The effect of base sequence on the stability of RNA and DNA single base bulges

Forty-eight RNA duplexes were constructed that contained all common single base bulges at six different locations. The stabilities of the RNAs were determined by temperature gradient gel electrophoresis (TGGE). The relative stability of a single base bulge was dependent on both base identity and the nearest neighbor context. The single base bulges were placed into two categories. A bulged base with no identical neighboring base was defined as a Group I base bulge. Group II-bulged bases had at least one neighboring base identical to it. Group II bulges were generally more stable than Group I bulges in the same nearest neighbor environments. This indicates that position degeneracy of an unpaired base enhances stability. Differences in the mobility transition temperatures between the RNA fragments with bulges and the completely base-paired reference RNAs were related to free energy differences. Simple models for estimating the free energy contribution of single base bulges were evaluated from the free energy difference data. The contribution of a Group I bulge 5'-(XNZ)-3'.5'-(Z'-X')-3' where N is the unpaired base and X.X' and Z.Z' the neighboring base pairs, could be well-represented (+/-0.34 kcal/mol) by the equation, DeltaG((X)(N)()(Z))(.)((Z)(')(-)(X)(')()) = 3.11 + 0. 40DeltaG(s)()((XZ))(.)((Z)(')(X)(')()). DeltaG(s)()((XZ))(. )((Z)(')(X)(')()) is the stacking energy of the closing base pair doublet. By adding a constant term, delta = -0.3 kcal/mol, to the right side of the above equation, free energies of Group II bulges could also be predicted with the same accuracy. The term delta represents the stabilizing effect due to position degeneracy. A similar equation/model was applied to previous data from 32 DNA fragments with single base bulges. It predicted the free energy differences with a similar standard deviation.     

Thermodynamic parameters for an expanded nearest-neighbor model for the formation of RNA duplexes with single nucleotide bulges

Thirty-four RNA duplexes containing single nucleotide bulges were optically melted, and the thermodynamic parameters deltaH degrees, deltaS degrees, deltaG degrees (37), and T(M) for each sequence were determined. Data from this study were combined with data from previous thermodynamic data [Longfellow, C. E., Kierzek, R., and Turner, D. H. (1990) Biochemistry 29, 278-85] to develop a model that will more accurately predict the free energy of an RNA duplex containing a single nucleotide bulge. Differences between purine and pyrimidine bulges as well as differences between Group I duplexes, those in which the bulge is not identical to either neighboring nucleotide, and Group II duplexes, those in which the bulge is identical to at least one neighboring nucleotide, were considered. The length of the duplex, non-nearest-neighbor effects, and bulge location were also examined. A model was developed which divides sequences into two groups: those with pyrimidine bulges and those with purine bulges. The proposed model for pyrimidine bulges predicts deltaG degrees (37,bulge) = 3.9 kcal/mol + 0.10deltaG degrees (37,nn) + beta, while the model for purine bulges predicts deltaG degrees (37,bulge) = 3.3 kcal/mol - 0.30deltaG degrees (37,nn) + beta, where beta has a value of 0.0 and -0.8 kcal/mol for Group I and Group II sequences, respectively, and deltaG degrees (37,nn) is the nearest-neighbor free energy of the base pairs surrounding the bulge. The conformation of bulge loops present in rRNA was examined. Three distinct families of structures were identified. The bulge loop was either extrahelical, intercalated, or in a "side-step" conformation.     

