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.     

Stability of XGCGCp, GCGCYp, and XGCGCYp helixes: an empirical estimate of the energetics of hydrogen bonds in nucleic acids

The stabilizing effects of dangling ends and terminal base pairs on the core helix GCGC are reported. Enthalpy and entropy changes of helix formation were measured spectrophotometrically for AGCGCU, UGCGCA, GGCGCCp, CGCGCGp, and the corresponding pentamers XGCGCp and GCGCYp containing the GCGC core plus a dangling end. Each 5' dangling end increases helix stability at 37 degrees C roughly 0.2 kcal/mol and each 3' end from 0.8 to 1.7 kcal/mol. The free energy increments for dangling ends on GCGC are similar to the corresponding increments reported for the GGCC core [Freier, S. M., Alkema, D., Sinclair, A., Neilson, T., & Turner, D. H. (1985) Biochemistry 24, 4533-4539], indicating a nearest-neighbor model is adequate for prediction of stabilization due to dangling ends. Nearest-neighbor parameters for prediction of the free energy effects of adding dangling ends and terminal base pairs next to G.C pairs are presented. Comparison of these free energy changes is used to partition the free energy of base pair formation into contributions of "stacking" and "pairing". If pairing contributions are due to hydrogen bonding, the results suggest stacking and hydrogen bonding make roughly comparable favorable contributions to the stability of a terminal base pair. The free energy increment associated with forming a hydrogen bond is estimated to be -1 kcal/mol of hydrogen bond.     

Sequence dependence for the energetics of terminal mismatches in ribooligonucleotides

Stability increments of terminal mismatches on the core helixes AUGCAU and UGCGCA are reported. Enthalpy, entropy, and free energy changes of helix formation were measured spectrophotometrically for 15 oligoribonucleotides containing the core sequences and various mismatches. Free energy increments for mismatches in this series range from -0.5 to -1.1 kcal/mol. These increments for mismatches on AU base pairs are smaller than those measured previously on GC base pairs [Freier, S.M., Kierzek, R., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Biochemistry 25, 3209-3213]. The terminal GU mismatches in the sequences GAUGCAUUp and UAUGCAUGp add approximately the same stability increment as the corresponding terminal AU mismatch. The stability increments for pyrimidine-pyrimidine and pyrimidine-purine mismatches can be approximated within 0.3 kcal/mol by adding the stability increments for the corresponding 3' and 5' unpaired nucleotides (dangling ends). Stability increments for purine-purine mismatches are approximated well by the stability increment for the corresponding 3' dangling end made more favorable by 0.2 kcal/mol. These approximations are used to provide a table of stability increments for all 48 possible sequences of mismatches.     

Contributions of dangling end stacking and terminal base-pair formation to the stabilities of XGGCCp, XCCGGp, XGGCCYp, and XCCGGYp helixes

The role of stacking in terminal base-pair formation was studied by comparison of the stability increments for dangling ends to those for fully formed base pairs. Thermodynamic parameters were measured spectrophotometrically for helix formation of the hexanucleotides AGGCCUp, UGGCCAp, CGGCCGp, GCCGGCp, and UCCGGAp and for the corresponding pentanucleotides containing a 5'-dangling end on the GGCCp or CCGGp core helix. In 1 M NaCl at 1 X 10(-4) M strands, a 5'-dangling nucleotide in this series increases the duplex melting temperature (Tm) only 0-4 degrees C, about the same as adding a 5'-phosphate. In contrast, a 3'-dangling nucleotide increases the Tm at 1 X 10(-4) M strands 7-23 degrees C, depending on the sequence [Freier, S. M., Burger, B. J., Alkema, D., Neilson, T., & Turner, D. H. (1983) Biochemistry 22, 6198-6206]. These results are consistent with stacking patterns observed in A-form RNA. The stability increments from terminal A.U, C.G, or U.A base pairs on GGCC or a terminal U.A pair on CCGG are nearly equal to the sums of the stability increments from the corresponding dangling ends. This suggests stacking plays a large role in nucleic acid stability. The stability increment from the terminal base pairs in GCCGGCp, however, is about 5 times the sum of the corresponding dangling ends, suggesting hydrogen bonding can also make important contributions.     

