Nucleic Acids Research, 2000, Vol. 28, No. 9 1929-1934
© 2000 Oxford University Press
Thermodynamic parameters for DNA sequences with dangling ends
Department of Chemistry, Wayne State University, Detroit, MI 48202, USA
Received January 7, 2000; Revised and Accepted March 7, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALREFERENCES |
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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 (
) 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/form1.cgi ) as well as a DNA hybridization prediction algorithm (HYT HERTM) (http://jsl1.chem.wayne.edu/Hyther/hythermenu.html ).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALREFERENCES |
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DNA nearest-neighbor parameters have already been determined for Watson–Crick base pairs (1,2) and all single internal mismatches (1,3–6). However, there is a significant lack of data in the literature for the stability of other DNA motifs, including dangling ends, terminal mismatches and various bulge and loop structures. Knowledge of the thermodynamic contribution of these motifs is important to accurately predict nucleic-acid hybridization and secondary structure (7). In this work, we report the thermodynamic contribution to duplex formation of the 32 DNA dangling ends with a closing Watson–Crick base pair. The available literature data indicate that dangling ends can significantly stabilize duplexes (8–11). Therefore, knowledge of the thermodynamic contributions of all dangling-end contexts will be useful to improve primer design and optimize the design of immobilized probes in DNA microarrays (12) and other diagnostics such as molecular beacons (13). Moreover, this data set will improve the accuracy of predictions of hybridization and of DNA secondary structure. The empirical data reported here also presents an opportunity to evaluate various theoretical models for nucleic-acid stacking (14–16).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALREFERENCES |
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DNA synthesis and purification
Oligonucleotides were synthesized on solid supports using standard phosphoroamidite chemistry (17). Oligonucleotides were detached from the solid support and the base blocking groups were removed by treatment with concentrated ammonia at 50°C overnight. Each oligonucleotide sample was dried and dissolved in 250 µl of water. The solution was then purified by TLC on a Si500F plate (Baker) using an elution mixture of n-propanol/ammonia/distilled deionized water in volumetric proportions 55:35:10, respectively (18). Bands were visualized under a UV lamp, the least mobile one was scraped off and oligonucleotides were extracted with distilled deionized water. The oligonucleotides were further purified and desalted with Sep-pak C-18 cartridges (Waters).
UV melting curves
Absorbance versus temperature curves were measured at 260 or 280 nm with a heating rate of 0.8°C min–1 using an AVIV 14DS UV-Vis spectrophotometer with a five cuvette thermoelectric controller as described previously (19). The buffer used for the melting curves was 1.0 M NaCl, 20 mM sodium cacodylate and 0.5 mM Na2EDTA, pH 7.0. Each duplex was melted at 8–10 different concentrations covering an 80- to 100-fold concentration range. Samples were annealed and degassed by raising the temperature to 85°C for 5 min and the absorbance of each sample (260 nm) was recorded to calculate total strand concentrations, CT, using the single-strand extinction coefficients calculated with the nearest-neighbor model (20).
Calculation of thermodynamic parameters
Thermodynamic parameters were determined from melting curves using the program MELTWIN v.3.0 (21). Thermodynamic parameters were calculated by two methods: (i) enthalpy and entropy changes from fits of individual melting curves at different concentrations were averaged (22); (ii) plots of reciprocal melting temperatures (Tm–1 versus lnCT) were fitted to the following equation (23) (all sequences in this study are self-complementary):
Tm–1 = (R/
H°) lnCT + S°/H° 1
Both methods assume a two-state model and Cp° = 0 for the transition equilibrium (22,24). The two-state approximation was assumed to be valid for sequences in which the H° values derived from the two methods agreed within 15% (25). Since the two methods are equally reliable, the error-weighted average of the data obtained by the two methods was calculated as follows and reported in Table 1 (26):
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G°37 = {[G°37(i)/
(i)2] + [G°37(ii)/(ii)2]}/{[1/(i)2] +
[1/(ii)2]} 2
where G°37(i) and (i) represent the free energy and the corresponding precision obtained by method (i) and G°37(ii) and (ii) represent the free energy and the corresponding precision obtained by method (ii). Errors in the weight-averaged free energies were calculated as follows (26):
weighted average = {1/[1/(i)2] + [1/(ii)2]} 3
where (i) and (ii) represent the precision of the data obtained by (i) average of enthalpy and entropy changes from fits of individual melting curves at different concentrations (22) and (ii) plots of reciprocal melting temperatures (Tm–1 versus lnCT).