Investigation of potential RNA bulge stabilizing elements

As a part of our interest in recognition and cleavage of RNA we carried out thermal melting studies with the aim of screening a number of simple oligonucleotide modifications for their potential as modifying elements for RNA bulge stabilizing oligonucleotides. A specific model system from our studies on oligonucleotide-based artificial nuclease (OBAN) systems was chosen and the bulge size was varied from one to five nucleotides. Introduction of single 2'-modified nucleoside moieties (2'-O-methyl, 2'-deoxy and 2'-deoxy-2'-amino) with different conformational preferences adjacent to the bulge revealed that a higher preference for the north conformers gave more stable bulges across the whole range of bulge sizes. Changing a bulge closing a G-U wobble base pair to a G-C pair resulted in the interesting observation that, although the fully complementary complex and small bulges were highly stabilized, there was little difference in the stability of the larger bulges. The wobble base pair even gave a slight stabilization of the 5 nt bulge system. Introduction of a uridine C-5 linker with a single ammonium group was clearly bulge stabilizing (DeltaT(m) + 4.6 to + 5.4 degrees C for the three most stabilized bulges), although with limited selectivity for different bulge sizes since the fully complementary duplex was also stabilized. Introduction of a naphthoyl group on a 2'-aminolinker mostly gave a destabilizing effect, while introduction of a 5-aminoneocuproine moiety on the same linker resulted in stabilization of all bulges, in particular those with two or four unpaired nucleotides (DeltaT(m) + 3.6 and + 2.9 degrees C respectively). The aromatic groups destabilize the fully complementary duplex, resulting in higher selectivity towards stabilization of bulges. A combination of the studied partial element should be suitable for future designs of modified oligonucleotides that, apart from standard base pairing, can also provide additional non-Watson-Crick recognition of RNA.     

Non-nearest-neighbor dependence of the stability for RNA bulge loops based on the complete set of group I single-nucleotide bulge loops

Fifty-nine RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters DeltaH degrees, DeltaS degrees, DeltaG 37 degrees, and TM for each sequence were determined. Sequences from this study were combined with sequences from previous studies [Longfellow, C. E., et al. (1990) Biochemistry 29, 278-285; Znosko, B. M., et al. (2002) Biochemistry 41, 10406-10417], thus examining all possible group I single-nucleotide bulge loop and nearest-neighbor sequence combinations. The free energy increments at 37 degrees C for the introduction of a group I single-nucleotide bulge loop range between 1.3 and 5.2 kcal/mol. The combined data were used to develop a model for predicting the free energy of a RNA duplex containing a single-nucleotide bulge. For bulge loops with adjacent Watson-Crick base pairs, neither the identity of the bulge nor the nearest-neighbor base pairs had an effect on the influence of the bulge loop on duplex stability. The proposed model for prediction of the stability of a duplex containing a bulged nucleotide was primarily affected by non-nearest-neighbor interactions. The destabilization of the duplex by the bulge was related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. The stability of a duplex containing a bulged nucleotide adjacent to a wobble base pair also was primarily affected by non-nearest-neighbor interactions. Again, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. However, when one or both of the bulge nearest neighbors was a wobble base pair, the free energy increment for insertion of a bulge loop is dependent upon the position and orientation of the wobble base pair relative the bulged nucleotide. Bulge sequences of the type ((5'UBX)(3'GY)), ((5'GBG)(3'UU)) and ((5'UBU)(3'GG)) are less destabilizing by 0.6 kcal/mol, and bulge sequences of the type ((5'GBX)(3'UY)) and ((5'XBU)(3'YG)) are more destabilizing by 0.4 kcal/mol than bulge loops adjacent to Watson-Crick base pairs.     

Bulge loops used to measure the helical twist of RNA in solution.

Bulge loops are commonly found in helical segments of cellular RNAs. When incorporated into long double-stranded RNAs, they may introduce points of flexibility or permanent bend that can be detected by the altered electrophoretic gel mobility of the RNA. We find that a single An or Un bulge loop near the middle of a long RNA helix significantly retards the RNA during polyacrylamide gel electrophoresis if n greater than or equal to 2. The mobility of an RNA containing two A2 bulges various periodically with the number of base pairs between the bulges. We interpret this to mean that A2 bulges varies periodically with the number of base pairs between the bulges. We interpret this to mean that Z2 bulges form torsionally stiff bends in the helix; the gel mobility reaches a minimum when the total helical twist between the bulges rotates the arms of the molecule into a cis conformation. The gel mobilities are proportional to the predicted end-to-end distance of the RNA if the average RNA helical repeat is 11.8 +/- 0.2 bp/turn and there is no helical twist (3 +/- 9 degrees) associated with the bulge (data obtained in 0.15 M Na+). Other sizes and sequences of bulges have very different effects on RNA helix conformation and flexibility. U2 bulges bend the helix to a much smaller degree than A2 bulges, while longer A or U bulge sequences probably allow bends of 90 degrees or more; all of these may be fairly flexible joints.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of bulge composition and flanking sequence on the kinking of DNA by bulged bases

