Nearest neighbor parameters for Watson–Crick

Nucleic Acids Research, 2006, Vol. 34, No. 13 3609–3614 doi:10.1093/nar/gkl232

Nearest neighbor parameters for Watson–Crick complementary heteroduplexes formed between 20-O-methyl RNA and RNA oligonucleotides Elzbieta Kierzek1,2, David H. Mathews3,4, Anna Ciesielska2, Douglas H. Turner1,3,* and Ryszard Kierzek2 1

Department of Chemistry, University of Rochester, RC Box 270216, Rochester, NY 14627, USA, 2Institute of Bioorganic Chemistry, Polish Academy of Sciences, 60-714 Poznan, Noskowskiego 12/14, Poland, 3Center for Pediatric Biomedical Research and 4Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA

Received January 20, 2006; Revised January 30, 2006; Accepted March 28, 2006

ABSTRACT Results from optical melting studies of Watson– Crick complementary heteroduplexes formed between 20 -O-methyl RNA and RNA oligonucleotides are used to determine nearest neighbor thermodynamic parameters for predicting the stabilities of such duplexes. The results are consistent with the physical model assumed by the individual nearest neighbor-hydrogen bonding model, which contains terms for helix initiation, base pair stacking and base pair composition. The sequence dependence is similar to that for Watson–Crick complementary RNA/RNA duplexes, which suggests that the sequence dependence may also be similar to that for other backbones that favor A-form RNA conformations.

INTRODUCTION Oligonucleotides are used for many applications, ranging from diagnostics (1–4) to therapeutics (5–9) to nanotechnology (10,11). The thermodynamics of nucleic acid duplex formation facilitates rational design of sequences for the various applications (12–14). The thermodynamics of duplex formation is dependent on the backbone of the nucleic acid. For example, the sequence dependence of the thermodynamics of DNA/DNA (15,16), RNA/RNA (17,18) and DNA/ RNA (19) duplexes differ. All, however, can be approximated well by nearest neighbor models when only Watson–Crick base pairs are formed. Thus, it is relatively easy to predict the thermodynamics of Watson–Crick paired duplexes from sequence (16,18–22). Here, optical melting studies are

analyzed to provide nearest neighbor thermodynamic parameters for formation of 20 -O-methyl RNA/RNA duplexes that are Watson–Crick complementary. The 20 -O-methyl RNA and other 20 -O-alkyl backbones are particularly useful for hybridization to RNA because they favor A-form helical structure and are more resistant than RNA or DNA to nuclease digestion (23–26).

MATERIALS AND METHODS Experimental Synthesis and purification of oligonucleotides was done as previously described (27). The buffer for melting experiments was 100 mM NaCl, 20 mM sodium cacodylate and 0.5 mM Na2EDTA, pH 7.0. Oligonucleotide single strand concentrations were determined from absorbances >80 C with extinction coefficients approximated by a nearest neighbor model (28,29). The sequence dependence of extinction coefficients for 20 -O-methyl and RNA strands was assumed to be identical. Melting curves were measured at 260 nm with a heating rate of 1 C/min from 0 to 90 C on a Beckman DU640 spectrophotometer with a water cooled Peltier thermoprogrammer. Melting curves were analyzed and thermodynamic parameters were calculated on the basis of a two-state model with the program MeltWin 3.5 (30). With one exception, agreement within 15% was found for thermodynamic parameters calculated from averaging parameters derived from the shapes of melting curves and from the following equation (20): 



T 1 M ¼ ðR/DH ÞlnðCT /4Þ þ DS /DH



1

This agreement is consistent with the two-state model.

*To whom correspondence should be addressed. Tel: +1 585 275 3207; Fax: +1 585 276 0205; Email: [email protected]  The Author 2006. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected]

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Parameter fitting The measured thermodynamic parameters were fit to the individual nearest neighbor-hydrogen bonding (INN-HB) model (18) by multiple linear regression with the program AnalyseIt v.1.71 (Analyse-It Software, Ltd, Leeds, England; www. analyse-it.com), which expands Microsoft Excel. Only duplexes that melted in a two-state manner were included in the fit. Measured parameters from T1 M versus ln(CT/4) plots were used as the data for the calculations. Error limits reported for the experimental data reflect the scatter in T1 M ’s when fit to Equation 1. Systematic errors are typically larger, however, and difficult to estimate (18). For example, the melting is not truly two-state because the stacking in the single strand conformations is dependent on temperature and sequence. Therefore, all duplexes included in the fit were given equal weight. RESULTS Table 1 lists measured thermodynamic parameters for several 20 -O-methyl RNA/RNA duplexes. Only the duplex m(50 -CGAAGUGAA)/r(30 -GCUUCACUU) does not melt in an apparent two-state manner as revealed by a >15% difference between the DH s derived from averaging fits to the shapes of melting curves and from the T1 M versus ln (CT/4)