Design of sequences
Oligonucleotides were designed to have a melting temperature between 30 and 65°C and to minimize formation of undesired hairpin or slipped-duplex conformations that might result in a non-two-state transition (1). Sequences were also designed to be self-complementary so that the error in the dangling-end contribution would be decreased by a factor of two compared to a duplex with only one dangling end (27).
Determination of dangling-end contributions to duplex stability
When thermodynamic data were available for core sequences (19; S.Varma and J.SantaLucia,Jr, unpublished results) dangling-end contributions were obtained by subtracting the core sequence thermodynamics from the sequence with dangling ends. This is equivalent to assuming that dangling-end stability can be approximated by a nearest-neighbor model (25). For instance the free energy for the dangling end
in the sequence (AGTAGCTAC)2 is calculated as follows:
G°37(
) = {G°37[(AGTAGCTAC)2] –
G°37[(GTAGCTAC)2]} 4
The factor of in equation 4 is introduced because two dangling ends are present in each duplex (27). When thermodynamic data were not available for core sequences the nearest-neighbor model was used to predict the stability of the core sequence (1,2). The error in the dangling-end parameters (Table 2) was calculated by standard error propagation methods (26). For instance, the error for the dangling end
free energy is calculated using the errors reported in Table 1 as follows:
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[G°37(
)] = {2[G°37((AAGAGCTCT)2)] +
2[G°37((AGAGCTCT)2)]}1/2 5
where [G°37((AAGAGCTCT)2)] and [G°37((AGAGCTCT)2)] are the errors reported in Table 1. Analogous equations apply for the error propagation for H° and S° of dangling-end formation. It is important to point out that the propagated errors obtained by these methods (equation 5) reflect the accuracy of the dangling-end parameters obtained for a given sequence context. Imposing the nearest-neighbor model itself, however, introduces additional uncertainty when the parameters are used to predict dangling-end stability in different sequence contexts (i.e. non-nearest-neighbor effects). Further, additional thermodynamic contributions from additional dangling bases can sometimes be significant (8,9) but are neglected with the model presented here.
Estimated errors
All of the sequences used in this study are 8 or 9 nt long and were specifically designed to avoid competing structures. As a result, the experimental precision of the data in Table 1 are generally within 1–2% for G°37, 5% for H° and 5–6% for S°. The accuracy of the measurements is probably slightly worse than this because of systematic errors such as temperature calibration (estimated to be correct within 0.3°C) and application of the two-state approximation and the assumption that Cp° = 0. Since the dangling-end and core sequences were both measured on the same instrument, however, systematic errors are likely to cancel so that errors in the calculated dangling-end contribution are largely reflected in the precision not accuracy of the measurements. Hence, when the core sequence corresponding to a given duplex with dangling ends was measured, errors reported in Table 1 for the duplex with dangling ends correspond to the precision of the data. When the core sequence thermodynamics were predicted using the nearest-neighbor model (2), errors reported in Table 1 for the corresponding sequence with dangling ends reflect the accuracy of the measurement and are conservatively estimated to be within 3, 7 and 8% for G°37, H° and S°, respectively.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALREFERENCES |
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Thermodynamic data
Plots of Tm–1 versus lnCT were linear with correlation coefficients
0.98 over the entire 80- to 100-fold range in concentration. Table S1 lists the thermodynamic parameters derived from fits of melting curves and from Tm–1 versus lnCT plots (see Supplementary Material). The agreement in H° values of the two methods is within 15% for all the sequences. Therefore the two-state approximation appears to be valid for these sequences (25). Since the data from both methods are equally reliable, the error-weighted average parameters (Table 1) were used in equation 4 to calculate the 32 dangling-end parameters shown in Table 2 (see Materials and Methods).
