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.     

Purine-purine mismatches in RNA helices: evidence for protonated G.A pairs and next-nearest neighbor effects.

Thermodynamic parameters are presented for 12 different RNA duplexes containing A.A, A.G, G.A and G.G mismatches flanked by C-G base pairs. UV melting studies were conducted under three different buffer conditions in order to evaluate the effects of salt concentration and pH on the stability of each mismatch-containing duplex. The main findings are: (i) the mismatches have a wide range of effects on duplex stability, decreasing delta G degrees 37 of denaturation by approximately 0-7 kcal/mol; (ii) the nearest-neighbor assumption commonly used to calculate helix stability breaks down for G.A mismatches; and (iii) G.A mismatches separated by 2 bp form a protonated structure.     

Thermodynamics of sequence-specific binding of PNA to DNA

For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and sequence specificity of binding (singly mismatched duplexes) using mainly absorption hypochromicity melting curves and isothermal titration calorimetry. For perfectly sequence-matched duplexes of varying lengths (6-20 bp), the average free energy of binding (DeltaG degrees ) was determined to be -6.5+/-0.3 kJ mol(-1) bp(-1), corresponding to a microscopic binding constant of about 14 M(-1) bp(-1). A variety of single mismatches were introduced in 9- and 12-mer PNA-DNA duplexes. Melting temperatures (T(m)) of 9- and 12-mer PNA-DNA duplexes with a single mismatch dropped typically 15-20 degrees C relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kJ mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only 0.02 M(-1) per mismatch. The impact of a mismatch was found to be dependent on the neighboring base pairs. To a first approximation, increasing the stability of the surrounding region, i.e., the distribution of A.T and G.C base pairs, decreases the effect of the introduced mismatch.     

Nucleic acid duplex stability: influence of base composition on cation effects.

The effects of counter ion on a nucleic acid duplex stability were investigated. Since a linear free energy relationship for the thermostability of oligonucleotide duplexes between those in 1 M and in 100 mM NaCl-phosphate buffer were observed regardless of whether they are DNA-DNA, RNA-RNA or RNA-DNA duplexes, simple prediction systems for [Delta] G degrees 37as well as T mvalues in 100 mM NaCl-phosphate buffer were established. These predictions were successful with an average error of only 2.4 degrees C for T mand 5. 7% for G degrees 37values. The number of Na+newly bound to a duplex when the duplex forms (-[Delta] n) was significantly influenced by the base composition, and -[Delta] n for d(GCCAGTTAA)/d(TTAACTGGC) was different for MgCl2, CaCl2, BaCl2and MnCl2(from 0.70 to 0.76 with the same order of the duplex stability). Almost no additive effects on the duplex stability was observed for NaCl and MgCl2, suggesting a competitive binding for these cations. The sequence-dependent manner of [Delta] n suggests the presence of preferential base pairs or nearest-neighbor base pairs for the cation binding, which would affect nearest-neighbor parameters.     

Stabilities of consecutive A.C, C.C, G.G, U.C, and U.U mismatches in RNA internal loops: Evidence for stable hydrogen-bonded U.U and C.C.+ pairs.

