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

Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson–Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson–Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson–Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3′ or 5′ of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.     

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.     

Thermodynamic parameters based on a nearest-neighbor model for DNA sequences with a single-bulge loop

All 64 possible thermodynamic parameters for a single-bulge loop in the middle of a sequence were derived from optical melting studies. The relative stability of a single bulge depended on both the type of bulged base and its flanking base pairs. The contribution of the single bulge to helix stability ranged from 3.69 kcal/mol for a TAT bulge to -1.05 kcal/mol for an ACC bulge. Thermodynamics for 10 sequences with a GTG bulge were determined to test the applicability of the nearest-neighbor model to a single-bulge loop. Thermodynamic parameters for the GTG bulge and Watson-Crick base pairs predict, DeltaH degrees, DeltaS degrees, and T(M)(50 microM) values with average deviations of 3.0%, 4.3%, 4.7%, and 0.9 degrees C, respectively. The prediction accuracy was within the limits of what can be expected for a nearest-neighbor model. This certified that the thermodynamics for single-bulge loops can be estimated adequately using a nearest-neighbor model.     

Stability of single-nucleotide bulge loops embedded in a GAAA RNA hairpin stem

Forty-six RNA hairpins containing combinations of 3' or 5' bulge loops and a 3' or 5' fluorescein label were optically melted in 1 M NaCl, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each hairpin were determined. The bulge loops were of the group I variety, in which the identity of the bulge is known, and the group II variety, in which the bulged nucleotide is identical to one of its nearest neighbors, leading to ambiguity as to the exact position of the bulge. The fluorescein label at either the 3' end or 5' end of the hairpin did not significantly influence the stability of the hairpin. As observed with bulge loops inserted into a duplex motif, the insertion of a bulge loop into the stem of a hairpin loop was destabilizing. The model developed to predict the influence of bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability. Specifically, the influence of the bulge is related to the stability of the hairpin stem distal from the hairpin loop.     

Thermodynamic and spectroscopic study of bulge loops in oligoribonucleotides

Thermodynamic parameters for bulge loops of one to three nucleotides in oligoribonucleotide duplexes have been measured by optical melting. The results indicate bulges Bn of An and Un have similar stabilities in the duplexes, GCGBmGCG + CGCCGC. The stability increment for a bulge depends on more than its adjacent base pairs. For example, the stability increment for a bulge is affected more than 1 kcal/mol by changing two nonadjacent base pairs or by adding terminal unpaired nucleotides (dangling ends) three base pairs away. Thus a nearest-neighbor approximation for helixes with bulges is oversimplified. Many of the non-self-complementary strands used in this study were observed to form homoduplexes. Such duplexes with GA mismatches were particularly stable.     

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.     

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.     

Sequence dependence of the stability of RNA hairpin molecules with six nucleotide loops

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequence of the types GCGXUAAUYCGC and GGUXUAAUYACC with Watson-Crick loop closure, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C varies from 3.1 to 5.1 kcal/mol. These results agree with the model previously developed [Vecenie, C. J., and Serra, M. J. (2004) Biochemistry 43, 11813] to predict the stability of RNA hairpin loops: DeltaG degrees (37L(n) = DeltaG degrees (37i(n) + DeltaG degrees (37MM) - 0.8 (if first mismatch is GA or UU) - 0.8 (if first mismatch is GG and loop is closed on the 5' side by a purine). Here, DeltaG degrees (37i(n) is the free energy for initiating a loop of n nucleotides, and DeltaG degrees (37MM) is the free energy for the interaction of the first mismatch with the closing base pair. Thermodynamic parameters are also reported for hairpin formation in 1 M NaCl by RNA sequence of the types GACGXUAAUYUGUC and GGUXUAAUYGCC with GU base pair closure, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C varies from 3.6 to 5.3 kcal/mol. These results allow the development of a model for predicting the stability of hairpin loops closed by GU base pairs. DeltaG degrees (37L(n) (kcal/mol) = DeltaG degrees (37i(n) - 0.8 (if the first mismatch is GA) - 0.8 (if the first mismatch is GG and the loop is closed on the 5' side by a purine). Note that for these hairpins, the stability of the loops does not depend on DeltaG degrees (37MM). For hairpin loops closed by GU base pairs, the DeltaG degrees (37i(n) values, when n = 4, 5, 6, 7, and 8, are 4.9, 5.0, 4.6, 5.0, and 4.8 kcal/mol, respectively. The model gives good agreement when tested against six naturally occurring hairpin sequences. Thermodynamic values for terminal mismatches adjacent to GC, GU, and UG base pairs are also reported.     

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.     

