The conformation and stability of ribonucleic acids: modeling base sequence effects in double stranded helices

Base sequence effects within double stranded RNA oligomers of A and Z conformations have been studied by molecular modeling using a methodological approach specifically adapted to nucleic acids. Calculations on symmetric oligomers having homonucleotide or dinucleotide repeating base sequences show that sequence changes can produce modifications in overall conformation, influence the degree of internal hydrogen bonding and strongly affect stability.     

Oligonucleotide-primed in situ DNA synthesis (PRINS): a method for chromosome mapping, banding, and investigation of sequence organization

Oligonucleotides were annealed to complementary sequences in fixed human metaphase chromosomes and extended with DNA polymerase. The newly synthesized fragments were labeled by incorporating bio-11-dUTP instead of TTP, and the sites of synthesis were detected by immunocytochemistry, using fluorochromes as the reporter molecules. We have obtained clear localization with oligonucleotides from alphoid (centromeric sequences), simple sequence (satellite) DNAs, a variety of Alu-dispersed repeated sequences, and oligonucleotides derived from the Tetrahymena and Trypanosoma telomere-specific sequences. The simple sequence and alphoid oligonucleotides gave results at least comparable to those obtained using the whole molecule as a probe for in situ hybridization, whereas the Alu oligonucleotides produced a diversity of results which depended on the absolute length and location of the oligonucleotide within the Alu sequence. The telomere-specific oligomers also produced a variety of results. The G-rich Trypanosoma oligomer and its complementary C-rich sequence produced strong telomeric signals and some interstitial signals on mouse chromosomes, but only weak telomeric signals on human chromosomes. The G-rich Tetrahymena oligomer produced detectable telomeric signals on human chromosomes. The technique appears to be a valuable extension of present tools for mapping and examining the organization of DNA sequences within chromosomes.     

Sequence dependent electrophoretic mobilities and melting temperatures for A-T containing oligodeoxyribonucleotides.

The electrophoretic mobilities and thermal melting properties of self complementary A-T containing dodecamer oligodeoxyribonucleotides have been investigated as a function of solution conditions. The oligomers contained tracts of nonalternating A-T base pairs of 2 (d(A2T2)3), 3 (d(A3T3)2), and 6 (d(A6T6] as well as the fully alternating (d(A-T)6) sequence. The melting temperature increased with the length of the nonalternating sequence and was approximately 12 degrees C higher in the d(A6T6) sequence than in the alternating oligomer. Under denaturing conditions all oligomers had the same electrophoretic mobility on acrylamide gels. Under conditions which favor duplex formation, the oligomers exhibited significant sequence dependent mobility differences. The mobilities of two oligomers, d(A-T)6 and d(A6-T6), were approximately equal and were less than those of the other oligonucleotides. The greatest mobility was observed for d(A2T2)3. These results are best explained by a model which requires bending at a junction of two or more continuous A or T bases with another sequence.     

Conformational sub-states in B-DNA.

Theoretical studies of the sequence-dependent conformation of B-DNA have been carried out using Jumna, a helicoidal co-ordinate minimization algorithm. The results obtained for a series of six oligomers with repetitive sequences show that, with the exception of the homopolymers (dA)n.(dT)n and (dG)n.(dC)n, all sequences can adopt a variety of conformations characterized by considerable changes in helicoidal parameters and also in sugar puckers which adopt C(2')-endo (falling into 2 classes) or, in the case of pyrimidine nucleotides, O(1')-endo forms. These studies lead to an improved understanding of the role of base sequence on DNA conformation and point to a number of interesting correlations between the various structural parameters describing the double helix.     

Sequence dependence of the B to Z transition in crystals and aqueous NaCl solutions for deoxyoligonucleotides containing all four canonical DNA bases

A laser Raman study has been made on the conformation of a series of self-complementary octameric deoxynucleotides that contain all four canonical deoxynucleotide bases [guanine (G), cytosine (C), adenine (A), and thymine (T)] in order to determine which sequences will crystallize in the Z form and which sequences will go into the Z form in aqueous solution at high salt concentrations (4-6 M NaCl). All four octadeoxynucleotides, d(TGCGCGCA) (I), d(CACGCGTG) (II), d(CGTGCACG) (III), and d(CGCATGCG) (IV), have been crystallized from low-salt solutions. The Raman spectra of microcrystals show that I, II, and IV crystallize in a rigorous Z form while III crystallizes in the B form. Sequences I and II go into a Z form in 4-6 M NaCl solution at 0 degrees C while sequences III and IV remain in the B form in 6 M salt. There are substantial differences in the Raman spectra of oligonucleotides in the Z form found in the crystal and in high-salt solutions. The Raman spectra of the Z forms in 6 M NaCl solution at 0 degrees C are not linear combinations of the Raman spectra of the complete Z form in the crystal and the complete B form in low-salt solutions. The terminal residues of these oligomers do not appear to be in a strict Z form. A detailed analysis of the ring puckers and syn/anti conformation for all of the residues both in solution and in the crystal has been made.(ABSTRACT TRUNCATED AT 250 WORDS)     