The stability and structure of RNA duplexes with consecutive A.C, C.A, C.C, G.G, U.C, C.U, and U.U mismatches were studied by UV melting, CD, and NMR. The results are compared to previous results for GA and AA internal loops [SantaLucia, J., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819; Peritz, A., Kierzek, R., & Turner, D.H. (1991) Biochemistry 30, 6428-6436)]. The observed order for stability increments of internal loop formation at pH 7 is AG = GA approximately UU greater than GG greater than or equal to CA greater than or equal to AA = CU = UC greater than or equal to CC greater than or equal to AC. The results suggest two classes for internal loops with consecutive mismatches: (1) loops that stabilize duplexes and have strong hydrogen bonding and (2) loops that destabilize duplexes and may not have strong hydrogen bonding. Surprisingly, rCGCUUGCG forms a very stable duplex at pH 7 in 1 M NaCl with a TM of 44.8 degrees C at 1 x 10(-4) M and a delta G degrees 37 of -7.2 kcal/mol. NOE studies of the imino protons indicate hydrogen bonding within the U.U mismatches in a wobble-type structure. Resonances corresponding to the hydrogen-bonded uridines are located at 11.3 and 10.4 ppm. At neutral pH, rCGCCCGCG is one of the least stable duplexes with a TM of 33.2 degrees C and delta G degrees 37 of -5.1 kcal/mol. Upon lowering the pH to 5.5, however, the TM increases by 12 degrees C, and delta G degrees 37 becomes more favorable by 2.5 kcal/mol. The pH dependence of rCGCCCGCG may be due to protonation of the internal loop C's, since no changes in thermodynamic parameters are observed for rCGCUUGCG between pH 7 and 5.5. Furthermore, two broad imino proton resonances are observed at 10.85 and 10.05 ppm for rCGCCCGCG at pH 5.3, but not at pH 6.5. This is also consistent with C.C+ base pairs forming at pH 5.5. rCGCCAGCG and rGGCACGCC have a small pH dependence, with TM increases of 5 and 3 degrees C, respectively, upon lowering the pH from 7 to 5.5. rCGCCUGCG and rCGCUCGCG also show little pH dependence, with TM increases of 0.8 and 1.4 degrees C, respectively, upon lowering the pH to 5.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches

Thermodynamic measurements are reported for 51 DNA duplexes with A.A, C.C, G.G, and T.T single mismatches in all possible Watson-Crick contexts. These measurements were used to test the applicability of the nearest-neighbor model and to calculate the 16 unique nearest-neighbor parameters for the 4 single like with like base mismatches next to a Watson-Crick pair. The observed trend in stabilities of mismatches at 37 degrees C is G.G > T.T approximately A.A > C.C. The observed stability trend for the closing Watson-Crick pair on the 5' side of the mismatch is G.C >/= C.G >/= A.T >/= T.A. The mismatch contribution to duplex stability ranges from -2.22 kcal/mol for GGC.GGC to +2.66 kcal/mol for ACT.ACT. The mismatch nearest-neighbor parameters predict the measured thermodynamics with average deviations of DeltaG degrees 37 = 3.3%, DeltaH degrees = 7. 4%, DeltaS degrees = 8.1%, and TM = 1.1 degrees C. The imino proton region of 1-D NMR spectra shows that G.G and T.T mismatches form hydrogen-bonded structures that vary depending on the Watson-Crick context. The data reported here combined with our previous work provide for the first time a complete set of thermodynamic parameters for molecular recognition of DNA by DNA with or without single internal mismatches. The results are useful for primer design and understanding the mechanism of triplet repeat diseases.     

Stability of DNA duplexes containing GG, CC, AA, and TT mismatches

We employed salt-dependent differential scanning calorimetric measurements to characterize the stability of six oligomeric DNA duplexes (5'-GCCGGAXTGCCGG-3'/5'-CCGGCAYTCCGGC-3') that contain in the central XY position the GC, AT, GG, CC, AA, or TT base pair. The heat-induced helix-to-coil transitions of all the duplexes are associated with positive changes in heat capacity, DeltaC(p), ranging from 0.43 to 0.53 kcal/mol. Positive values of DeltaC(p) result in strong temperature dependences of changes in enthalpy, DeltaH degrees, and entropy, DeltaS degrees , accompanying duplex melting and cause melting free energies, DeltaG degrees, to exhibit characteristically curved shapes. These observations suggest that DeltaC(p) needs to be carefully taken into account when the parameters of duplex stability are extrapolated to temperatures distant from the transition temperature, T(M). Comparison of the calorimetric and van't Hoff enthalpies revealed that none of the duplexes studied in this work exhibits two-state melting. Within the context of the central AXT/TYA triplet, the thermal and thermodynamic stabilities of the duplexes in question change in the following order: GC > AT > GG > AA approximately TT > CC. Our estimates revealed that the thermodynamic impact of the GG, AA, and TT mismatches is confined within the central triplet. In contrast, the thermodynamic impact of the CC mismatch propagates into the adjacent helix domains and may involve 7-9 bp. We discuss implications of our results for understanding the origins of initial recognition of mismatched DNA sites by enzymes of the DNA repair machinery.     