We recently showed that bulged bases kink duplex DNA, with the degree of kinking increasing in roughly equal increments as the number of bases in the bulge increases from one to four [Hsieh, C.-H., & Griffith, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837]. Here we have examined the kinking of DNA by single A, C, G, or T bulges with different neighboring base pairs. Synthetic 30 base pair (bp) duplex DNAs containing 2 single-base bulges spaced by 10 bp were ligated head to tail, and their electrophoretic behavior in highly cross-linked gels was examined. All bulge-containing DNAs showed marked electrophoretic retardations as compared to non-bulge-containing DNA. Regardless of the sequence of the flanking base pairs, purine bulges produced greater retardations than pyrimidine bulges. Furthermore, C and T bulges produced the same retardations as did G and A bulges. Bulged DNA containing different flanking base pairs showed marked differences in electrophoretic mobility. For C-bulged DNA, the greatest retardations were observed with G.C neighbors, the least with T.A neighbors, and an intermediate amount with a mixture of neighboring base pairs. For A-bulged DNA, the retardations were greatest with G.C neighbors, less with T.A neighbors, even less with a mixture of neighboring base pairs, and finally least with C.G neighbors. Thus flanking base pairs affect C-bulged DNA and A-bulged DNA differently, and G.C and C.G flanking base pairs were seen to have very different effects. These results imply an important role of base stacking in determining how neighboring base pairs influence the kinking of DNA by a single-base bulge.     

Effect of single-residue bulges on RNA double-helical structures: crystallographic database analysis and molecular dynamics simulation studies

Asymmetric bulge loop motifs are widely dispersed in all types of functional RNAs. They are frequently occurring structural motifs in folded RNA structures and appear commonly in pre-microRNA and ribosomes, where they are involved in specific RNA–RNA and RNA–protein interactions. It is therefore necessary to understand such motifs from a structural point of view. We analyzed all available RNA structures and identified quite a few fragments of double helices that contain bulges. We found that these discontinuities often introduce kinks into the double helices, which also affects the stacking overlap between the base pairs across the irregularity. In order to understand the influence of these bulges on stability and flexibility, we carried out molecular dynamics simulations of three different single-residue bulge-containing RNA helices using the CHARMM36 force field. The structural variability at the junctions of RNA bulges is expected to differ from that in continuous double-helical stretches. The structural features of the junction region were observed to vary noticeably depending on the orientation of the bulge residue. When the base of the bulge residue is looped out, the RNA stretch behaves like a standard long A-form RNA double helix, whereas the entire RNA behaves differently when the base of the bulge residue is intercalated between base pairs inside the RNA stem. Such single-base intercalation was found to introduce a permanent kink into the composite double helix, which could be a recognition element for Dicer during the maturation of miRNA.     

Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.     

Stabilization factors affecting duplex formation of peptide nucleic acid with DNA.

Peptide nucleic acid (PNA) is an oligonucleotide analogue in which the sugar-phosphate backbone is replaced by an N-(2-aminoethyl)glycine unit to which the nucleobases are attached. We investigated the thermodynamic behavior of PNA/DNA hybrid duplexes with identical nearest neighbors but with different sequences and chain lengths (5, 6, 7, 8, 10, 12, and 16 mers) to reveal whether the nearest-neighbor model is valid for the PNA/DNA duplex stability. CD spectra of 6, 7, and 8 mer PNA/DNA duplexes showed similar signal, while 10, 12, and 16 mer duplexes did not. The average difference in Delta G degrees (37) for short PNA/DNA duplexes with identical nearest-neighbor pairs was only 3.5%, whereas that of longer duplexes (10, 12, and 16 mers) was 16.4%. Therefore, the nearest-neighbor model seems to be useful at least for the short PNA/DNA duplexes. Thermodynamics of PNA/DNA duplexes containing 1--3 bulge residues were also studied. While the stability of the 12 mer DNA/DNA duplex decreased as the number of bulge bases increases, the number of bulge bases in PNA/DNA unchanged the duplex stability. Thus, the influence of bulge insertion in the PNA/DNA duplexes is different from that of a DNA/DNA duplex. This might be due to the different base geometry in a helix which may potentially make hydrogen bonds in a base pair and stacking interaction unfavorable compared with DNA/DNA duplexes.     

Solution structure of a trinucleotide A-T-A bulge loop within a DNA duplex.