Folding of alpha(r)beta and epsilonbeta reverse turns; a nanosecond molecular dynamics simulation of the hexapeptide MSALNT and the octapeptide NMSALNTL in water.

Folding of the hexapeptide MSALNT and the octapeptide NMSALNTL were investigated using 2.8 ns molecular dynamics (MD) simulations in aqueous solution. In the simulation, the central sequence SALN of the hexapeptide folded rapidly within 200 ps into an alpha(r)beta turn conformation (type VIII conformation) and remained in this conformation for the rest of the trajectory. The sequence SALN of the octapeptide needed 2 ns to fold via epsilonbeta conformations into a similar conformation. The results join the sequences into a growing group of sequences which have a tendency to form secondary structures and thereby to direct protein folding. The structures of the reverse turn conformations were in accordance with the experimental results (Hakalehto et al., Eur J. Biochem. 250, 19-29 (1997)). The main driving force of folding seems to be the hydrophobic interaction between the side chains of Ala and Leu at the i+1 and i+2 positions of the beta-turn.     

Neighboring nucleotide interactions during DNA sequencing gel electrophoresis.

Electrophoretic separation of oligonucleotides in denaturing polyacrylamide gels is primarily a function of length-dependent mobility. The 3' terminal nucleotide sequence of the oligonucleotide is a significant, secondary determinant of mobility and separation. Oligomers with 3'-ddT migrate more slowly than expected on the basis of length alone, and thus are better separated from the preceding, shorter oligomers in the sequencing ladder. Oligomers with 3'-ddC are relatively faster than expected, and are therefore less separated. At the 3' penultimate position, -dC- increases and -dT- reduces separation. Purines at the 3' terminal or penultimate positions of oligonucleotides affect separation less than the pyrimidines. These results suggest specific interactions among neighboring nucleotides with important effects on the conformation of oligonucleotides during electrophoresis. These interactions are compared to compression artifacts, which represent more extreme anomalies of length-dependent separation of oligonucleotides. Knowledge of base-specific effects on electrophoretic behavior of DNA oligomers supplements the usual information available for determination of sequences; additionally it provides an avenue to thermodynamic and hydrodynamic investigations of DNA structure.     

Raman studies of nucleic acids. XII. Conformations of oligonucleotides and deuterated polynucleotides

Laser Raman spectra of the trinucleoside diphoshate ApApA and dinucleoside phosphates ApU, UpA, GpC, CpG, and GpU are reported and discussed. Assignments of conformationally sensitive frequencies are-facilitated by comparison with spectra reported here of poly(rA), poly(rC), and poly(rU) in deuterium oxide solutions. The significant spectral differences between ApU and UpA, and between GpC and CpG, reveal that the sequence isomers have nonidentical conformations in aqueous solution. In UpA at low temperature the bases are stacked and the backbone conformation is similar to that found in ordered polynucleotide structures and RNA. In ApU no base stacking can be detected and the backbone conformation differs from that found in UpA, both in the orientation of phosphodiester linkages and in the internal conformation of ribose. At the conditions employed neither ApU nor UpA exhibits base pairing in aqueous solutions. In both GpC and CpG the bases are stacked and the phosphodiester conformations are similar to those encountered for UpA and RNA. However, major differences between spectra of GpC and CpG indicate that the geometries of stacking and ribosyl conformations are different. In GpC the Raman data favor the formation of hydrogen bonded dimers containing GC pairs. Protonation of C in GpC is sufficient to eliminate the ordered conformation detected by Raman spectroscopy. Despite the ordered backbone conformation evident in GpU, this dinucleoside apparently contains neither stacked nor hydrogen bonded bases at the conditions employed here. The Raman data also confirm the stacking interactions in ApApA, poly(rA), and poly(rC) but suggest that the backbone conformation in poly(rC) differs qualitatively from that found in most ordered polynucleotide structures and is thermally more stable. The present results demonstrate the sensitivity of the Raman technique to sequence-related structural differences in oligonucleotides and provide additional spectra–structure correlations for future conformational studies of RNA by laser Raman spectroscopy.     

