Oligodeoxynucleotide Duplexes Containing (5′S)-5′-C-Alkyl-Modified 2′-Deoxynucleosides: Can an Alkyl Zipper across the DNA Minor-Groove Enhance Duplex Stability?

10.1002/hlca.200390311.abs A series of oligonucleotides containing (5′S)-5′-C-butyl- and (5′S)-5′-C-isopentyl-substituted 2′-deoxyribonucleosides were designed, prepared, and characterized with the intention to explore alkyl-zipper formation between opposing alkyl chains across the minor groove of oligonucleotide duplexes as a means to modulate DNA-duplex stability. From four possible arrangements of the alkyl groups that differ in the density of packing of the alkyl chains across the minor groove, three (duplex types I–III, Fig. 2) could experimentally be realized and their duplex-forming properties analyzed by UV-melting curves, CD spectroscopy, and isothermal titration calorimetry (ITC), as well as by molecular modeling. The results show that all arrangements of alkyl residues within the minor groove of DNA are thermally destabilizing by 1.5–3°/modification in Tm. We found that, within the proposed duplexes with more loosely packed alkyl groups (type-III duplexes), accommodation of alkyl residues without extended distorsion of the helical parameters of B-DNA is possible but does not lead to higher thermodynamic stability. The more densely packed and more unevenly distributed arrangement (type-II duplexes) seems to suffer from ecliptic positioning of opposite alkyl groups, which might account for a systematic negative contribution to stability due to steric interactions. The decreased stability in the type-III duplexes described here may be due either to missing hydrophobic interactions of the alkyl groups (not bulky enough to make close contacts), or to an overcompensation of favorable alkyl-zipper formation presumably by loss of structured H2O in the minor groove.

Pentafluorophenyl phenyl interaction in biphenyl DNA

We prepared and investigated oligonucleotide duplexes of the sequence d(GATGAC(X)(n)GCTAG)d(CTAGC(Y)(n)GTCATC), in which X and Y designate biphenyl- (bph) and pentafluorobiphenyl- ((5F)bph) C-nucleotides, respectively, and n varies from 0-4. These hydrophobic base substitutes are expected to adopt a zipperlike, interstrand stacking motif, in which not only bph/bph or (5F)bph/(5F)bph homo pairs, but also (5F)bph/bph mixed pairs can be formed. By performing UV-melting curve analysis we found that incorporation of a single (5F)bph/(5F)bph pair leads to a duplex that is essentially as stable as the unmodified duplex (n=0), and 2.4 K more stable than the duplex with the nonfluorinated bph/bph pair. The T(m) of the mixed bph/(5F)bph pair was in between the T(m) values of the respective homo pairs. Additional, unnatural aromatic pairs increased the T(m) by +3.0-4.4 K/couple, irrespective of the nature of the aromatic residue. A thermodynamic analysis using isothermal titration calorimetry (ITC) of a series of duplexes with n=3 revealed lower (less negative) duplex formation enthalpies (DeltaH) in the (5F)bph/(5F)bph case than in the bph/bph case, and confirmed the higher thermodynamic stability (DeltaG) of the fluorinated duplex, suggesting it to be of entropic origin. Our data are compatible with a model in which the stacking of (5F)bph versus bph is dominated by dehydration of the aromatic units upon duplex formation. They do not support a model in which van der Waals dispersive forces (induced dipoles) or electrostatic (quadrupole) interactions play a dominant role

Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes

A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy DeltaH. Arranging the tc-residues in a continuous fashion rescues T(m) and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in T(m) when paired to complementary DNA and leads to substantial increases in T(m) when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therapy.

Impaired protein stability of 11beta-hydroxysteroid dehydrogenase type 2: a novel mechanism of apparent mineralocorticoid excess

Apparent mineralocorticoid excess (AME) is a severe form of hypertension that is caused by impaired activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts biologically active cortisol into inactive cortisone. Mutations in HSD11B2 result in cortisol-induced activation of mineralocorticoid receptors and cause hypertension with hypokalemia, metabolic alkalosis, and suppressed circulating renin and aldosterone concentrations. This study uncovered the first patient with AME who was described in the literature, identified the genetic defect in HSD11B2, and provided evidence for a novel mechanism of reduced 11beta-HSD2 activity. This study identified a cluster of amino acids (335 to 339) in the C-terminus of 11beta-HSD2 that are essential for protein stability. The cluster includes Tyr(338), which is mutated in the index patient, and Arg(335) and Arg(337), previously reported to be mutated in hypertensive patients. It was found that wild-type 11beta-HSD2 is a relatively stable enzyme with a half-life of 21 h, whereas that of Tyr(338)His and Arg(337)His was 3 and 4 h, respectively. Enzymatic activity of Tyr(338)His was partially retained at 26 degrees C or in the presence of the chemical chaperones glycerol and dexamethasone, indicating thermodynamic instability and misfolding. The results provide evidence that the degradation of both misfolded mutant Tyr(338)His and wild-type 11beta-HSD2 occurs through the proteasome pathway. Therefore, impaired 11beta-HSD2 protein stability rather than reduced gene expression or loss of catalytic activity seems to be responsible for the development of hypertension in some individuals with AME.

