insertion analyzed. No homology between the inverted repeat sequences at the ends of IS1 and the sequences of the target sites is observed. Models for the mechanism of IS1 insertion are proposed.