Selective binding of looped oligonucleotides to a single-stranded DNA and its influence on replication in vitro.

Complexing of looped and circular oligonucleotides, composed of either 2'-deoxyribo- or 2'-O-methylribonucleoside units, with completely matching or partially mismatching complementary DNA sequences was studied. Melting experiments revealed considerable differences among the stabilities of these hybrid complexes. Maximum stability and selectivity was displayed by oligomers 2 and 5. It was concluded that a linear stretch, attached to 1'-O- of 3'-deoxypsicothymidine unit (Z) increases the selectivity of hybridisation and stability of the complex as a whole. This allows one to aim the target DNA very precisely at its polyadenine part as well as at adjacent sequence simultaneously. Experiments on termination of primer extension catalysed by different DNA-polymerases--Sequenase, Klenow fragment and Tth--have demonstrated that looped oligomer 5, composed of 2'-O-methylribonucleosides appears to be a highly selective and potent inhibitor of replication in vitro. Features of looped oligonucleotides, composed of 2'-O-methylribonucleosides seem to be useful for design of highly specific antigene oligonucleotides.     

Derivation of nearest-neighbor properties from data on nucleic acid oligomers. I. Simple sets of independent sequences and the influence of absent nearest neighbors

The constraints on combinations of nearest neighbors in nucleic acid sequences and the numbers of independent sequences needed to describe nearest-neighbor properties of oligomers and polymers are derived and summarized. It has been pointed out in previous work [D. M. Gray and I. Tinoco, Jr. (1970) Biopolymers, Vol. 9, pp. 223-244; R. F. Goldstein and A. S. Benight (1992) Biopolymers, Vol. 32, pp. 1679-1693] that these constraints restrict the information available from measurements of properties of sequence combinations. The emphasis in this paper is on the properties of oligomer sequences that vary in length, where each nucleotide or base pair at the end of the sequence makes a significant contribution to the measured property by interacting with its boundary of fixed sequence or solvent. In such cases it is not be possible to determine values of properties of individual nearest neighbors, except for the like neighbors [e.g., d(A-A), d(G-G), d(T-T), and d(C-C) nucleotide neighbors in single-stranded DNA or d(A-A)/d(T-T) and d(G-G)/d(C-C) base pair neighbors in double-stranded DNA], solely from measurements of properties of different sequences. Even values for properties of the like neighbors cannot be determined from such oligomeric sequences if the sequences are all of the same length. Nearest-neighbor properties of oligomer sequences that vary in length can be summarized in terms of the values for independent sets of sequences that are nearest neighbors and monomers all with boundaries of the fixed sequence or solvent. Straightforward combinations of the values for the independent sequences will give the values of the property for any dependent sequence, without explicit knowledge of the individual nearest-neighbor values. These considerations have important consequences for the derivation of widely used thermodynamic parameters, as discussed in the following paper.     

Detection of native-like models for amino acid sequences of unknown three-dimensional structure in a data base of known protein conformations.

We present an approach which can be used to identify native-like folds in a data base of protein conformations in the absence of any sequence homology to proteins in the data base. The method is based on a knowledge-based force field derived from a set of known protein conformations. A given sequence is mounted on all conformations in the data base and the associated energies are calculated. Using several conformations and sequences from the globin family we show that the native conformation is identified correctly. In fact the resolution of the force field is high enough to discriminate between a native fold and several closely related conformations. We then apply the procedure to several globins of known sequence but unknown three dimensional structure. The homology of these sequences to globins of known structures in the data base ranges from 49 to 17%. With one exception we find that for all globin sequences one of the known globin folds is identified as the most favorable conformation. These results are obtained using a force field derived from a data base devoid of globins of known structure. We briefly discuss useful applications in protein structural research and future development of our approach.     

Hairpin and parallel quartet structures for telomeric sequences.