plot. With the exception of m(50 -CGAAGUGAA)/ r(30 -GCUUCACUU), results in Table 1 were combined with previously reported results (27) listed in Table 2 and fit to the INN-HB nearest neighbor model (18) to give the nearest neighbor thermodynamic parameters listed in Table 3. On a percentage basis, the errors in nearest neighbor parameters for DG 37 are much smaller than for DH and DS . This is expected from the high correlation of errors in DH and DS , typically with R2 > 0.99 (18,31,32). While the individual errors in nearest neighbor parameters for DH and DS are large, the percentage errors in predicting DH and DS for duplex formation are smaller. This is because the values of DH and DS are given by the sums of the nearest neighbors, but the errors propagate as the square root of the sum of the squares of the errors. Table 2 lists the predicted values of DG 37, DH and DS and their percentage differences from measured values for the oligonucleotides studied. The range of percentage differences is 0–15, 0.04–23 and 0.1–26% for DG 37, DH and DS , respectively, while the average differences are, respectively, 2.4, 6.7 and 7.7%. The worst percentage prediction of DG 37 differs from the measured value by 0.73 kcal/mol, which translates to a difference of 3-fold in measured and predicted association constants. The thermodynamics for the duplex m(50 -CGAAGUGAA)/ r(30 -GCUUCACUU) (Table 1) are also predicted reasonably well even though it does not melt in a two-state manner and

Table 1. Thermodynamic parameters of heteroduplex formation between 20 -O-methyl RNA and oligoribonucleotides in 0.1 M NaCl, pH 7a 20 -O-methyl RNA (50 !30 )

RNA (50 !30 )

CAUGGG CCCAUG ACAACCA UGGUUGU ACACCCA UGGGUGU ACAGCCA UGGCUGU ACCACCA UGGUGGU ACCGCCA UGGCGGU ACGACCA UGGUCGU ACGCCCA UGGGCGU ACGGCCA UGGCCGU ACGUACA UGUACGU ACGUGCA UGCACGU ACGUUCA UGAACGU ACUACAU AUGUAGU ACUACUU AAGUAGU AUUACCA UGGUAAU CGGCAUG CAUGCCG CUUACCA UGGUAAG GCUAAGG CCUUAGC GUUACCA UGGUAAC UUUACCA UGGUAAA CGAGCAAG CUUGCUCG CGUUGAAG CUUCAACG GAGUGAAG CUUCACUC AGAAGUAAG CUUACUUCU CCAAGAUUG CAAUCUUGG CGAAAGAUG CAUCUUUCG GAAGAUUCG CGAAUCUUC GAUGUAAGU ACUUACAUC GGAAUGUAG CUACAUUCC Non-two-state duplex CGAAGUGAA UUCACUUCG a

Average of curve fits DS (eu) DH (kcal/mol)

DG 37 (kcal/mol)

TbM ( C)

T1 M versus log(CT/4) plots DH DS (eu) (kcal/mol)

DG 37 (kcal/mol)

TbM ( C)

61.6 49.4 51.5 54.4 51.1 60.0 53.5 54.4 57.5 52.5 58.3 52.9 46.5 52.1 47.8 69.0 54.9 66.5 55.3 43.2 76.3 67.1 78.3 84.5 94.2 78.3 79.1 81.7 87.0

7.72 6.20 8.52 8.70 8.62 10.86 8.40 10.32 10.71 7.09 9.20 7.05 5.41 5.78 5.34 9.59 6.89 8.15 6.88 5.27 10.15 8.09 9.33 8.49 9.95 8.87 9.21 8.76 10.19

0.34 0.13 0.15 0.14 0.18 0.23 0.14 0.11 0.30 0.11 0.10 0.14 0.10 0.10 0.11 0.15 0.08 0.11 0.10 0.21 0.35 0.09 0.21 0.11 0.23 0.12 0.22 0.25 0.24