The dynamic structural basis of differential enhancement of conformational stability by 5'- and 3'-dangling ends in RNA

Unpaired bases at the end of an RNA duplex (dangling ends) can stabilize the core duplex in a sequence-dependent manner and are important determinants of RNA folding, recognition, and functions. Using 2-aminopurine as a dangling end purine base, we have employed femtosecond time-resolved fluorescence spectroscopy, combined with UV optical melting, to quantitatively investigate the physical and structural nature of the stacking interactions between the dangling end bases and the terminal base pairs. A 3'-dangling purine base has a large subpopulation that stacks on the guanine base of the terminal GC or UG pair, either intrastrand or cross-strand depending on the orientation of the pair, thus providing stabilization of different magnitudes. On the contrary, a 5'-dangling purine base only has a marginal subpopulation that stacks on the purine of the same strand (intrastrand) but has little cross-strand stacking. Thus a 5'-dangling purine does not provide significant stabilization. These stacking structures are not static, and a dangling end base samples a range of stacked and unstacked conformations with respect to the terminal base pair. Femtosecond time-resolved anisotropy decay reveals certain hindered base conformational dynamics that occur on the picosecond to nanosecond time scales, which allow the dangling base to sample these substates. When the dangling purine is opposite to a U and is able to form a potential base pair at the end of the duplex, there is an interplay of base stacking and hydrogen-bonding interactions that depends on the orientation of the base pair relative to the adjacent GC pair. By resolving these populations that are dynamically exchanging on fast time scales, we elucidated the correlation between dynamic conformational distributions and thermodynamic stability.     

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.     

Low temperature solution structures and base pair stacking of double helical d(CGTACG)(2)

Solution structures and base pair stacking of a self- complementary DNA hexamer d(CGTACG)(2) have been studied at 5, 10 and 15 degrees C, respectively. The stacking interactions among the center base pair steps of the DNA duplex are found to improve when the terminal base pairs became less stable due to end fraying. A new structural quantity, the stacking sum (Sigma(s)), is introduced to indicate small changes in the stacking overlaps between base pairs. The improvements in the stacking overlaps to maintain the double helical conformation are probably the cause for the observed temperature dependent structural changes in double helical DNA molecule. A detailed analysis of the helical parameters, backbone torsion angles, base orientations and sugar conformations of these structures has been performed.     

A uniform mechanism correlating dangling-end stabilization and stacking geometry

The geometry of the dangling base in 105 published structures (from X-ray/NMR) containing single-stranded overhangs has been analyzed and correlated to the thermodynamic stabilization found (UV) for the corresponding dangling base/closing basepair combination in short oligonucleotides. The study considers most combinations of closing basepairs, sequence and dangling base residue type, attached in both the 3'- and 5'-ends of both DNA and RNA. Linear regression analysis showed a straightforward correlation (R = 0.873) between the degree of screening for the hydrogen bonds of the closing basepair provided by the dangling base and the resulting thermodynamic stabilization in both DNA and RNA series with dangling ends either at the 3'- or at the 5'-terminus. Regression analysis of only the datasets from RNA gives an improved correlation, R = 0.934, showing that dangling ends on RNA are more ordered than the dangling ends on DNA, R = 0.376. This study highlights the gain in the free energy of stabilization owing to the favorable stacking between the dangling nucleobase and the neighboring basepair and the resulting strengthening of the hydrogen bond of the closing basepair. By acting as a hydrophobic cap on the terminal of the DNA or RNA duplex, the dangling-end residue restricts the bulk water access to the terminal basepair, thereby providing it with a microenvironment devoid of water, which consequently enhances its thermodynamic stability, making it energetically comparable to the corresponding internal basepair. Thus, one single structural model consisting of the interplay of the above electrostatic interactions can be used to explain the molecular basis of the observed thermodynamic effects for dangling-end attachment to the 3'- and 5'-ends of both DNA and RNA duplexes, which is a key step toward accurate dangling-end effect prediction.     

Sequence specificity of the non-natural pyrido[2,3-d]pyrimidine nucleoside in triple helix formation.

The non-natural pyrido[2,3-d]pyrimidine nucleoside F, which pairs preferentially with guanine (G) and adenine (A) within double-helical DNA, recognizes with high selectivity AT base pairs within triple-helical complexes. These observations suggest that F may exist in different tautomeric forms within double-helical and triple-helical complexes. Analysis of the base stacking properties of this extended ring system using two oligodeoxyribonucleotides containing terminal thymines and/or pyrido[2,3-d]pyrimidines bound to adjacent sites showed a decrease in free energy of binding in a triple-helical complex in the order (5'-3') TT > FT > TF > FF.     