The role of thymine residues in the formation of G-quartet structures for telomeric sequences has been investigated using model oligonucleotides of the type d(G4TnG4), with n = 1-4. Sequences d(G4T3G4) and d(G4T4G4) adopt a G-quartet structure formed by hairpin dimerization in 70 mM NaCl as judged by a characteristic circular dichroism signature with a 295 nm positive and 265 nm negative bands while d(G4TG4) adopts a parallel G-quartet structure like d(G12) which exhibits a strong positive band at 260 nm and a negative band at 240 nm. The sequence d(G4T2G4) exhibits a mixture of both conformations. The stability of hairpin G-quartet structures decreases with decrease in the number of intervening thymine residues. Potassium permanganate, a single strand specific probe has been used to establish the presence of loops composed of T residues in the hairpin G quartet structures formed by the oligonucleotides d(G4TnG4) with n = 2-4 in 70 mM NaCl. The formation of hairpin G quartet structure for the above sequences is further supported by the enhanced electrophoretic mobility observed on non-denaturing polyacrylamide gels. Human telomeric sequence d(TTAGGG)4 which showed enhanced electrophoretic mobility like Tetrahymena telomeric sequence d(T2G4)4 also exhibited a characteristic CD spectrum for a folded-back G-quartet structure. A detailed model for G-quartet structure involving hairpin dimer with alternating syn-anti-syn-anti conformation for the guanine residues both along the chain as well as around the G tetrad with at least two thymine residues in the loop is proposed. Intermolecular association of short telomeric sequences reported here provides a possible model for chromosomal pairing.     

DNA flexibility as a function of allomorphic conformation and of base sequence.

Systematic theoretical modeling of symmetric DNA oligomers, carried out earlier for the B conformation, is now extended to A-DNA. In contrast to the previous results, it is found that A-DNA shows no multiplicity of low-energy substate conformations. The possibilities of the Jumna algorithm are subsequently applied to studying deformations of the oligomers. Controlled winding and stretching deformations are used to study how the two allomorphs and different base sequences absorb such external stress. The results help explain the internal mechanics of the DNA double helix and the extent to which fine structure influences this behavior. The results point to some differences between the A and B double helices, but also to many similarities. Sequence effects on flexibility are relatively limited compared to their impact on optimal energy conformations. It is also shown that the conformational substates detected for B-DNA oligomers are preserved under deformation, but have little influence on its energetics.     

B-Z Transition of Synthetic Oligonucleotides Containing Non-Z Forming Elements

Abstract

Ten oligonucleotides of the length 8–12 base pairs have been synthesized, which contain, in addition to the obligatory sequences CG/CG, sequences not favorable for the transition to the Z conformation (AT pairs, GG/CC or AA/TT sequences). Conformational transitions of these oligonucleotides in high concentrations of NaClO₄ in the absence and in the presence of Ni²⁺ were investigated using CD spectroscopy.

The B-Z transition is affected by the length and sequence of the oligonucleotide. Increasing the NaC1O₄ concentration alone the transition of only one of the oligonucleotides studied, (CGCGCGTGC ACGCGCG)₂, can be induced. Other oligonucleotides remain in the B conformation or only partial transition to the Z conformation can be observed.

Most other oligonucleotides can be converted into the Z conformation at intermediate concentrations of NaC1O₄ (2.0?3.2 M) by an addition of Ni²⁺ ions. In some cases, however, Ni²⁺ can destabilize the double stranded structure of the sample. We have studied the effect of the presence of A.T pairs in the G.C containing oligonucleotides and the effect of the presence of pu-pu/pyr-pyr sequences. The presence of the latter sequences in the Z form implicates the formation of a Z-Z′ junction which makes the transition quite difficult. Despite the fact that some oligonucleotides contained several structural elements not favorable for the transition, we did not find any sequence which would completely block the ability of the oligonucleotide to adopt the Z conformation.     

Sequence dependent conformations of oligomeric DNA's in aqueous solutions and in crystals

In order to determine the sequence dependence of the conformation of deoxynucleotides, Raman spectra have been obtained for the following oligodeoxynucleotides in aqueous salt solutions and in crystals: d(CpG)(I), d(TGCGCGCA)(II), d(CACGCGTG)(III), d(CGTGCACG)(IV), d(CGCATGCG)(V), d(ACGCGCGT)(VI), d(CGCGTACGCG)(VII), d(CGCACGTGCG)(VIII) and d(CGTGCGCACG)(IX), d(GCTATAGC) (X), d(GCATATGC) (XI), d(GGTATACC) (XII) and d(GGATATCC) (XIII). The normal B type conformation is observed for all the oligomer DNA's at low salt (0.1-1.0 M NaCl) concentration in the temperature range of 0-25 degrees C. It was considered possible that all of the first nine oligomers could go into the Z form in aqueous high salt (5.0-6.0 M NaCl) solutions, and under these conditions the last four were considered candidates to go into the A form. The B-type conformation was found to exist in high salt solutions for (I), (IV), (V), (VI), (X), (XI) and (XIII); the Z or partial Z conformation appears in high salt solution for the oligomers, (II), (III), (VII), (VIII) and (IX); an A or partial A conformation appears in high salt solution for (XII). In the crystalline state, (IV), (VIII), (X), and (XI) stay in the B-form and all of the other oligomers adopt the complete Z-form except for (XII) which crystallizes in the A form. In both the crystal and in aqueous solutions, the identification of the conformation genus was made by means of Raman spectroscopy. In the crystal of (I), grown at pH7.0, guanosine is found to be in C3'-endo/syn conformation and cytidine in C2'-endo/anti, which may be taken as the ideal building block of the typical Z conformation. At pH4, (I) crystallizes in a conformation similar to the B genus. A study of the thermally induced B to Z transition has been carried out for (II) and (III). Based on the analysis of Raman spectra of the alternating pyrimidine-purine oligomers which might be expected to go into the Z form, the tendency for these oligonucleotides to adopt the Z form can be ranked as: d(CGCGCGCG) greater than (II) greater than (III) greater than (V) approximately (VI) greater than (IV) for octamers and (VII) greater than (VIII) greater than (IX) for the decamers. Similarly, those oligomers which might have a tendency to go into the A form could be ranked as (XII) greater than (XIII) approximately (X) greater than (XI). These data should provide help in formulating rules for predicting the sequence dependence of the B to A and B to Z transitions. Some possible rules are explored, but precautions should be taken.     

