CYCLOHEXENE NUCLEIC ACIDS (CeNA) FORM STABLE DUPLEXES WITH RNA AND INDUCE RNASE H ACTIVITY

Cyclohexene nucleic acids (CeNA) were synthesized using classical phosporamidite chemistry. Incorporation of a cyclohexene nucleo-side in a DNA chain leads to an increase in stability of the DNA/RNA duplex. CeNA is stable against degradation in serum. A CeNA/RNA hybrid is able to activate E. Coli RNase H, resulting in cleavage of the RNA strand.     

Cyclohexene nucleic acids (CeNA) form stable duplexes with RNA and induce RNase H activity

Cyclohexene nucleic acids (CeNA) were synthesized using classical phosporamidite chemistry. Incorporation of a cyclohexene nucleo-side in a DNA chain leads to an increase in stability of the DNA/RNA duplex. CeNA is stable against degradation in serum. A CeNA/RNA hybrid is able to activate E. Coli RNase H. resulting in cleavage of the RNA strand.     

Cyclohexenyl nucleic acids: conformationally flexible oligonucleotides

Cyclohexenyl nucleic acid (CeNA) is a nucleic acid mimic, where the (deoxy)ribose sugar has been replaced by cyclohexenyl moieties. In order to study the conformation of cyclohexenyl nucleosides by NMR, the HexRot program was developed to calculate conformations from scalar coupling constants of cyclohexenyl compounds, analogous to the methods applied for (deoxy)ribose nucleosides. The conformational equilibria and the values of the thermodynamic parameters are very similar between a cyclohexenyl nucleoside [energy difference between ₂H³ (N-type) and ²H₃ (S-type) is 1.8 kJ/mol and equilibrium occurs via the eastern hemisphere with a barrier of 10.9 kJ/mol] and a natural ribose nucleoside (energy difference between N-type and S-type is 2 kJ/mol and equilibrium occurs via the eastern hemisphere with a barrier of 4–20 kJ/mol). The flexibility of the cyclohexenyl nucleoside was demonstrated by the fast equilibrium between two conformational states that was observed in a CeNA-U monomer, combined with the ₂H³ conformation of the cyclohexene moiety when incorporated into a Dickerson dodecamer and the ²H₃ conformation when incorporated in a d(5′-GCGT*GCG-3′)/d(5′-CGCACGC-3′) duplex, as determined by the NMR spectroscopy. This represents the first example of a synthetic nucleoside that adopts different conformations when incorporated in different double-stranded DNA sequences.     

Novel antisense oligonucleotides containing hydroxamate linkages: targeted iron-triggered chemical nucleases

Antisense oligonucleotides with iron binding hydroxamate linkages are designed to act as sequence-selective cleaving agents of complementary nucleic acids through Fenton chemistry. Oligothymidylate analogs with hydroxamate linkages were efficiently synthesized from coupling of nucleoside intermediates, activated as p-nitrophenyl carbonates, with hydroxylamine derivatized nucleosides. Iron binding studies showed that hydroxamate linked oligonucleotides are effective iron chelators when there are three nonadjacent internucleosidic hydroxamate linkages available in the same oligonucleotide molecule. However, analysis of the CD spectra of an oligothymidylate 16mer, which contained complete substitution of all phosphates with hydroxamates, indicated that the hydroxamate linkage was too rigid to allow the analog to base pair with the complementary DNA d(A₁₆). Syntheses of mix-linked thymidine oligomers with up to three hydroxamate linkages incorporated in the center of the sequence are also reported. Iron binding of the thymidine oligomer with hydroxamate linkages was confirmed by matrix assisted laser desorption mass spectrometry analysis. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides. A thymidine 16mer with three hydroxamate linkages incorporated in the center of the sequence was shown to be able to bind with both iron and its complementary polyA strand. A small destablizing effect was observed when the phosphodiester linkage was changed to the hydroxamate linkage. Under Fenton chemistry conditions, this novel iron binding oligothymidylate analog cleaved the complementary DNA strand sequence-selectively.     