Thermodynamics of unpaired terminal nucleotides on short RNA helixes correlates with stacking at helix termini in larger RNAs.

Free energies for stacking of unpaired nucleotides (dangling ends) at the termini of oligoribonucleotide Watson-Crick helixes (DeltaG(0)37,stack) depend on sequence for 3' ends but are always small for 5' ends. Here, these free energies are correlated with stacking at helix termini in a database of 34 RNA structures determined by X-ray crystallography and NMR spectroscopy. Stacking involving GA pairs is considered separately. A base is categorized as stacked by its distance from (相似文献   

Proton nuclear magnetic resonance investigations of fraying in double-stranded d-ApTpGpCpApT in H2O solution.

The chemical shifts and line widths of the Watson-Crick ring NH resonances of the self-complementary duplex of d-ApTpGpApT have been monitored at low ionic strength and in the presence of Mg ions at neutral pH in aqueous solution to determine the thermodynamic parameters associated with fraying (D. J. Patel (1974), Biochemistry 13, 2396) at the terminal and internal base pairs as a function of temperature and pH. From studies in H2O-MeOH (3:2), the fraying process persists down to approximately -20 degrees for the internal TA base pair and down to and probably beyond -30 degrees for the terminal AT base pair. The observed average chemical shift at each of these base pairs as a function of temperature suggests rapid exchange on the nuclear magnetic resonance (NMR) time scale between helix and coil (chemical shift separation of 3.2 ppm) and have been utilized to determine the dissociation constant at the terminal and internal base pairs. Comparison of the reaction enthalpies elucidated from the chemical shift parameters with those reported from optical studies suggests that the symmetry related internal TA base pairs break in a coupled manner at low ionic strength, with the coupling removed in the presence of Mg ions and high salt. By contrast, the symmetry related terminal AT base pairs break independently of each other in the absence and presence of Mg ions and high salt. The terminal base pair exhibits a Tm of 10-15 degrees lower than that of the internal base pair in the hexanucleotide, with divalent Mg ions and high salt stabilizing the double helix as reflected in the Tm values of these base pairs. The observed line width changes as a function of temperature provide an estimate of the exchange rate of the proton from the coil form with water. The exchange reaction from the coil state is base catalyzed with rate constants in the diffusion limit.     

Thermodynamic analysis of stacking hybridization of oligonucleotides with DNA template.

Contiguous stacking hybridization of oligodeoxyribonucleotides with DNA as template was investigated using three types of complexes: oligonucleotide contiguously stacked with the stem of the preformed minihairpin (complexes I), oligonucleotide tandems containing two (complexes II) or three (complexes III) short oligomers with a common DNA template. Enthalpy Delta H degrees and entropy Delta S degrees of the coaxial stacking of adjacent duplexes were determined for GC/G*pC, GT/A*pC, AC/G*pT, AT/A*pT, CT/A*pG, AG/C*pT, AA/T*pT and TT/A*pA nicked (*) dinucleotide base pairs. The maximal efficiency of co-operative interaction was found for the GC/G*pC interface (Delta G degrees(NN/N*pN)=-2.7 kcal/mol) and the minimal one for the AA/T*pT interface (Delta G degrees(NN/N*pN)=-1.2 kcal/mol) at 37 degrees C. As a whole, the efficiency of the base pairs interaction Delta G degrees(NN/N*pN) in the nick is not lower than that within the intact DNA helix (Delta G degrees(NN/NN)).These observed Delta G degrees(NN/N*pN) values are proposed may include the effect of the partial removal of fraying at the adjacent helix ends additionally to the effect of the direct stacking of the terminal base pairs in the duplex junction (Delta G degrees(NN/NN). The thermodynamic parameters have been found to describe adequately the formation of all tandem complexes of the II and III types with oligonucleotides of various length and hybridization properties. The performed thermodynamic analysis reveals features of stacking oligonucleotide hybridization which allow one to predict the temperature dependence of association of oligonucleotides and the DNA template within tandem complexes as well as to determine optimal concentration for formation of these complexes characterized by high co-operativity level.     