Conformational studies of oligomeric oxetane-based dipeptide isosteres derived from L-rhamnose or D-xylose.

Conformational investigations have been undertaken on oligomers (dimers, tetramers, hexamers) of five closely related oxetane-based dipeptide isosteres. All the oligomers were subjected to a range of studies by NMR, FT-IR and CD spectroscopy. The oligomers derived from methyl 2,4-anhydro-5-azido-3-O-tert-butyldimethylsilyl-5-deoxy-L-rhamnonate 'monomer' all exhibited evidence of ordered conformations in chloroform and 2,2,2-trifluoroethanol (TFE) solution. 5-Acetamido and N-methylamide derivatives of the L-rhamnonate 'monomer', along with a 'dimer' lacking silyl protection at C-3, were synthesized to ascertain the role of intramolecular interactions. This led to the conclusion that, for the L-rhamnonate oligomers, steric interactions govern the conformational preference observed. The equivalent silyl-protected D-lyxonate oligomers gave ordered CD spectra in TFE solution, but NMR and FT-IR spectroscopy in chloroform solution suggested an irregular, non-hydrogen bonded system. The remaining silyl-protected 6-deoxy-L-altronate, 6-deoxy-D-gulonate and D-fuconate oligomers appear to be characterized by their lack of ordered conformation in TFE and chloroform solution.     

The conformations of locked nucleic acids (LNA)

We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2'-O, 4'-C-methylene bridge. We have investigated a modified 9-mer single stranded oligonucleotide as well as three 9- and 10-mer modified oligonucleotides hybridized to unmodified DNA. The single-stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson-Crick base-pairing with all the nucleotides in anti-conformation. By use of selective DQF-COSY spectra we determined the ratio between the N-type (C3'-endo) and S-type (C2'-endo) sugar conformations of the nucleotides. In contrast to the corresponding single-stranded DNA (ssDNA), we found that the sugar conformations of the single-stranded LNA oligonucleotide (ssLNA) cannot be described by a major S-type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N-type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N-type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S-type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N-type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases.     

Searching for folding initiation sites of staphylococcal nuclease: a study of N-terminal short fragments

The N-terminal short fragments of staphylococcal nuclease (SNase), SNase20, SNase28, and SNase36, corresponding to the sequence regions, Ala1-Gly20, Ala1-Lys28, and Ala1-Leu36, respectively, as well as an 8-residue peptide (Ala17-Ile18-Asp19-Gly20-Asp21-Thr22-Val23-Lys24) have been synthesized. The conformational states of these fragments were investigated using CD and NMR spectroscopy in aqueous solution and in trifluoroethanol (TFE)-H(2)O mixture. SNase20 containing a sequence corresponding to a bent peptide in native SNase shows a transient population of bend-like conformation around Ala12-Thr13-Leu14 in TFE-H(2)O mixture. The sequence region of Ala17-Thr22 of SNase28 displays a localized propensity for turn-like conformation in both aqueous solution and TFE-H(2)O mixture. The conformational ensemble of SNase36 in aqueous solution includes populated turn-like conformations localized in sequence regions Ala17-Thr22 and Tyr27-Gln30. The analysis suggests that these sequence regions, which form the regular secondary structures in native protein, may serve as the folding nucleation sites of SNase fragments of different chain lengths starting from the N-terminal end. Thus, the formation of bend- and turn-like conformations of these sequence regions may be involved in the early folding events of the SNase polypeptide chain in vitro.