Modification of ribonucleic acid by chemical carcinogens. VI. Effect of N-2-acetylaminofluorene modification of guanosine on the codon function of adjacent nucleosides in oligonucleotides

Oligonucleotides containing a guanosine residue on the 5′ or the 3′ side of tri- and tetranucleotides were prepared. The guanosine residue was modified with the chemical carcinogen N-2-acetylaminofluorene and the control and modified oligonucleotides were tested for their ability to stimulate ¹⁴C-labeled amino-acyl-tRNA binding to ribosomes. The effects of the modification are twofold. The first is that if the guanosine residue to which the drug is eovalently bound is part of a codon the oligonucleotide is completely inactive in the ribosomal binding assay. The second is that if an adenosine residue is adjacent to either the 5′ or 3′ side of the modified guanosine, as in (Ap)₃G or G(pA)₃, there is partial inhibition of ¹⁴C-labeled lysyl-tRNA binding to ribosomes. This inhibitory effect extends only to the function of the immediately adjacent adenosine since the chemical modification of guanosine residues in (Ap)₄G or G(pA)₄ did not impair their ability to code for lysine. In contrast to these findings if there is a uridine residue adjacent to the modified guanosine, as in (Up)₃G or G(pU)₃ there is no effect on ¹⁴C-labeled phenylalanyl-tRNA binding to ribosomes. Proton magnetic resonance spectra of UpG, GpU and the corresponding dinners in which the guanosine residue was modified with the drug failed to indicate a stacking interaction between the fluorene moiety and the adjacent uridine residue. This is in contrast to previous studies demonstrating a strong stacking interaction between fluorene and adjacent adenosine residues. Taken together these results indicate that acetylaminofluorene modification of guanosine next to an adenosine residue in oligonucleotide inhibits its ribosomal binding capacity. The stacking interaction with adjacent adenosine, and not with adjacent uridine residues, in oligonucleotides probably accounts for the effects observed in the ribosomal binding assay. These data are consistent with our previously described “base displacement” model.     

Chemical probes of the conformation of DNA modified by cis-diamminedichloroplatinum(II)

The purpose of this work was to analyze at the nucleotide level the distortions induced by the binding of cis-diamminedichloroplatinum(II) (cis-DDP) to DNA by means of chemical probes. In order to test the chemical probes, experiments were first carried out on two platinated oligonucleotides. It has been verified by circular dichroism and gel electrophoresis that the binding of cis-DDP to an AG or to a GTG site within a double-stranded oligonucleotide distorts the double helix. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers strongly suggests that the platinated oligonucleotides are bent. The reactivity of the oligonucleotide platinated at the GTG site with chloroacetaldehyde, diethyl pyrocarbonate, and osmium tetraoxide, respectively, suggests a local denaturation of the double helix. The 5'G residue and the T residue within the adduct are no longer paired, while the 3'G residue is paired. The double helix is more distorted (but not denatured) at the 5' side of the adduct than at the 3' side. In the case of the oligonucleotide platinated at the AG site, the double helix is also more distorted at the 5' side of the adduct than at the 3' side. The G residue within the adduct is paired. The reactivities of the chemical probes with six platinated DNA restriction fragments show that even at a relatively high level of platination only a few base pairs are unpaired but the double helix is largely distorted. No local denaturation has been detected at the GG sites separated from the nearest GG or AG sites by at least three bases pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNase H mediated cleavage of RNA by cyclohexene nucleic acid (CeNA)

Cyclohexene nucleic acid (CeNA) forms a duplex with RNA that is more stable than a DNA–RNA duplex (ΔT_m per modification: +2°C). A cyclohexenyl A nucleotide adopts a 3′-endo conformation when introduced in dsDNA. The neighbouring deoxynucleotide adopts an O4′-endo conformation. The CeNA:RNA duplex is cleaved by RNase H. The V_max and K_m of the cleavage reaction for CeNA:RNA and DNA:RNA is in the same range, although the k_cat value is about 600 times lower in the case of CeNA:RNA.     

Synthesis and Application of Negatively Charged PNA Analogues

Negatively charged DNA mimics containing phosphonate analogues of peptide nucleic acids were designed, and their physicochemical and biological properties were evaluated in the comparison with natural oligonucleotides, classical peptide nucleic acids, and morpholino phosphorodiamidate oligonucleotide analogues. The results obtained revealed a high potential of phosphonate-containing PNA derivatives for a number of biological applications, such as diagnostic, nucleic acids analysis, and inhibition of gene expression.     