43.1 34.9 49.5 49.9 50.2 61.1 48.2 60.2 61.3 40.3 51.9 40.1 29.7 32.6 29.5 51.4 39.0 44.7 39.0 28.2 52.4 44.4 48.5 44.4 48.7 46.6 47.9 45.7 50.6

60.9 42.7 47.7 50.7 47.4 57.7 49.6 58.9 57.5 49.2 59.1 47.2 45.9 46.9 44.5 65.5 50.2 71.1 49.9 43.0 70.8 76.4 79.6 85.6 92.5 75.8 76.1 79.9 79.9

7.52 6.31 8.38 8.56 8.50 10.69 8.28 10.56 10.68 7.09 9.22 7.09 5.45 5.91 5.47 9.48 6.68 8.17 6.85 5.28 10.01 8.27 9.37 8.49 9.89 8.80 9.11 8.71 9.98

0.01 0.02 0.04 0.02 0.09 0.16 0.03 0.10 0.10 0.02 0.05 0.01 0.07 0.04 0.05 0.04 0.01 0.04 0.01 0.11 0.17 0.39 0.18 0.17 0.17 0.02 0.21 0.19 0.56

42.1 35.4 49.5 49.9 50.3 61.1 48.3 59.8 61.1 40.5 51.8 40.7 29.9 32.9 29.8 51.6 39.1 44.3 39.0 28.3 53.0 44.2 48.5 44.3 48.7 46.6 47.9 45.7 51.0

10.56 ± 0.23

50.5

77.2 ± 2.4

9.99 ± 0.07

51.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

17.7 2.7 5.3 2.3 3.1 3.2 3.6 2.3 5.2 3.3 1.5 0.43 2.6 1.9 1.6 3.6 3.8 6.4 3.5 3.7 8.9 11.5 5.1 6.8 4.9 3.4 5.1 8.4 4.8

96.6 ± 6.1

173.9 139.1 138.5 147.4 137.0 158.3 145.5 142.2 150.9 146.5 158.4 147.9 132.3 149.3 137.0 191.7 154.7 188.2 156.0 122.2 213.2 190.3 22.6 245.0 271.6 223.9 225.3 235.1 247.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

56.1 8.7 16.7 6.9 9.4 9.6 11.1 7.1 15.9 10.5 4.6 14.1 8.7 5.9 5.2 11.1 12.1 20.7 11.1 12.4 27.6 36.9 16.1 22.4 15.3 10.6 16.0 26.7 14.9

277.6 ± 19.1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Solutions are 100 mM NaCl, 20 mM sodium cacodylate and 0.5 mM Na2EDTA, pH 7. Calculated for 104 M total strand concentration.

b

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.5 0.8 1.5 0.8 2.6 2.7 1.3 1.7 1.7 1.6 1.4 0.8 1.7 1.2 1.3 1.1 0.8 2.0 1.2 2.1 4.4 13.1 5.9 7.6 5.8 1.6 6.6 7.4 13.0

172.3 117.3 126.9 135.7 126.5 151.4 133.3 155.9 151.0 135.8 160.8 129.3 130.4 132.3 125.8 180.7 139.6 202.0 138.6 121.7 196.0 219.6 226.4 248.5 266.4 216.2 216.1 229.6 225.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.9 2.6 4.6 2.5 8.1 8.2 4.1 5.2 5.0 5.2 4.4 2.7 5.8 4.1 4.5 3.6 2.6 6.4 3.9 7.0 13.7 41.4 18.8 24.1 18.2 5.0 20.8 23.7 40.6

216.8 ± 7.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

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Table 2. Measured and predicted (in parenthesis) DG 37, DH , DS and the percentage difference between measured and predicted values for Watson–Crick complementary 20 -O-methyl RNA/RNA duplexes in 0.1 M NaCl, pH 7 20 -O-methyl RNA (50 !30 )

DG 37 (kcal/mol)

% of difference

DH (kcal/mol)

Percentage of difference

DS (eu)