Stability and kinetics of nucleic acid triplexes with chimaeric DNA/RNA third strands

A series of chimaeric DNA/RNA triplex-forming oligonucleotides (TFOs) with identical base-sequence but varying sequential composition of the sugar residues were prepared. The structural, kinetic and thermodynamic properties of triplex formation with their corresponding double-helical DNA target were investigated by spectroscopic methods. Kinetic and thermodynamic data were obtained from analysis of non-equilibrium UV-melting- and annealing curves in the range of pH 5.1 to 6.7 in a 10 mM citrate/phosphate buffer containing 0.1M NaCl and 1 mM EDTA. It was found that already single substitutions of ribo- for deoxyribonucleotides in the TFOs greatly affect stability and kinetics of triplex formation in a strongly sequence dependent manner. Within the sequence context investigated, triplex stability was found to increase when deoxyribonucleotides were present at the 5'-side and ribonucleotides in the center of the TFO. Especially the substitution of thymidines for uridines in the TFO was found to accelerate both, the association and dissociation process, in a strongly position-dependent way. Differential structural information on triplexes and TFO single-strands was obtained from CD-spectroscopy and gel mobility experiments. Only minor changes were observed in the CD spectra of the triplexes at all pH values investigated, and the electrophoretic mobility was nearly identical in all cases, indicating a high degree of structural similarity. In contrast, the single-stranded TFOs showed high structural variability as determined in the same way. The results are discussed in the context of the design of TFOs for therapeutic or biochemical applications.

Synthesis and Thermodynamic and Biophysical Properties of Tricyclo-DNA

The DNA analogue tricyclo-DNA, built from conformationally rigid nucleoside analogues that were linked via tertiary phosphodiester functions, can efficiently be synthesized from the corresponding phosphoramidites by conventional solid-phase cyanoethyl phosphoramidite chemistry. 5'-End phosphorylated tricyclo-DNA sequences are chemically stable in aqueous, pH-neutral media at temperatures from 0 to 90 C. Tricyclo-DNA sequences resist enzymatic hydrolysis by the 3'-exonuclease snake venom phosphodiesterase. Homobasic adenine- and thymine-containing tricyclo-DNA octa- and nonamers are extraordinarily stable A-T base-pairing systems, not only in their own series but also with complementary DNA and RNA. Base mismatch formation is strongly destabilized. As in bicyclo-DNA, the tricyclo-DNA purine sequences preferentially accept a complementary strand on the Hoogsteen face of the base. A thermodynamic analysis reveals entropic benefits in the case of hetero-backbone duplex formation (tricyclo-DNA/DNA duplexes) and both an enthalpic and entropic benefit for duplex formation in the pure tricyclo-DNA series compared to natural DNA. Stability of tricyclo-DNA duplex formation depends more strongly on monovalent salt concentration compared to natural DNA. Homopyrimidine DNA sequences containing tricyclothymidine residues form triplexes with complementary double-stranded DNA. Triple-helix stability depends on the sequence composition and can be higher when compared to that of natural DNA. The use of one tricyclothymidine residue in the center of the self-complementary dodecamer duplex (d(CGCGAAT t CGCG), t = tricyclothymidine) strongly stabilizes its monomolecular hairpin loop structure relative to that of the corresponding pure DNA dodecamer ( T m = +20 C), indicating (tetra)loop-stabilizing properties of this rigid nucleoside analogue.

A thermodynamic model for di-trioctahedral chlorite from experimental and natural data in the system MgO-FeO-Al2O3-SiO2-H2O. Applications to P-T sections and geothermometry

We present a new thermodynamic activity-composition model for di-trioctahedral chlorite in the system FeO–MgO–Al2O3–SiO2–H2O that is based on the Holland–Powell internally consistent thermodynamic data set. The model is formulated in terms of four linearly independent end-members, which are amesite, clinochlore, daphnite and sudoite. These account for the most important crystal-chemical substitutions in chlorite, the Fe–Mg, Tschermak and di-trioctahedral substitution. The ideal part of end-member activities is modeled with a mixing-on-site formalism, and non-ideality is described by a macroscopic symmetric (regular) formalism. The symmetric interaction parameters were calibrated using a set of 271 published chlorite analyses for which robust independent temperature estimates are available. In addition, adjustment of the standard state thermodynamic properties of sudoite was required to accurately reproduce experimental brackets involving sudoite. This new model was tested by calculating representative P–T sections for metasediments at low temperatures (<400 °C), in particular sudoite and chlorite bearing metapelites from Crete. Comparison between the calculated mineral assemblages and field data shows that the new model is able to predict the coexistence of chlorite and sudoite at low metamorphic temperatures. The predicted lower limit of the chloritoid stability field is also in better agreement with petrological observations. For practical applications to metamorphic and hydrothermal environments, two new semi-empirical chlorite geothermometers named Chl(1) and Chl(2) were calibrated based on the chlorite + quartz + water equilibrium (2 clinochlore + 3 sudoite = 4 amesite + 4 H2O + 7 quartz). The Chl(1) thermometer requires knowledge of the (Fe3+/ΣFe) ratio in chlorite and predicts correct temperatures for a range of redox conditions. The Chl(2) geothermometer which assumes that all iron in chlorite is ferrous has been applied to partially recrystallized detrital chlorite from the Zone houillère in the French Western Alps.