Nucleic Acid Conformation: Crystal Structure of a Naturally Occurring Dinucleoside Phosphate (UpA)

THEORIES of the molecular structure of nucleic acids have so far been based on evidence from the crystal structures of monomeric units such as nucleosides and mononucleotides, the interpretation of diffraction patterns of oriented nucleic acid fibres and molecular model building^1–6. Such approaches can help to suggest structures of periodic molecules such as helices, but they are insufficient for predicting and understanding nonrepetitive structures such as the loops in transfer RNA (tRNA), presumably associated with many of the functions of tRNA. To understand the geometry of nucleic acids and possible constraints on their conformation, it is therefore essential to know the detailed conformation of the sugar residues and the conformational relationship between the sugar residue, the base and the phosphate group^7–9. The simplest molecule which contains this information is a 3´5´-dinucleoside phosphate. We now report the structure of uridine-3´,5´-adenosine phosphate (UpA). This is the first naturally occurring dinucleoside phosphate whose crystal structure has been determined by X-ray diffraction. The only other dinucleoside phosphate with known crystal structure is adenosine-2´,5´-uridine phosphate¹⁰, but it does not have the naturally occurring 3´5´ sugar phosphate linkage.     

Characterization of fluorescein–oligonucleotide conjugates and measurement of local electrostatic potential

The properties of fluorescein are substantially altered upon conjugation to nucleic acids, affecting not only the molar absorptivities and fluorescence quantum yields but also the protolytic equilibrium constant and fluorescence lifetimes. Around neutral pH, the fluorescein moiety is present as both mono- and dianion, and the pK_a relating them is increased from 6.43 for free fluorescein to about 6.90 for fluorescein attached to both single- and double-stranded oligonucleotides of at least 12 bases/base pairs. This difference reflects the local electrostatic potential around the nucleic acid, which is calculated to −28 mV. The molar absorptivities and spectral responses of the conjugated fluorescein protolytic species are also determined, from which the concentrations of fluorescein–oligonucleotide conjugates can be calculated by assuming: ε₄₉₄ = 62000/[1 + 10^{−(pH−6.90)}] + 12000/[1 + 10^(pH−6.90)] (M⁻¹ cm⁻¹). The fluorescence quantum yield of the conjugates depends, in a complex way, on temperature, environment and oligonucleotide length, sequence and conformation, and must be determined for each experimental situation. © 1998 John Wiley & Sons, Inc. Biopoly 46: 445–453, 1998     

The structure of helix III in Xenopus oocyte 5 S rRNA: an RNA stem containing a two-nucleotide bulge

The solution structure of an oligonucleotide containing the helix III sequence from Xenopus oocyte 5 S rRNA has been determined by NMR spectroscopy. Helix III includes two unpaired adenosine residues, flanked on either side by G:C base-pairs, that are required for binding of ribosomal protein L5. The consensus conformation of helix III in the context provided by this oligonucleotide has the two adenosine residues located in the minor groove and stacked upon the 3' flanking guanosine residue, consistent with biochemical studies of free 5 S rRNA in solution. A distinct break in stacking that occurs between the first adenosine residue of the bulge and the flanking 5' guanosine residue exposes the base of the adenosine residue in the minor groove and the base of the guanosine residue in the major groove. The major groove of the helix is widened at the site of the unpaired nucleotides and the helix is substantially bent; nonetheless, the G:C base-pairs flanking the bulge are intact. The data indicate that there may be conformational heterogeneity centered in the bulge region. The corresponding adenosine residues in the Haloarcula marismortui 50 S ribosomal subunit form a dinucleotide platform, which is quite different from the motif seen in solution. Thus, the conformation of helix III probably changes when 5 S rRNA is incorporated into the ribosome.     

Base pairing properties of D- and L-cyclohexene nucleic acids (CeNA)

Cyclohexene nucleic acids (CeNA) with a D-like configuration form very stable self-complementary duplexes and stable duplexes with RNA. An increased duplex stability with Delta T(m)/mod of +1.2 degrees C is observed. The duplex with DNA is less stable. Excellent mismatch discrimination has been observed as well for the duplex with DNA as for the duplex with RNA. The results obtained with mixed CeNA sequences warrant antisense studies with CeNA. The CeNAs of opposite chirality constitute a self-pairing system on their own, resembling L-RNA sequences.     

Conformational change of the triple-helical structure. IV. Kinetics of the helix-folding of (Pro-Pro-Gly)n (n = 10, 12, and 15)

The kinetic curves of the helix-refolding of (PPG)_n (n = 10, 12, and 15) were analyzed with an all-or-none model. The Arrhenius plot of the overall rate constant of the helixfolding k_F showed a negative activation energy at high temperature. With the aid of a sequential model, it was concluded that the reason for the anomaly was the instability of short helices (shorter than seven helical units in a trimeric molecule), and/or the more rapid rates of helix-folding and helix-opening for shorter helices. The rate constant of the formation of one helical unit composed of three tripeptides at an end of a long helix was calculated to be 10^2–4 sec^?1. It was much smaller than that for other kinds of helices, such as an α helix (10¹⁰ sec^?1) or a double helix of nucleic acids (10^7–9sec^?1).     