CMGMGMCMAM UMCMGMGMCM CMAMUMGMGMGM CMGMGMCMAMUM GMCMAMUMGMGM GMUMUMCMGMGM GMGMCMAMUMGM UMCMGMGMCMAM UMUMCMGMGMCM AMCMAMAMCMCMAM AMCMAMCMCMCMAM AMCMAMGMCMCMAM AMCMAMUMCMCMAM AMCMCMAMCMCMAM AMCMCMGMCMCMAM AMCMCMUMCMCMAM AMCMGMAMCMCMAM AMCMGMCMCMCMAM AMCMGMGMCMCMAM AMCMGMUMAMCMAM AMCMGMUMCMCMAM AMCMGMUMGMCMAM AMCMGMUMUMCMAM AMCMUMAMAMCMAM AMCMUMAMCMAMUM AMCMUMAMCMCMAM AMCMUMAMCMCMCM AMCMUMAMCMCMGM AMCMUMAMCMCMUM AMCMUMAMCMGMUM AMCMUMAMCMUMUM AMCMUMAMGMCMAM AMCMUMAMUMCMAM AMCMUMCMAMCMAM AMCMUMCMCMCMAM AMCMUMCMGMCMAM AMCMUMCMUMCMAM AMCMUMGMAMCMAM AMCMUMGMCMCMAM AMCMUMGMGMCMAM AMCMUMGMUMCMAM AMCMUMUMAMCMAM AMCMUMUMCMCMAM AMCMUMUMGMCMAM AMCMUMUMUMCMAM AMGMUMAMCMCMAM AMUMUMAMCMCMAM CMCMUMAMCMCMAM CMGMGMCMAMUMGM CMUMUMAMCMCMAM GMCMUMAMAMGMGM GMCMUMAMCMCMAM GMCMUMAMCMUMGM GMGMCMAMUMGMGM GMGMUMAMUMGMGM GMUMUMAMCMCMAM UMCMUMAMCMCMAM UMUMUMAMCMCMAM UMUMUMCMAMCMUM CMGMAMGMCMAMAMGM CMGMUMUMGMAMAMGM GMAMGMUMGMAMAMGM AMGMAMAMGMUMAMAMGM CMCMAMAMGMAMUMUMGM CMGMAMAMAMGMAMUMGM GMAMAMGMAMUMUMCMGM GMAMUMGMUMAMAMGMUM GMGMAMAMUMGMUMAMGM

6.19 (6.46) 6.38 (6.47) 7.52 (6.99) 6.98 (7.30) 7.19 (7.19) 6.95 (6.86) 7.19 (7.19) 8.06 (8.06) 7.76 (7.41) 6.31 (6.39) 8.38 (8.62) 8.56 (9.07) 6.86 (6.98) 8.50 (8.62) 10.69 (10.50) 9.20 (9.20) 8.28 (8.36) 10.56 (10.50) 10.68 (10.54) 7.09 (6.89) 8.96 (8.77) 9.22 (9.05) 7.09 (6.93) 5.23 (5.09) 5.45 (5.38) 7.13 (7.32) 8.29 (8.51) 8.04 (8.08) 7.37 (7.60) 7.05 (7.17) 5.91 (5.76) 8.22 (7.77) 5.82 (5.68) 7.22 (7.13) 9.51 (9.20) 9.04a (9.01) 7.66 (7.71) 7.36 (7.34) 9.34 (9.48) 9.76 (9.52) 7.83 (7.75) 5.65 (5.48) 7.36 (7.36) 7.59 (7.64) 5.76 (5.52) 7.33 (7.53) 5.47 (5.33) 9.07 (8.80) 9.48 (9.54) 6.86 (6.96) 8.17 (8.25) 9.78 (9.04) 8.51 (8.78) 9.69 (10.01) 8.34 (8.47) 6.85 (6.60) 7.18 (7.62) 5.28 (5.43) 4.79 (5.52) 10.01 (10.17) 8.27 (8.50) 9.37 (9.08) 8.49 (8.34) 9.89 (9.49) 8.80 (8.84) 9.11 (9.19) 8.71 (9.12) 9.98 (10.07)

4.36 1.41 7.05 4.58 0.00 1.29 0.00 0.00 4.51 1.27 2.86 5.96 1.75 1.41 1.78 0.00 0.97 0.57 1.31 2.82 2.12 1.84 2.26 2.68 1.28 2.66 2.65 0.50 3.12 1.70 2.54 5.47 2.41 1.25 3.26 0.33 0.65 0.27 1.50 2.46 1.02 3.01 0.00 0.66 4.17 2.73 2.56 2.98 0.63 1.46 0.98 7.57 3.17 3.30 1.56 3.65 6.13 2.84 15.24 1.60 2.78 3.09 1.77 4.04 0.45 0.88 4.71 0.90