We have synthesized an oligodeoxynucleotide duplex, d(G-C-A-T-C-G-A-T-A-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-C-G), with a three-base bulge loop (A-T-A) at a central site in the first strand. Nuclear Overhauser experiments (NOESY) in H2O indicate that the GC base pairs flanking the bulge loop are intact between 0 and 25 degrees C. Nuclear Overhauser effects in both H2O and D2O indicate that all bases within the bulge loop are stacked into the helix. These unpaired bases retain an anti conformation about their glycosidic bonds as they stack within the duplex. The absence of normal sequential connectivities between the two cytosine residues flanking the bulge site on the opposite strand indicates a disruption in the geometry of this base step upon insertion of the bulged bases into the helix. This conformational perturbation is more akin to a shearing apart of the bases, which laterally separates the two halves of the molecule, rather than the "wedge" model often invoked for single-base bulges. Using molecular dynamics calculations, with both NOE-derived proton-proton distances and relaxation matrix-calculated NOESY cross peak volumes as restraints, we have determined the solution structure of an A-T-A bulge loop within a DNA duplex. The bulged bases are stacked among themselves and with the guanine bases on either side of the loop. All three of the bulged bases are displaced by 2-3 A into the major groove, increasing the solvent accessibility of these residues. The ATA-bulge duplex is significantly kinked at the site of the lesion, in agreement with previously reported electron microscopy and gel retardation studies on bulge-containing duplexes [Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A 86, 4833-4837; Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840]. Bending occurs in a direction away from the bulge-containing strand, and we find a significant twist difference of 84 degrees between the two base pairs flanking the bulge loop site. This value represents 58% of the twist difference for base pairs four steps apart in B-DNA. These results suggest a structural mechanism for the bending of DNA induced by unpaired bases, as well as accounting for the effect bulge loops may have on the secondary and tertiary structures of nucleic acids.     

Conformational transitions in RNA single uridine and adenosine bulge structures: a molecular dynamics free energy simulation study

Extra unmatched nucleotides (single base bulges) are common structural motifs in folded RNA molecules and can participate in RNA-ligand binding and RNA tertiary structure formation. Often these processes are associated with conformational transitions in the bulge region such as flipping out of the bulge base from an intrahelical stacked toward a looped out state. Knowledge of the flexibility of bulge structures and energetics of conformational transitions is an important prerequisite to better understand the function of this RNA motif. Molecular dynamics simulations were performed on single uridine and adenosine bulge nucleotides at the center of eight basepair RNA molecules and indicated larger flexibility of the bulge bases compared to basepaired regions. The umbrella sampling method was applied to study the bulge base looping out process and accompanying conformational and free energy changes. Looping out toward the major groove resulted in partial disruption of adjacent basepairs and was found to be less favorable compared to looping out toward the minor groove. For both uridine and adenosine bulges, a positive free energy change for full looping out was obtained which was approximately 1.5 kcal mol-1 higher in the case of the adenosine compared to the uridine bulge system. The simulations also indicated stable partially looped out states with the bulge bases located in the RNA minor groove and forming base triples with 5'-neighboring basepairs. In the case of the uridine bulge this state was more stable than the intrahelical stacked bulge structure. Induced looping out toward the minor groove involved crossing of an energy barrier of approximately 3.5 kcal mol-1 before reaching the base triple state. A continuum solvent analysis of intermediate bulge states indicated that electrostatic interactions stabilize looped out and base triple states, whereas van der Waals interactions and nonpolar contributions favor the stacked bulge conformation.     

Base pairing geometry in GA mismatches depends entirely on the neighboring sequence.

We have synthesized nine self-complementary DNA oligomers containing different flanking sequences adjacent to a pair of contiguous GA mismatches, and have used high resolution nuclear magnetic resonance (n.m.r.) to investigate the GpA phosphodiester backbone conformation and mismatch pairing schemes in these duplexes. We found dramatic effects of the flanking base pair on the hydrogen bonding and backbone conformation, which appear to be coupled. Thus the Ganti-Aanti base pairing scheme in a NAGATN sequence switches to a more stable sheared GA base pairing scheme in a NCGAGN or NTGAAN context, while no duplex is formed (or only GA bulges occur) when NAGATN is changed to NGGACN. Furthermore, the more stable sheared GA pairing in NPyGAPuN sequences is associated with a BII rather than BI backbone conformation for the phosphodiester between the adjacent mismatched GA pairs. The overall stability of these adjacent GA mismatches as measured by imino proton n.m.r. studies is Py-GA-Pu > A-GA-T > G-GA-C.     