Effects of GA mismatches on the structure and thermodynamics of RNA internal loops

UV melting, CD and NMR studies indicate rGCGAGCG and rGCAGGCG from unusually stable duplexes of type a and b. The observed delta G degree 37's in 1 M NaCl are -6.7 and -6.3 kcal/mol, respectively. For the related duplex, c, delta G degree 37 is -4.2 kcal/mol. The predicted delta G degree 37 from nearest-neighbor parameters (formula; see text) for all three duplexes is -4.7 kcal/mol (Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377). The results suggest a special interaction in the duplexes containing GA mismatches. Presumably, this is hydrogen bonding between G and A. While the thermodynamics for (rGCGAGCG)2 and (rGCAGGCG)2 are similar, CD and the imino region of the proton NMR spectra indicate their structures are different. In particular, (rGCAGGCG)2 exhibits a CD spectrum typical of A-form geometry with a weak negative band at 280 nm. In contrast, the CD spectrum for (rGCGAGCG)2 has an intense positive band at 285 nm. The NMR spectrum of (rGCAGGCG)2 has a resonance corresponding to a hydrogen-bonded GA mismatch, while for (rGCGAGCG)2 no hydrogen-bonded imino proton is observed for the mismatch. The glycosidic torsion angles of the bases in the GA mismatches of (rGCAGGCG)2 and (rCGCAGGCG)2 are anti. Duplexes of type d, where X is A, G, or U, are more stable than e, and the stability differences are similar to those (formula; see text) observed for f versus g. Thus, 3'-dangling ends in this system make contributions to duplex stability that are similar to contributions observed with fully paired duplexes.     

Thermodynamic characterization of single mismatches found in naturally occurring RNA

Many naturally occurring RNA structures contain single mismatches. However, the algorithms currently used to predict RNA structure from sequence rely on a minimal set of data for single mismatches, most of which occur rather infrequently in nature. As a result, several approximations and assumptions are used to predict the stability of RNA duplexes containing the most common single mismatches. Therefore, the relative frequency of single mismatches was determined by compiling and searching a database of 955 RNA secondary structures. Thermodynamic parameters for duplex formation, derived from optical melting experiments, are reported for 28 oligoribonucleotides containing frequently occurring single mismatches. These data were then combined with previous data to construct a dataset of 64 single mismatches, including the 30 most common in the database. Because of this increase in experimental thermodynamic parameters for single mismatches that occur frequently in nature, more accurate free energy calculations have resulted. To improve the prediction of the thermodynamic parameters for duplexes containing single mismatches that have not been experimentally measured, single mismatch-specific nearest neighbor parameters were derived. The free energy of an RNA duplex containing a single mismatch that has not been thermodynamically characterized can be calculated by: DeltaG degrees 37,single mismatch = DeltaG degrees 37,mismatch nt + DeltaG degrees 37,mismatch-NN interaction + DeltaG degrees 37,AU/GU. Here, DeltaG degrees 37,mismatch is -0.4, -2.1, and -0.3 kcal/mol for A.G, G.G, and U.U mismatches, respectively; DeltaG degrees 37,mismatch-NN interaction is 0.7, -0.5, 0.4, -0.4, and -1.0 kcal/mol for 5'YRR3'/3'RRY5', 5'RYY3'/3'YYR5', 5'YYR3'/3'RYY5', 5'YRY3'/3'RYR5', and 5'RRY3'/3'YYR5' mismatch-nearest neighbor combinations, respectively, when A and G are categorized as purines (R) and C and U are categorized as pyrimidines (Y); and DeltaG degrees 37,AU/GU is a penalty of 1.2 kcal/mol for replacing a G-C base pair with either an A-U or G-U base pair. Similar predictive models were also derived for DeltaH degrees single mismatch and DeltaS degrees single mismatch. These new predictive models, in conjunction with the reported thermodynamics for frequently occurring single mismatches, should allow for more accurate calculations of the free energy of RNA duplexes containing single mismatches and, furthermore, allow for improved prediction of secondary structure from sequence.     