46.0 51.5 60.9 45.3 51.9 54.6 65.2 65.5 53.5 42.7 47.7 50.7 43.5 47.4 57.7 50.9 49.6 58.9 57.5 49.2 55.8 59.1 47.2 48.9 45.9 44.4 61.2 53.0 62.2 50.9 46.9 75.2 52.1 63.6 55.9 56.0 53.0 48.2 58.7 59.6 52.5 46.7 47.6 52.7 39.6 44.5 44.5 60.5 65.5 50.2 71.1 54.3 66.2 64.1 57.2 49.9 45.2 43.0 56.4 70.8 76.4 79.6 85.6 92.5 75.8 76.1 79.9 79.9

1.76 3.63 8.37 13.73 10.46 1.78 12.07 21.07 2.92 7.59 0.75 12.96 10.62 0.13 2.13 8.15 2.24 4.13 0.43 4.61 5.61 2.76 4.28 2.02 1.87 11.06 8.30 7.00 13.68 0.81 7.12 21.22 3.86 23.24 1.52 3.48 6.66 7.61 1.96 1.73 6.76 1.80 8.40 7.02 21.59 21.26 1.48 9.07 1.98 2.71 0.04 5.56 2.87 4.51 11.33 2.91 16.46 2.93 14.63 12.71 3.60 1.43 0.35 15.70 7.10 0.71 6.10 1.64

128.4 145.4 172.3 123.5 144.1 153.8 187.1 185.1 147.3 117.3 126.9 135.7 118.2 126.5 151.4 134.6 133.3 155.9 151.0 135.8 151.1 160.8 129.3 140.9 130.4 120.2 170.4 145.3 176.8 141.3 132.3 216.0 149.2 181.7 149.5 152.5 146.1 131.8 159.2 160.7 144.1 132.2 129.6 145.3 109.2 119.9 125.8 165.9 180.7 139.6 202.0 146.7 186.0 175.6 157.4 138.6 122.5 121.7 166.3 196.0 219.6 226.4 248.5 266.4 216.2 216.1 229.6 225.5

a

(45.19) (49.63) (55.80) (51.52) (57.33) (53.63) (57.33) (51.70) (55.06) (45.94) (47.34) (57.27) (48.12) (47.34) (56.47) (55.05) (48.49) (56.47) (57.25) (46.93) (52.67) (57.47) (49.22) (47.91) (46.76) (49.31) (56.12) (56.71) (53.69) (51.31) (50.24) (59.24) (50.09) (48.82) (55.05) (57.95) (56.53) (51.87) (59.85) (60.63) (56.05) (45.86) (51.60) (56.40) (48.15) (53.96) (45.16) (55.01) (66.80) (51.56) (71.13) (57.32) (68.10) (66.99) (63.68) (48.45) (52.64) (44.26) (48.15) (79.80) (73.65) (78.46) (85.30) (77.98) (81.18) (76.64) (75.03) (81.21)

The error in this value is 0.07 kcal/mol, but was originally reported as 0.7 kcal/mol (27).

Percentage of difference (125.0) (139.3) (157.5) (142.7) (161.7) (150.9) (161.7) (140.9) (153.8) (127.6) (125.0) (155.5) (132.8) (125.0) (148.4) (148.0) (129.5) (148.4) (150.8) (129.2) (141.7) (156.2) (136.5) (138.1) (133.5) (135.5) (153.6) (156.9) (148.7) (142.4) (143.5) (166.0) (143.3) (134.5) (148.0) (157.9) (157.5) (143.6) (162.5) (164.9) (155.8) (130.3) (142.8) (157.3) (137.6) (149.8) (128.6) (149.1) (184.7) (143.9) (202.7) (155.7) (191.2) (183.8) (178.1) (135.0) (145.3) (125.4) (137.6) (224.4) (210.0) (223.5) (248.0) (220.8) (233.1) (217.4) (212.4) (229.3)

2.65 4.20 8.59 15.55 12.21 1.89 13.58 23.88 4.41 8.78 1.50 14.59 12.35 1.19 1.98 9.96 2.85 4.81 0.13 4.86 6.22 2.86 5.57 1.99 2.38 12.73 9.86 7.98 15.89 0.78 8.47 23.15 3.95 25.98 1.00 3.54 7.80 8.95 2.07 2.61 8.12 1.44 10.19 8.26 26.01 24.94 2.23 10.13 2.21 3.08 0.35 6.13 2.80 4.67 13.15 2.60 18.61 3.04 17.26 14.49 4.37 1.28 0.20 17.12 7.82 0.60 7.49 1.69