Distortions induced in double-stranded oligonucleotides by the binding of cis- or trans-diammine-dichloroplatinum(II) to the d(GTG) sequence.

Conformational changes induced in double-stranded oligonucleotides by the binding of trans- or cis-diamminedichloro platinum(II) to the d(GTG) sequence have been characterized by means of melting temperatures, electrophoretic migrations in non-denaturing polyacrylamide gels, reactivities with the artificial nuclease Phenanthroline-copper and with chemical probes. The cis-platinum adduct behaves more as a centre of directed bend than as a hinge joint, the induced bend angle being of the order of 25-30 degrees. The double helix is locally denatured over 2 base pairs (corresponding to the platinated 5'G residue and the central T residue) and is distorted over 4-5 base pairs. The trans-platinum adduct behaves also more as a centre of directed bend than as a hinge joint, the induced bend angle being of the order of 60 degrees. The double helix is locally denatured over 4 base pairs (corresponding to the immediately 5'T residue adjacent to the adduct and to the three base residues of the adduct). Both the cis- and trans-platinum adducts decrease the thermal stability of the double helix.     

Synthesis of Oligonucleotide Derivatives with Aryl(trifluoromethyl)diazirine Moiety for the Photoaffinity Modification of Proteins and Nucleic Acids

1-(4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)benzamido)-3-O-(4,4"-dimethoxytrityl)-2,3-propanediol phosphoramidite was synthesized and used as a modified unit in the automatic synthesis of oligodeoxyribonucleotides. Pentadecathymidylates with various numbers of 2,3-propanediol moieties substituted with aryl(trifluoromethyl)diazirinyl (ATFMD) were obtained, and the thermal stability of their duplexes with (dA)₁₅ were studied. One ATFMD-propanediol residue was shown to reduce the thermal stability of the duplex by 8–9°C. The irradiation of the ATFMD-containing duplexes by UV light with the wavelength of 350 nm was found to cause the cross-linking reaction of the ATFMD-containing strand with the complementary strand and the formation of the cross-linked duplexes. The photomodification efficiency was independent of the oligonucleotide sequence, with each ATFMD group providing for 5% cross-linking. The irradiation of an ATFMD-containing duplex, a substrate of the HIV-1 integrase, in the presence of this enzyme resulted in the covalent DNA–protein complex. The oligonucleotides with the 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-propanediol moiety in their chains can be used for the photoaffinity modification of both nucleic acids and proteins that recognize them.     

Synthesis of cationic glucosamino nucleic acids for stabilizing oligonucleotides

Glucosamino nucleic acids (GANAs) bearing a β-N-glycoside bond between carbon 1 of the glucosamine and the nucleobase nitrogen were synthesized and incorporated into oligonucleotides (4′,6′-GANA and 3′,6′-GANA). The thermal stability of oligonucleotide duplexes containing the GANA zwitterionic nucleotides was also investigated.     

Synthesis of steroid-containing oligonucleotides and their alkylating derivatives

New alkylating derivatives of oligonucleotides carrying a steroid (cholesterol, testosterone or ergosterol) residue have been synthesized, the residue being introduced via its hydroxyl group into the triester oligonucleotide block in the presence of triisopropylbenzenesulphonyl chloride and N-methylimidazole. Covalent attachment of steroids to oligonucleotides increases their hydrophobicity and does not influence the melting temperature of their complementary complexes. The data obtained showed that the oligonucleotide derivatives, bearing both an alkylating group of nitrogen mustard and a steroid residue, can be used as reagents for specific modification of nucleic acids.     

KINETICALLY SELECTIVE BINDING OF SINGLE STRANDED RNA OVER DNA BY A PYRROLIDINE-AMIDE OLIGONUCLEOTIDE MIMIC (POM)

Replacing the sugar-phosphodiester backbone of nucleic acids with a pyrrolidine-amide backbone results in an oligonucleotide mimic POM 1 which binds with high affinity and specificity to complementary DNA and RNA. Unlike other modified oligonucleotides, POM binds much more rapidly to single stranded RNA than DNA.