Free energy contributions of G.U and other terminal mismatches to helix stability

Thermodynamic parameters of helix formation were measured spectroscopically for seven hexaribonucleotides containing a GC tetramer core and G.U or other terminal mismatches. The free energies of helix formation are compared with those for the tetramer core alone and with those for the hexamer with six Watson-Crick base pairs. In 1 M NaCl, at 37 degrees C, the free energy of a terminal G.U mismatch is about equal to that of the corresponding A.U pair. Although other terminal mismatches studied add between -1.0 and -1.6 kcal/mol to delta G0 37 for helix formation, all are less stable than the corresponding Watson-Crick pairs. Comparisons of the stability increments for terminal G.U mismatches and G.C pairs suggest when stacking is weak the additional hydrogen bond in the G.C pair adds roughly -1 kcal/mol to the favorable free energy of duplex formation.     

RNA hairpin loop stability depends on closing base pair.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXAUAAUAYCC, where X and Y are CG, GC, AU, UA, GU, or UG. A nearest neighbor analysis of the data indicates the free energy change for loop formation at 37 degrees C, delta degrees Gl,37, averages 3.4 kcal/mol for hairpin loops closed with C.G, G.C, and G.U pairs. In contrast, delta G degree l,37 averages 4.6 kcal/mol for loops closed with A.U, U.A, or U.G pairs. Thus the stability of an RNA hairpin depends on the closing base pair. The hairpin with a GA mismatch that is formed by GGCGUAAUAGCC is more stable than the corresponding hairpin with an AA mismatch. Thus hairpin stability also depends on loop sequence. These effects are not included in current algorithms for prediction of RNA structure from sequence.     

Thermodynamic and structural properties of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of identical sequence

Four pentamers with the general sequence 5'CU(T)GU(T)G/5'CACAG have been prepared by chemical synthesis in order to generate duplex structures with common sequences. The four duplexes studied include the DNA.DNA duplex (5'dCACAG/5'dCTGTG) and the RNA.RNA duplex (5'rCUGUG/5'rCACAG) as well as the two corresponding DNA.RNA heteroduplexes (5'rCUGUG/5'dCACAG and 5'CACAG/5'dCTGTG). The measured entropy, enthalpy, and free energy changes upon melting are reported for each pentamer and compared to the predicted values where possible. Results show that the two DNA.RNA heteroduplexes are destabilized (delta G degrees 25 = -4.2 +/- 0.4 kcal/mol) relative to either the DNA.DNA duplex (delta G degrees 25 = -4.8 +/- 0.5 kcal/mol) or the RNA.RNA duplex (delta G degrees 25 = -5.8 +/- 0.6 kcal/mol). Circular dichroism spectra indicate that the RNA and the two heteroduplexes adopt an A-form conformation, while the DNA conformation is B-form. Imino proton NMR spectra also show that the heteroduplex structures resemble the RNA.RNA duplex.     

Watson-Crick base-pairing properties of bicyclo-DNA.

A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly discriminates for base mismatches. Duplexes are always inferior in stability compared with the natural ones. A detailed analysis of the thermodynamic properties was performed with the sequence 5'-GGATGGGAG-3'x 5'-CTCCCATCC-3' in both backbone systems. Comparison of the pairing enthalpy and entropy terms shows an enthalpic advantage for DNA association (delta deltaH = -18 kcal x (mol)-1)) and an entropic advantage for bicyclo-DNA association (delta deltaS = 49 cal x K(-1) x mol(-1), leading to a delta deltaG 25 degrees C of -3.4 kcal x mol(-1) in favor of the natural duplex. The salt dependence of Tm for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of 8 compared with DNA.     

Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops.

We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.     

Comparison of the base pairing properties of a series of nitroazole nucleobase analogs in the oligodeoxyribonucleotide sequence 5'-d(CGCXAATTYGCG)-3'.