Solution structure of dAATAA and dAAUAA DNA bulges

The NMR structure analysis is described for two DNA molecules of identical stem sequences with a five base loop containing a pyrimidine, thymin or uracil, in between purines. These five unpaired nucleotides are bulged out and are known to induce a kink in the duplex structure. The dAATAA bulge DNA is kinked between the third and the fourth nucleotide. This contrasts with the previously studied dAAAAA bulge DNA where we found a kink between the fourth and fifth nucleotide. The total kinking angle is ~104° for the dAATAA bulge. The findings were supported by electrophoretic data and fluorescence resonance energy transfer measurements of a similar DNA molecule end-labeled by suitable fluorescent dyes. For the dAAUAA bulge the NMR data result in a similar structure as reported for the dAATAA bulge with a kinking angle of ~87°. The results are discussed in comparison with a rAAUAA RNA bulge found in a group I intron. Generally, the sequence-dependent structure of bulges is important to understand the role of DNA bulges in protein recognition.     

Non-nearest-neighbor dependence of the stability for RNA group II single-nucleotide bulge loops

Thirty-one RNA duplexes containing single-nucleotide bulge loops were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each sequence were determined. The bulge loops were of the group II variety, where the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The data were used to develop a model to predict the free energy of an RNA duplex containing a single-nucleotide bulge. The destabilization of the duplex by the bulge was primarily related to the stability of the stems adjacent to the bulge. Specifically, there was a direct correlation between the destabilization of the duplex and the stability of the less stable duplex stem. Since there is an ambiguity of the bulge position for group II bulges, several different stem combinations are possible. The destabilization of group II bulge loops is similar to the destabilization of group I bulge loops, if the second least stable stem is used to predict the influence of the group II bulge. In-line structure probing of the group II bulge loop embedded in a hairpin indicates that the bulged nucleotide is the one positioned farther from the hairpin loop.     

Sequence dependence for the energetics of dangling ends and terminal base pairs in ribonucleic acid

Stability increments of terminal unpaired nucleotides (dangling ends) and terminal base pairs on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 18 oligoribonucleotides containing the core sequences. The results indicate 3' dangling purines add more stability than 3' dangling pyrimidines. In most cases, the additional stability from a 3' dangling end on an AU base pair is less than that on a GC base pair [Freier, S.M., Burger, B.J., Alkema, D., Neilson, T., & Turner, D.H. (1985) Biochemistry 22, 6198-6206]. The sequence dependence provides a test for the importance of dangling ends for various RNA interactions. Correlations are suggested with codon context effects and with the three-dimensional structure of yeast phenylalanine transfer RNA. In the latter case, all terminal unpaired nucleotides having stability increments more favorable than -1 kcal/mol are stacked on the adjacent base pair. All terminal unpaired nucleotides having stability increments less favorable than -0.3 kcal/mol are not stacked on the adjacent base pair. In several cases, this lack of stacking is associated with a turn in the sugar-phosphate backbone. This suggests stability increments measured on oligoribonucleotides may be useful for predicting tertiary structure in large RNA molecules. Comparison of the stability increments for terminal dangling ends and base pairs, and of terminal GC and AU base pairs, indicates the free energy increment associated with forming a hydrogen bond can be about -1 kcal/mol of hydrogen bond.     

Mechanics of DNA flexibility visualized by selective 2'-amine acylation at nucleotide bulges

We used selective acylation of 2'-amine-substituted nucleotides to visualize local backbone conformations that occur preferentially at bulged sites in DNA duplexes. 2'-Amine acylation reports local nucleotide flexibility because unconstrained 2'-amino nucleotides more readily reach a reactive conformation in which the amide-forming transition state is stabilized by interactions between the amine nucleophile and the adjacent 3'-phosphodiester group. Bulged 2'-amine-substituted cytidine nucleotides react approximately 20-fold more rapidly than nucleotides constrained by base-pairing at 35 degrees C. In contrast, base-paired 2'-amine-substituted nucleotides flanked by a 5' or 3' bulge react two- or six-fold more rapidly, respectively, than the perfectly paired duplex. The relative lack of 2'-amine reactivity for nucleotides adjacent to a DNA bulge emphasizes, first, that structural perturbations do not extend significantly into the flanking duplex structure. Second, the exquisite sensitivity towards very local perturbations in nucleic acid structure suggests that 2'-amine acylation can be used to chemically interrogate deletion mutations in DNA. Finally, these data support the mechanical interpretation that the reactive ribose conformation for 2'-amine acylation requires that the base lies out of the helix and in the major groove, a mechanistic insight useful for designing 2'-amine-based sensors.     