Thermodynamics of RNA duplexes with tandem mismatches containing a uracil-uracil pair flanked by C.G/G.C or G.C/A.U closing base pairs

The thermodynamics governing the denaturation of RNA duplexes containing 8 bp and a central tandem mismatch or 10 bp were evaluated using UV absorbance melting curves. Each of the eight tandem mismatches that were examined had one U-U pair adjacent to another noncanonical base pair. They were examined in two different RNA duplex environments, one with the tandem mismatch closed by G.C base pairs and the other with G.C and A.U closing base pairs. The free energy increments (Delta Gdegrees(loop)) of the 2 x 2 loops were positive, and showed relatively small differences between the two closing base pair environments. Assuming temperature-independent enthalpy changes for the transitions, (Delta Gdegrees(loop)) for the 2 x 2 loops varied from 0.9 to 1.9 kcal/mol in 1 M Na(+) at 37 degrees C. Most values were within 0.8 kcal/mol of previously estimated values; however, a few sequences differed by 1.2-2.0 kcal/mol. Single strands employed to form the RNA duplexes exhibited small noncooperative absorbance increases with temperature or transitions indicative of partial self-complementary duplexes. One strand formed a partial self-complementary duplex that was more stable than the tandem mismatch duplexes it formed. Transitions of the RNA duplexes were analyzed using equations that included the coupled equilibrium of self-complementary duplex and non-self-complementary duplex denaturation. The average heat capacity change (DeltaC(p)) associated with the transitions of two RNA duplexes was estimated by plotting DeltaH degrees and DeltaS degrees evaluated at different strand concentrations as a function of T(m) and ln T(m), respectively. The average DeltaC(p) was 70 +/- 5 cal K(-)(1) (mol of base pairs)(-)(1). Consideration of this heat capacity change reduced the free energy of formation at 37 degrees C of the 10 bp control RNA duplexes by 0.3-0.6 kcal/mol, which may increase Delta Gdegrees(loop) values by similar amounts.     

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.     

DNA bending by the bulge defect

Comparative gel electrophoresis measurements were used to characterize DNA bending in molecules containing an extra adenosine on one strand, the so-called bulge defect. We used oligomers containing A6 tracts separated from the bulged base by varying numbers of nucleotides to determine the direction and magnitude of the bulge bend. Helix unwinding by the bulge was determined from the electrophoretic anomaly as a function of the size of the repeated monomers. We conclude that the bulge bend is 21 degrees +/- 3 degrees, primarily in the direction of tilt away from the bulged base. The total helical advance of the DNA at the bulge site is smaller than would be the case if the complementary T were present, corresponding to an unwinding by 25 degrees +/- 6 degrees. These values are in good agreement with the results of NMR and energy minimization studies of the bulged base in double-helical deoxyoligonucleotides [Woodson, S. A., & Crothers, D.M. (1988) Biochemistry 27, 3130-3141]     

Bulged adenosine influence on the RNA duplex conformation in solution

The RNA single bulge motif is an unpaired residue within a strand of several complementary base pairs. To gain insight into structural changes induced by the presence of the adenosine bulge on RNA duplex, the solution structures of RNA duplex containing a single adenine bulge (5'-GCAGAAGAGCG-3'/5'-CGCUCUCUGC-3') and a reference duplex with all Watson-Crick base pairs (5'-GCAGAGAGCG-3'/5'-CGCUCUCUGC-3') have been determined by NMR spectroscopy. The reference duplex structure is a regular right-handed helix with all of the attributes of an A-type helix. In the bulged duplex, single adenine bulge stacks into the helix, and the bulge region forms a well-defined structure. Both structures were analyzed by the use of calculated helical parameters. Distortions induced by the accommodation of unpaired residue into the helical structure propagate over the entire structure and are manifested as the reduced base pairs inclination and x-displacement. Intrahelical position of bulged adenine A5 is stabilized by efficient stacking with 5'-neighboring residues G4.     

Stability of RNA hairpin loops closed by AU base pairs

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequence of the type GCAXUAAUYUGC, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates that the free energy of loop formation at 37 degrees C varies from 3.2 to 5.0 kcal/mol. These results combined with the model previously developed [Dale et al. (2000) RNA 6, 608] allow improvements in the model to predict the stability of RNA hairpin loops: DeltaG degrees (37L(n) = DeltaG degrees (37i(n)) + DeltaG degrees (37MM) - 0.8 (if first mismatch is GA or UU) - 0.8 (if first mismatch is GG and loop is closed on 5' side by a purine). Here, DeltaG degrees (37i(n) is the free energy for initiating a loop of n nucleotides, and DeltaG degrees (37MM) is the free energy for the interaction of the first mismatch with the closing base pair. Hairpins with GG first mismatches were found to vary in stability depending upon the orientation of the closing base pair (5' or 3' purine relative to the loop). The model gives good agreement when tested against four naturally occurring hairpin sequences.     

Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs

Bulge loops are common features of RNA structures that are involved in the formation of RNA tertiary structures and are often sites for interactions with proteins and ions. Minimal thermodynamic data currently exist on the bulge size and sequence effects. Using thermal denaturation methods, thermodynamic properties of 1- to 5-nt adenine and guanine bulge loop constructs were examined in 10 mM MgCl₂ or 1 M KCl. The ΔG37∘ loop parameters for 1- to 5-nt purine bulge loops in RNA constructs were between 3.07 and 5.31 kcal/mol in 1 M KCl buffer. In 10 mM magnesium ions, the ΔΔG° values relative to 1 M KCl were 0.47–2.06 kcal/mol more favorable for the RNA bulge loops. The ΔG37∘ loop parameters for 1- to 5-nt purine bulge loops in DNA constructs were between 4.54 and 5.89 kcal/mol. Only 4- and 5-nt guanine constructs showed significant change in stability for the DNA constructs in magnesium ions. A linear correlation is seen between the size of the bulge loop and its stability. New prediction models are proposed for 1- to 5-nt purine bulge loops in RNA and DNA in 1 M KCl. We show that a significant stabilization is seen for small bulge loops in RNA in the presence of magnesium ions. A prediction model is also proposed for 1- to 5-nt purine bulge loop RNA constructs in 10 mM magnesium chloride.     

Thermal stability of RNA hairpins containing a four-membered loop and a bulge nucleotide

Fourteen RNA hairpins containing a four-membered loop and a bulge nucleotide were synthesized and their thermal stabilities determined. The combined contribution of a four-membered loop and bulge A to the free energy of a hairpin is calculated to be 9.3 kcal/mol at 37 degrees C and successfully predicts the stability of an independent RNA hairpin. The introduction of a bulge nucleotide to the helical stem of an RNA hairpin destabilizes the molecule in a sequence-dependent manner. The individual thermodynamic contributions of a four-membered loop and bulge A, G, and U residues to the stability of an RNA hairpin loop are presented.     

Positional effect of single bulge nucleotide on PNA(peptide nucleic acid)/DNA hybrid stability.

We report positional effect of bulge nucleotide on PNA/DNA hybrid stability. CD spectra showed that PNA/DNA hybrids required at least seven base pairings at a stem region to form a bulged structure. On the other hand, DNA/DNA could form bulged structure when there are only four base pairings adjacent to the bulge nucleotide. We discuss why PNA requests such a many base pairings to form bulged structure from a nearest neighbor standpoint.     

Thermodynamic parameters for DNA sequences with dangling ends

The thermodynamic contributions to duplex formation of all 32 possible single-nucleotide dangling ends on a Watson-Crick pair are reported. In most instances, dangling ends are stabilizing with free energy contributions ranging from +0.48 (GT(A)) to-0.96 kcal/mol (). In comparison, Watson-Crick nearest-neighbor increments range from -0. 58 (TA/AT) to -2.24 (GC/CG) kcal/mol. Hence, in some cases, a dangling end contributes as much to duplex stability as a Watson-Crick A-T base pair. The implications of these results for DNA probe design are discussed. Analysis of the sequence dependence of dangling-end stabilities show that the nature of the closing base pair largely determines the stabilization. For a given closing base pair, however, adenine dangling ends are always more or equally as stable as the other dangling nucleotides. Moreover, 5' dangling ends are more or equally as stabilizing as their 3' counterparts. Comparison of DNA with RNA dangling-end motifs shows that DNA motifs with 5' dangling ends contribute to stability equally or more than their RNA counterparts. Conversely, RNA 3' dangling ends contribute to stability equally or more than their DNA counterparts. This data set has been incorporated into a DNA secondary structure prediction algorithm (DNA MFOLD) (http://mfold2.wustl.edu/mfold/dna/for m1.cgi) as well as a DNA hybridization prediction algorithm (HYTHERtrade mark) (http://jsl1.chem.wayne.edu/Hyther/hythermenu .html).     

Analysis of the stability and flexibility of RNA complexes containing bulge loops of different sizes

Molecular dynamics simulations of RNA molecules consisting of an antisense oligonucleotide forming a complex with a target strand thereby creating an internal bulge-loop with 3, 4, or 5 nucleotides have been performed with and without O2' methylation of the antisense strand. The methylation influcences minor groove hydration, in particular near guanines but also around the methylated O2', and it also reduces the flexibility of both RNA strands. A G.U wobble pair adjacent to the bulge-loop is also found to increase the flexibility of the bulge nucleotides, compared to the situation with a standard Watson-Crick G-C base-pair in the same position.     

Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion

Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, noncanonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype–phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction‐dependent destabilization of the underlying double helix, and a self‐structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self‐structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size‐dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 1–12, 2014.