The nucleoside analogs 1-(2'-deoxy-beta-D-ribofuranosyl)- 3-nitropyrrole (9), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitropyrazole (10), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitroimidazole (11) and 1-(2'-deoxy-beta-D-ribofuranosyl)-5-nitroindole (21) were incorporated into the oligonucleotide 5'-d(CGCXAATTYGCG)-3'in the fourth position from the 5'-end. Procedures for synthesis of two of the nitroazole nucleosides, 10 and 11, were developed for this study. Each of the nitroazoles was converted into a 3'-phosphoramidite for oligonucleotide synthesis by conventional automated protocols. Four oligonucleotides were synthesized for each modified nucleoside in order to obtain duplexes in which each of the four natural bases was placed opposite (position 9) the nitroazole. In order to assess the role of the nitro group on base stacking interaction, sequences were also synthesized in which the fourth base was 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole. Corresponding sequences containing an abasic site, as well as sequences containing inosine, were synthesized for comparison. Thermal melting studies yielded T m values and thermodynamic parameters. Each nucleoside analog displayed a unique pattern of base pairing preferences. The least discriminating analog was 3-nitropyrrole, for which T m values differed by 5 degrees C and Delta G 25 degrees C ranged from -6.1 to -6.5 kcal/mol. 5-Nitroindole gave duplexes with significantly higher thermal stability, with Tm values varying from 35.0 to 46.5 degrees C and -Delta G 25 degrees C ranging from 7.7 to 8.5 kcal/mol. Deoxyinosine (22), a natural analog which has found extensive use as a universal nucleoside, is far less non-discriminating than any of the nitroazole derivatives. Tm values ranged from 35.4 degrees C when paired with G to 62.3 degrees C when paired with C. The significance of the nitro substituent was determined by comparison of the base pairing properties of a simple azole nucleoside, 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole (12). The pyrazole-containing sequences melt at 10-20 degrees C lower than the corresponding nitropyrazole-containing sequences. On average, the pyrazole-containing sequences were equivalent in stability (average Delta G = -4.8 kcal/mol) to the sequences containing an abasic site (average Delta G = -4.7 kcal/mol).     

The thermodynamics of 3'-terminal pyrene and guanosine for the design of isoenergetic 2'-O-methyl-RNA-LNA chimeric oligonucleotide probes of RNA structure

To facilitate design of short isoenergetic hybridization probes for RNA, we report the influence of adding 5'- or 3'-terminal 2'-O-methylguanosine (GM), LNA-guanosine (GL), or 3'-terminal pyrene pseudo-nucleotide (PPN) on the thermodynamic stability of 2'-O-methyl-RNA/RNA (2'-O-Me-RNA/RNA) duplexes with sequences 5'CMGMGMCMAM/3'AAXGCCGUXAA, where X is A, C, G, or U. A 3'-terminal GM or GL added to the 2'-O-Me-RNA strand to form a G-A, G-G or G-U mismatch enhances thermodynamic stability (DeltaDeltaG degrees 37) of the 2'-O-Me-RNA/RNA duplexes on average by 0.7 and 1.5 kcal/mol, respectively. A 3'-terminal GM or GL in a GM-C or GL-C pair stabilizes the 2'-O-Me-RNA/RNA duplex by 2.6 and 3.4 kcal/mol, respectively. A 5'-terminal GM or GL in a G-A or G-G mismatch provided less stabilization in comparison with a 3'-terminal G-A or G-G mismatch, but more stabilization in a G-C or G-U pair. In contrast to guanosine derivatives, pyrene residue (P) as PPN at the 3'-terminal position enhances thermodynamic stability of the 2'-O-Me-RNA/RNA duplexes on average by 2.3 +/- 0.1 kcal/mol, relatively independent of the type of ribonucleotide placed in the opposite strand. The thermodynamic data can be applied to design 2'-O-Me-RNA/RNA duplexes with enhanced thermodynamic stability that is also sequence independent. This is useful for design of hybridization probes to interrogate RNA structure and/or expression by microarray and other methods.     