Consecutive terminal GU pairs stabilize RNA helices

Consecutive GU pairs at the ends of RNA helices provide significant thermodynamic stability between -1.0 and -3.8 kcal/mol at 37 °C, which is equivalent to approximately 2 orders of magnitude in the value of a binding constant. The thermodynamic stabilities of GU pairs depend on the sequence, stacking orientation, and position in the helix. In contrast to GU pairs in the middle of a helix that may be destabilizing, all consecutive terminal GU pairs contribute favorable thermodynamic stability. This work presents measured thermodynamic stabilities for 30 duplexes containing two, three, or four consecutive GU pairs at the ends of RNA helices and a model to predict the thermodynamic stabilities of terminal GU pairs. Imino proton NMR spectra show that the terminal GU nucleotides form hydrogen-bonded pairs. Different orientations of terminal GU pairs can have different conformations with equivalent thermodynamic stabilities. These new data and prediction model will help improve RNA secondary structure prediction, identification of miRNA target sequences with GU pairs, and efforts to understand the fundamental physical forces directing RNA structure and energetics.     

Solution structure of the aminoacyl-capped oligodeoxyribonucleotide duplex (W-TGCGCAC)(2).

Reported here is the solution structure of the aminoacyl-DNA duplex (W-TGCGCAC)(2). This duplex forms a continuously pi-stacked helix consisting of both nucleobases and amino acid side chains. According to NMR and UV analyses, the duplex melts in a cooperative transition and with 1.3-1.8% greater hyperchromicity than the control duplex (TGCGCAC)(2). A van't Hoff analysis of UV melting points at different concentrations shows that the two tryptophan residues contribute 4.8 kcal/mol to the DeltaH degrees of complex formation at 10 mM salt concentration and less than 1 kcal/mol at 150 mM salt. The entropic cost for duplex association in the presence of the amino acid residues is 13 cal/molK greater than that for the control at 10 mM salt concentration, and 3 cal/molK lower than that of the control at 0.15 ionic strength. The conformation of W-TGCGCAC in duplex form, determined via restrained torsion angle molecular dynamics, shows an undisturbed B-form DNA duplex with dangling 3'-termini. The tryptophanyl residue at the 5'-terminus packs tightly against T2 and the proximal part of adenine, without engaging in hydrogen bonding. While not providing strong enthalpic net stabilization of the duplex, the tryptophan "cap" on the duplex does seem to reduce the fraying at the termini, indicating a subtle balance of entropic and enthalpic factors contributing to the molecular dynamics. The structure also shows that, at least in the present sequence context, stacking on the terminal base pair is more favorable than intercalation, probably because the enthalpic cost associated with breaking up the stacking between DNA base pairs cannot be paid for by favorable pi-stacking interactions with the indole ring of tryptophan. These results are of importance for understanding stacking interactions in protein-DNA complexes, particularly those in enzyme-substrate complexes involving exposed nucleobases.     

1H-NMR study of the OR1 operon in bacteriophage lambda. I. Assignment of signals from imino protons and adenine C2 protons

An oligodeoxyribonucleotide composed of 17 residues, d(TATCACCGCCAGAGGTA), and a complementary chain were synthesized. Their duplex was identical with the operator OR1, the binding site for bacteriophage lambda cro and c1 repressors. The 1H NMR spectra (500 MHz) of the duplex imino and aromatic protons were studied at 10, 20 and 25 degrees C. Signals from the imino protons of complementary base pairs and from the C2 protons of adenine (with the exception of the duplex terminal nucleotides) were assigned using the NOE technique and the known characteristics of short DNA fragment melting. No signals from the imino protons of the terminal base pairs were detected even at 10 degrees C due to fraying which increased as the temperature was raised. The assignment of signals can be used to identify centers of interaction between the operator OR1 and repressors, as well as to study possible local changes in DNA geometry.     

Stabilization of duplex DNA and RNA by dangling ends studied by free energy simulations

Single unpaired nucleotides at the end of double‐stranded nucleic acids, termed dangling ends, can contribute to duplex stability. Umbrella sampling free energy simulations of dangling cytosine and guanine nucleotides at the end of duplex and single stranded RNA and DNA molecules have been used to investigate the molecular origin of dangling end effects. In unrestraint simulations, the dangling end nucleotides stayed close to placements observed in experimental structures. Calculated free energy contributions associated with the presence of dangling nucleotides were in reasonable agreement with experiment predicting the general trend of a more stabilizing effect of purine vs. pyrimidine dangling ends. In addition, the calculations indicate a more significant stabilizing effect of dangling ends at the 5′‐end vs. 3′‐end in case of DNA and the opposite trend in case of RNA. Both electrostatic and van der Waals interactions contribute to the duplex stabilizing effect of dangling end nucleotides. The free energy simulation scheme could also be used to design dangling end nucleotides that result in enhanced duplex stabilization. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 418–427, 2014.     