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Table 3. Thermodynamic parameters for INN-HB nearest neighbor model applied to 20 -O-methyl RNA/RNA heteroduplexes in 0.1 M NaCl, pH 7 Parameters 0

0

m(5 -AA)/r(3 -UU) m(50 -AU)/r(30 -UA) m(50 -UU)/r(30 -AA) m(50 -UA)/r(30 -AU) m(50 -AC)/r(30 -UG) m(50 -AG)/r(30 -UC) m(50 -CA)/r(30 -GU) m(50 -UC)/r(30 -AG) m(50 -UG)/r(30 -AC) m(50 -GA)/r(30 -CU) m(50 -CU)/r(30 -GA) m(50 -GU)/r(30 -CA) m(50 -CG)/r(30 -GC) m(50 -CC)/r(30 -GG) m(50 -GG)/r(30 -CC) m(50 -GC)/r(30 -CG) Initiation Per terminal AU a

DG a37 (kcal/mol)

DH (kcal/mol)

DS b (eu)

Number of occurrences

0.55 0.84 0.94 1.20 1.60 1.81 1.89 1.90 1.94 2.06 2.17 2.17 2.35 2.78 2.82 3.02 3.32 0.30

7.48 6.33 5.43 6.47 6.32 13.94 5.21 9.65 12.14 5.77 9.59 6.62 9.47 8.88 9.66 11.19 12.80 3.14

22.3 17.7 14.5 17.0 15.2 39.1 10.7 25.0 32.9 11.9 23.9 14.3 23.0 19.7 22.1 26.3 52.0 9.1

14 17 19 25 62 17 56 20 19 14 34 16 22 35 19 23 68 92

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.15 0.13 0.11 0.15 0.16 0.16 0.15 0.16 0.16 0.18 0.16 0.16 0.16 0.10 0.17 0.18 0.55 0.08

(0.93) (1.10) (0.93) (1.33) (2.24) (2.08) (2.11) (2.35) (2.11) (2.35) (2.08) (2.24) (2.36) (3.26) (3.26) (3.42) (4.09) (0.45)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.20 2.85 2.41 3.31 3.47 3.47 3.33 3.51 3.56 3.90 3.40 3.37 3.48 2.15 3.69 3.90 11.98 1.82

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

9.8 8.8 7.4 10.2 10.7 10.7 10.2 10.8 10.9 12.0 10.4 10.4 10.7 6.6 11.4 12.0 36.8 5.6

Values in parentheses are for RNA/RNA duplexes in 1 M NaCl [Xia et al. (18)]. Calculated from DS ¼ (DH  DG 37)/310.15 and given in eu ¼ cal K1 mol1.

b

was not used in fitting the nearest neighbor parameters. The predicted values for DG 37, DH and DS are 9.87 kcal/ mol, 78.3 kcal/mol and 220.6 eu while the measured values are 10.56 kcal/mol, 96.6 kcal/mol and 277.6 eu, respectively.

DISCUSSION Thermodynamic parameters for nucleic acid duplexes are useful for designing sequences for many applications, including diagnostics, therapeutics and nanotechnology (12–14,33). The 20 -O-methyl backbone and other 20 -O-alkyl backbones are particularly useful for binding to RNA because they favor A-form helixes and are chemically stable relative to DNA and RNA backbones. Thus for example, 20 -O-alkyl backbones have been used to flank a ‘gap’ DNA sequence in order to decrease nuclease digestion of the oligonucleotide while providing a long enough pairing between DNA and RNA to induce RNase H to cleave an RNA target (34). Reduction of gene expression by an RNA interference mechanism has been demonstrated with siRNA duplexes having completely 20 -O-methyl modified sense strands (35). The 20 -O- methyl modification is also used in aptamers (7), including the commercially successful therapeutic, Macugen (8). There are several ways to analyze the data in Table 2. In principle, it is possible to fit the data to 20 parameters each for DG 37, DH and DS (21,22). We chose, however, to fit the data to the 18 parameters of the INN-HB model, which ascribes separate parameters to the 16 different nearest neighbor stacks, to the average difference between a CG and UA pair, and to duplex initiation (18). This simplifies the 20 parameter model by assuming that the parameters for initiation are independent of the nature of the terminal base pairs and that terminal base pairs are equivalent to internal base pairs. With the 18 parameter fit, the highest P-values for DG 37 were 0.0007 and 0.0005 for the terminal AU and

m(50 -AA)/r(30 -UU) parameters, respectively. All other P-values were