Application of dangling-end parameters
In this work, we assume that the nearest-neighbor model is an accurate approximation for sequences with terminal dangling ends. For instance, the thermodynamics for the sequence (AAGAGCTCT)2 is decomposed as follows:
G°37[(AAGAGCTCT)2] = 2G°37 initiation with terminal AT +
G°37 sym + 2G°37(
) + 4G°37(
) + 2G°37(
) +
G°37(
) 6
Stability trends
Dangling ends are stabilizing in 24 out of 32 cases and destabilizing in only three out of 32 cases with G°37 values ranging from +0.48 to –0.96 kcal/mol (Fig. 1 and Table 2). Stabilization depends on four factors: the base type (A, C, G or T) and position (5' versus 3') of the dangling nucleotide and the type and orientation of the closing base pair (A-T, T-A, C-G or G-C). For a given closing base pair of given orientation, adenine dangling ends are always more or equally as stabilizing as the other dangling ends. However, for a given closing base pair, C, G and T dangling nucleotides show stabilizations that do not differ significantly from one another. Therefore, with the exception of A dangling ends, the dangling nucleotide identity plays only a minor role in the stabilization. The only exceptions to this trend are observed for
(G°37 = –0.01 kcal/mol), which is less stable than
(G°37 = –0.52 kcal/mol), and for
(G°37 = +0.48 kcal/mol), which is less stable than both
(G°37 = –0.02 kcal/mol) and
(G°37 = –0.10 kcal/mol). To ensure that the observation for
is not the result of an experimental artifact, two different sequences bearing this dangling-end motif were melted. Both sequences [(GTTAGCTAA)2 and (GTGAGCTCA)2] showed identical contributions for the dangling-end motif (Table 1). This result suggests that the unusual behavior of
is not due to next nearest-neighbor effects or due to unusual single strand stability. At present we do not have a good explanation for why
is destabilizing. The 3' versus 5' position of a dangling end can result in stabilization of up to 0.98 kcal/mol (
versus
). Moreover, 5' dangling ends are more or equally as stable as their 3' counterparts. This observation confirms previous results obtained by Senior et al. (8). The only significant exceptions to this trend are observed for
(+0.48 kcal/mol) versus
(–0.50 kcal/mol) and
(–0.58 kcal/mol) versus
(–0.92 kcal/mol). Furthermore, changing the closing base pair type and orientation can lead to stabilization by up to 1.20 kcal/mol (
versus
).
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Comparison with literature data
A few thermodynamic measurements of DNA sequences with dangling ends are reported in the literature. Guckian et al. (10) studied
and
dangling-end motifs and reported stabilizations of –1.0 ± 0.2 and –0.6 ± 0.2 kcal/mol, respectively. These measurements are in excellent agreement with our data of –0.96 ± 0.05 kcal/mol for
and –0.58 ± 0.05 kcal/mol for
. In another study, Doctycz et al. (9) studied hairpins with 5 base overhangs on their 5'-side. They observed that the stabilization depended mainly on the dangling nucleotide adjacent to the duplex, consistent with the nearest-neighbor approximation. To allow comparison with our data, the data of Doktycz et al. were converted from G°25 to G°37. Averages of the free energy measurements obtained for all overhangs with an identical dangling nucleotide adjacent to the duplex (
, –0.58 kcal/mol;
, –0.53 kcal/mol;
, –0.59 kcal/mol;
, –0.54 kcal/mol) are within the experimental error of the results presented in this work (
, –0.58 kcal/mol;
, –0.56 kcal/mol;
, –0.34 kcal/mol;
, –0.61 kcal/mol). Since the data of Guckian et al. (10) and Doktycz et al. (9) were measured in different contexts than the sequences in this study, these data suggest that the nearest-neighbor model is a good approximation for dangling ends. Senior et al. (8) also reported thermodynamic measurements of duplexes with two T nucleotide overhangs. To allow comparison with our work, the data of Senior et al. were converted from G°25 to G°37 (
, –0.93 kcal/mol;
, –0.45 kcal/mol;
, –1.01 kcal/mol;
, –0.35 kcal/mol). These data of Senior et al. (8) are basically within the experimental error of our measurements (
, –0.61 kcal/mol;
, –0.52 kcal/mol;
, –0.58 kcal/mol;
, –0.35 kcal/mol). It is possible that the slight differences are due either to experimental error or due to the effect of tandem TT dangling ends that are not present in our sequences.