C5-(1-propynyl)-2'-deoxy-pyrimidines enhance mismatch penalties of DNA:RNA duplex formation

UV melting experiments show that C5-(1-propynyl)ation of seven pyrimidines to give a fully propynylated oligodeoxynucleotide (PrODN) heptamer increases the thermodynamic stability of six Watson-Crick paired DNA:RNA duplexes by 8.2 kcal/mol, on average, at 37 degrees C. About 2.5 kcal/mol of this enhancement is due to long-range cooperativity between the propynylated pyrimidines, Y(p)'s. On average, penalties for dU(p):rG, dC(p):rA, dU(p):rC, and dC(p):rC mismatches are enhanced by 2.9 kcal/mol in PrODN:RNA duplexes over those in unmodified duplexes. This results in penalties as large as 10 kcal/mol for a single mismatch. Removing a single propyne two base pairs away from a mismatch in a PrODN:RNA duplex eliminates the enhancement in specificity. Evidently, enhanced specificity is directly linked to long-range cooperativity between Y(p)'s. In most cases, the enhanced specificity is larger for internal than for terminal mismatches. PrODN:RNA duplexes are destabilized by full phosphorothioate backbone substitution to give S-PrODN:RNA duplexes. The S-PrODN:RNA duplexes retain enhanced mismatch penalties, however. These results provide insight for utilizing long-range cooperativity and enhanced specificity to improve nucleic acid based probe and drug design.     

Synthesis and high stability of complementary complexes of N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides.

Two simple methods for the synthesis of oligonucleotides bearing a N-(2-hydroxyethyl)phenazinium (Phn) residue at the 5'- and/or 3'-terminal phosphate groups are proposed. By forming complexes between a dodecanucleotide d(pApApCpCpTpGpTpTpTpGpGpC), a heptanucleotide d(pCpCpApApApCpA), and Phn derivatives of the latter, it is shown that the introduction of a dye at the end of an oligonucleotide chain strongly stabilizes its complementary complexes. The Tmax and the thermodynamic parameters (delta H, delta S, delta G) of complex formation were determined. According to these data, coupling of a dye with the 5'-terminal phosphate group is the most advantageous: delta G(37 degrees C) is increased by 3.59 +/- 0.04 kcal/mol compared to 2.06 +/- 0.04 kcal/mol for 3'-Phn derivatives. The elongation of the linker, which connects the dye to the oligonucleotide, from a dimethylene up to a heptamethylene usually leads to destabilization of the oligonucleotide complex. The complementary complex formed by the 3',5'-di-Phn derivative of the heptanucleotide was found to be the most stable among all duplexes investigated. Relative to the unmodified complex the increase in free energy was 4.96 +/- 0.04 kcal/mol. The association constant of this modified complex at 37 degrees C is 9.5 x 10(6) M-1, whereas the analogous value for the unmodified complex is only 3 x 10(3) M-1.     

Physicochemical studies of processes involving potential photodynamic drugs on route to their targets: self-aggregation and membrane-binding of Zn-hematoporphyrin

The thermodynamics of zinc hematoporphyrin (ZnHP) dimerization and ZnHP-membrane binding were studied. The dimerization equilibrium was determined over the temperature range 19-40 degrees C, using fluorometric techniques. The dimerization constant obtained at 37 degrees C (neutral pH in phosphate-buffered saline) is 4.6 (+/- 0.6) X 10(4) M-1. The dimerization was found to decrease with temperature over the range 19-36 degrees C, the data allowing the extraction of the following thermodynamic parameters for the temperature range 19-31 degrees C: delta G0 = -9.3 kcal/mol, delta H0 = -7.4 kcal/mol, delta S0 = -6.4 eu. For temperatures above 36 degrees C the dimerization was found to be temperature independent, giving the following parameters: delta G0 = -6.6 kcal/mol, delta H0 = 0 kcal/mol, delta S0 = 21.2 eu. On the basis of the data the case is made for the existence of two types of ZnHP dimers, differing in the location of the fifth Zn2+ ligand and in the nature of the contribution of the solvent to the dimerization. For the membrane binding, large unilamellar liposomes served to model biological membranes. The binding of ZnHP to the liposomes was found to be similar, quantitatively, to the corresponding metal-free molecule, namely, fitting a case of one type of site and giving a binding constant of 1600 +/- 160 M (neutral pH and 37 degrees C) which is independent of the length of the porphyrin-liposome.