Predicting stability of DNA bulge at mononucleotide microsatellite

Mononucleotide microsatellites are clinically and forensically crucial DNA sequences due to their high mutability and abundance in the human genome. As a mutagenic intermediate of an indel in a microsatellite and a consequence of probe hybridization after such mutagenesis, a bulge with structural degeneracy sliding within a microsatellite is formed. Stability of such dynamic bulges, however, is still poorly understood despite their critical role in cancer genomics and neurological disease studies. In this paper, we have built a model that predicts the thermodynamics of a sliding bulge at a microsatellite. We first identified 40 common bulge states that can be assembled into any sliding bulges, and then characterized them with toehold exchange energy measurement and the partition function. Our model, which is the first to predict the free energy of sliding bulges with more than three repeats, can infer the stability penalty of a sliding bulge of any sequence and length with a median prediction error of 0.22 kcal/mol. Patterns from the prediction clearly explain landscapes of microsatellites observed in the literature, such as higher mutation rates of longer microsatellites and C/G microsatellites.     

Comparative NMR study of A(n)-bulge loops in DNA duplexes: intrahelical stacking of A, A-A, and A-A-A bulge loops.

We have prepared a series of deoxyoligonucleotide duplexes of the sequence d(G-C-A-T-C-G-X-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-G-C), in which X represents either one (A), two (A-A), or three (A-A-A) unpaired adenine basis. Using two-dimensional proton and phosphorus NMR spectroscopy, we have characterized conformational features of these bulge-loop duplexes in solution. We find that Watson-Crick hydrogen bonding is intact for all 12 base pairs, including the GC bases that flank the bulge loop. Observation of NOE connectivities in both H2O and D2O allows us to unambiguously localize all of the bulged adenine residues to intrahelical positions within the duplex. This is in contrast to an earlier model for multiple-base bulge loops in DNA [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840], in which all but the most 5' bulged base are looped out into solution. We find that insertion of two or three bases into the duplex results in the disruption of specific sequential NOEs for the base step across from the bulge loop site on the opposite strand. This disruption is characterized by a partial shearing apart of these bases, such that certain sequential NOEs for this base step are preserved. We observe a downfield-shifted phosphorus resonance, which we assign in the A-A-A bulge duplex to the 3' side of the last bulged adenine residue. Proton and phosphorus chemical shift trends within the An-bulge duplex series indicate that there is an additive effect on the structural perturbations caused by additional unpaired bases within the bulge loop. This finding parallels previous observations [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840; Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837] on the magnitude of the induced bending of DNA duplexes by multiple-base bulge loops.     

Conformational transitions in thymidine bulge-containing deoxytridecanucleotide duplexes. Role of flanking sequence and temperature in modulating the equilibrium between looped out and stacked thymidine bulge states

Structural features at extra thymidine bulge sites in DNA duplexes have been elucidated from a two-dimensional NMR analysis of through-bond and through-space connectivities in the otherwise self-complementary d(C-C-G-T-G-A-A-T-T-C-C-G-G) (GTG 13-mer) and d(C-C-G-G-A-A-T-T-C-T-C-G-G) (CTC 13-mer) duplexes in aqueous solution. These studies establish that the extra thymidine flanked by guanosines in the GTG 13-mer duplex is in a conformational equilibrium between looped out and stacked states. The looped-out state is favored at low temperature (0 degrees C), whereas the equilibrium shifts in favor of the stacked state at elevated temperatures (35 degrees C) prior to the onset of the duplex-strand transition. By contrast, the extra thymidine flanked by cytidines in the CTC 13-mer duplex is looped out independent of temperature in the duplex state. Our results demonstrate that temperature and flanking sequence modulate the equilibrium between looped-out and stacked conformations of single base thymidine bulges in DNA oligomer duplexes.