Comparison with terminal mismatch data
Dangling-end parameters were also compared with the 48 terminal mismatch parameters (S.Varma and J.SantaLucia,Jr, unpublished results) to test if the stability contribution of a terminal mismatch equals the sum of the contributions of the two corresponding dangling ends. For instance, the terminal mismatch motif
can be expressed as the sum of the two dangling end motifs
and
. In eight of the 48 cases, the sums of the dangling-end free energy values are >0.4 kcal/mol more stable than the corresponding terminal-mismatch free-energy values. This observation can be explained by considering the possibility for optimal stacking in the case of dangling ends, which is sterically precluded for terminal mismatches. In 29 of the 48 cases, the sums of the dangling-end free-energy contributions are within 0.4 kcal/mol of the corresponding terminal-mismatch free-energy contributions. Three explanations for this observation are: (i) neither dangling-end motif yields any significant stabilization and hydrogen bonding in the terminal mismatch is negligible; (ii) the terminal-mismatch and dangling-end structures stack with equal efficiency in some cases; (iii) stacking of the terminal nucleotides is less efficient in the terminal-mismatch structure than in the dangling-end structures, but there is compensating favorable hydrogen bonding or electrostatic interactions between the bases of the terminal nucleotides. In 11 of the 48 cases, the sums of the dangling-end free-energy values are >0.4 kcal/mol less stable than the corresponding terminal-mismatch free-energy values. In these cases, the higher stability of the terminal-mismatch parameters may be due to interactions between the terminal bases, such as hydrogen bonding or electrostatic interactions. Further structural data on terminal mismatches are required to determine the exact nature of these interactions. Similar cases in which the sum of the dangling-end stabilities are either equal to or higher than the corresponding terminal-mismatch free energy were also observed for RNA (28). Solvation and conformational entropy may also be important determinants of dangling-end and terminal-mismatch stability (29).
Comparison with RNA
Comparison with the RNA dangling-end parameters compiled by Freier et al. (25,28) show that DNA motifs with 5' dangling ends are more or equally as stable as their RNA counterparts. The only exception to this trend is observed for
. On the other hand, RNA motifs with 3' dangling ends are more or equally as stable as their DNA counterparts. A geometric explanation for this difference in stability of 3' versus 5' RNA dangling ends is that for A-form RNA structures 3' nucleotides are well stacked on top of the adjacent base pair whereas 5' dangling ends display poor stacking. For B-form DNA, however, dangling ends show efficient geometric intrastrand stacking. Thus, DNA trends (dictated by B-form geometry) are expected to differ from RNA trends (dictated by A-form geometry) (28). This expectation is verified by our DNA measurements that show 5' dangling ends are always more or equally as stable as their 3' counterparts.
Physical significance of dangling-end parameters
Each dangling-end thermodynamic parameter reflects the stacking ability of the dangling base on the duplex closing base pair. The fundamental interactions responsible for the sequence dependence of stacking are, however, not clear and probably include dipole-induced dipole (14,30) or induced dipole-induced dipole interactions (31,32) as well as solvophobic effects (30,33). Further understanding of the physical chemistry of dangling-end stabilization might be achieved by solving the structures of several duplexes with dangling ends. Theoretical approaches such as ab initio quantum mechanical calculations could also be used to determine the origin of the stabilization. To date, however, the various studies available have focused on base/base stacking (15,16,31,32) and none have studied stacking of an unpaired base on top of a base pair. Thus, we are currently unable to correlate the literature data (15,31,32,34) with our thermodynamic parameters. With the development of improved computational techniques, it is likely that in the future such theoretical approaches will bring insights into the structural basis of the thermodynamic data.