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).     

Moloney murine leukemia virus integration protein produced in yeast binds specifically to viral att sites.

The integration protein (IN) of Moloney murine leukemia virus (MuLV), purified after being produced in yeast cells, has been analyzed for its ability to bind its putative viral substrates, the att sites. An electrophoretic mobility shift assay revealed that the Moloney MuLV IN protein binds synthetic oligonucleotides containing att sequences, with specificity towards its cognate (MuLV) sequences. The terminal 13 base pairs, which are identical at both ends of viral DNA, are sufficient for binding if present at the ends of oligonucleotide duplexes in the same orientation as in linear viral DNA. However, only weak binding was observed when the same sequences were positioned within a substrate in a manner simulating att junctions in circular viral DNA with two long terminal repeats. Binding to att sites in oligonucleotides simulating linear viral DNA was dependent on the presence of the highly conserved CA residues preceding the site for 3' processing (an IN-dependent reaction that removes two nucleotides from the 3' ends of linear viral DNA); mutation of CA to TG abolished binding, and a CA to TA change reduced affinity by at least 20-fold. Removal of either the terminal two base pairs from both ends of the oligonucleotide duplex or the terminal two nucleotides from the 3' ends of each strand did not affect binding. The removal of three 3' terminal nucleotides, however, abolished binding, suggesting an essential role for the A residue immediately upstream of the 3' processing site in the binding reaction. These results help define the sequence requirements for att site recognition by IN, explain the conservation of the subterminal CA dinucleotide, and provide a simple assay for sequence-specific IN activity.     

Stability of 3' double nucleotide overhangs that model the 3' ends of siRNA

Thermodynamic parameters are reported for duplex formation in 1 M NaCl for 16 RNA sequences, each containing a core tetramer duplex, GGCC, and a 3' overhang consisting of two bases. The results indicate additional double-helical stability is conferred by the double 3' terminal overhang relative to the single 3' terminal overhang. A nearest-neighbor analysis of the data indicates that the free energy contribution at 37 degrees C of the second base in the double 3' terminal overhang varies from 0 to 0.7 kcal/mol. The second base in the 3' double overhang can contribute nearly the same stability to a duplex as a base pair or a 3' dangling overhang. Stability contribution of a dangling base, two nucleotides removed from the 3' end of a duplex, is dependent upon both the identity of the base as well as that of the dangling base that it neighbors. A second dangling base only increases the stability of the duplex when it is neighboring a 3' purine dangling nucleotide. Furthermore, a second dangling pyrimidine provides a greater contribution to duplex stability than a purine. A nearest-neighbor model was developed to predict the influence of 3' double overhang on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against six sequences with different core duplexes.     

Solution conformation of a deoxynucleotide containing tandem G.A mismatched base pairs and 3'-overhanging ends in d(GTGAACTT)2.

We have used 31P and 1H NMR spectroscopy and circular dichroism to define the solution conformation of d(GTGAACTT)2 which contains tandem G.A mismatched base pairs and 3'-overhanging TT ends. Measurements of coupling constants and NOE intensities show that the sugar puckers of the nucleotides are predominantly in the south domain (i.e., near C2'-endo) and that the glycosidic torsion angles are anti. The sequential NOE intensities indicate the presence of a right-handed helix. Analysis of the 31P and 1H NMR spectra of the duplex shows that the tandem mismatch forms a block in which there are unusual backbone torsion angles (i.e., in the BII state), within an otherwise B-like structure. The chemical shift of the N1H of the mismatched guanosine and NOEs between the mismatched base pairs and their nearest neighbors are inconsistent with the imino pairing present in single A.G mismatches or in the X-ray structure of a tandem mismatch [Privé, G. G., et al. (1987) Science 238, 498-503] but the data are consistent with the amino pairing found by Li et al. (1991) [Li, Y., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The strong base-base stacking both within the tandem G.A block and between the G.A mismatches and their other nearest neighbors offsets the intrinsic destabilizing effects of the mismatch. Further, the 3'-TT overhangs stack onto the ends of the helix and stabilize the duplex against fraying, which accounts for the observed increase in the melting temperature compared with the flush-ended duplex.