Implications for DNA probe design
Oligonucleotide probe hybridization to a long target DNA involves not only base pairing, but also two dangling-end contributions. This work demonstrates that the free-energy contribution of each dangling end ranges from +0.48 (
) to –0.96 kcal/mol (
). Thus, depending on the sequence, two dangling ends can affect probe–target hybridization by as much as +0.96 to –1.92 kcal/mol. By comparison, the stabilization conferred by addition of one Watson–Crick base pair to a probe–target duplex ranges from –0.58 (TA/AT) to –2.24 kcal/mol (GC/CG) (2). Therefore, accounting for dangling-end effects is crucial to accurately calculate probe/target binding strength and dangling-end parameters should be incorporated into current probe design programs.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALREFERENCES |
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The set of measurements reported in this paper is the first systematic analysis of dangling end contributions to DNA duplex stability. The data show that in many cases dangling ends stabilize duplexes as much as an additional A-T base pair would. These parameters will be useful to optimize probe–target-based biochemical techniques as well as DNA secondary structure prediction. The dangling end parameters reported here are now implemented in the DNA MFOLD secondary structure prediction algorithm (35; J.SantaLucia,Jr and M.Zuker, unpublished results; http://mfold2.wustl.edu/~mfold/dna/form1.cgi ) as well as in the HYTHERTM hybridization optimization algorithm (N.Peyret and J.SantaLucia,Jr, unpublished results; http://jsl1.chem.wayne.edu/Hyther/hythermenu.html ).
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SUPPLEMENTARY MATERIAL |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALREFERENCES |
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See Supplementary Material available at NAR Online.
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ACKNOWLEDGEMENTS |
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We thank Dr Christine Chow and May Meroueh for assistance with DNA synthesis. We thank Dr Shikha Varma for providing unpublished data on terminal mismatches and for useful discussions. This work was supported by NIH grant HG02020.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 313 577 0101; Fax: +1 313 577 8822; Email: jsl@chem.wayne.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSSUPPLEMENTARY MATERIALREFERENCES |
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P. Yakovchuk, E. Protozanova, and M. D. Frank-Kamenetskii Base-stacking and base-pairing contributions into thermal stability of the DNA double helix Nucleic Acids Res., January 31, 2006; 34(2): 564 - 574. [Abstract] [Full Text] [PDF]
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S.-i. Nakano, Y. Uotani, K. Uenishi, M. Fujii, and N. Sugimoto DNA base flipping by a base pair-mimic nucleoside Nucleic Acids Res., December 15, 2005; 33(22): 7111 - 7119. [Abstract] [Full Text] [PDF]
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N. E. Watkins Jr and J. SantaLucia Jr Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes Nucleic Acids Res., November 1, 2005; 33(19): 6258 - 6267. [Abstract] [Full Text] [PDF]
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 R. T. Koehler and N. Peyret Thermodynamic properties of DNA sequences: characteristic values for the human genome Bioinformatics, August 15, 2005; 21(16): 3333 - 3339. [Abstract] [Full Text] [PDF]
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R. E. Johnson, L. Prakash, and S. Prakash Biochemical evidence for the requirement of Hoogsteen base pairing for replication by human DNA polymerase {iota} PNAS, July 26, 2005; 102(30): 10466 - 10471. [Abstract] [Full Text] [PDF]
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M. Leber, L. Kaderali, A. Schonhuth, and R. Schrader A fractional programming approach to efficient DNA melting temperature calculation Bioinformatics, May 15, 2005; 21(10): 2375 - 2382. [Abstract] [Full Text] [PDF]
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P. Mignon, S. Loverix, J. Steyaert, and P. Geerlings Influence of the {pi}-{pi} interaction on the hydrogen bonding capacity of stacked DNA/RNA bases Nucleic Acids Res., March 23, 2005; 33(6): 1779 - 1789. [Abstract] [Full Text] [PDF]
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F. Tanaka, A. Kameda, M. Yamamoto, and A. Ohuchi Design of nucleic acid sequences for DNA computing based on a thermodynamic approach Nucleic Acids Res., February 8, 2005; 33(3): 903 - 911. [Abstract] [Full Text] [PDF]
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 J. Winshell, B. A. Paulson, B. D. Buelow, and J. J. Champoux Requirements for DNA Unpairing during Displacement Synthesis by HIV-1 Reverse Transcriptase J. Biol. Chem., December 17, 2004; 279(51): 52924 - 52933. [Abstract] [Full Text] [PDF]
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A. Halperin, A. Buhot, and E. B. Zhulina Hybridization Isotherms of DNA Microarrays and the Quantification of Mutation Studies Clin. Chem., December 1, 2004; 50(12): 2254 - 2262. [Abstract] [Full Text] [PDF]
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P. M. K. Gordon and C. W. Sensen Osprey: a comprehensive tool employing novel methods for the design of oligonucleotides for DNA sequencing and microa |