Looped oligonucleotides form stable hybrid complexes with a single-stranded DNA.

Several new branched (1, 2), circular (9) and looped oligonucleotides (14-17) were synthesized. 3'-Deoxypsicothymidine was employed to create the site of branching when required. The circular and looped structures were obtained by oxidative disulfide bond formation between mercaptoalkyl tether groups. All the oligonucleotides prepared contained two T11 sequences, and the branched and looped oligomers an additional alternating CT sequence. The melting experiments revealed that the branched oligonucleotides form relatively weak hybrid (double/triple helix) complexes with the single-stranded oligodeoxyribonucleotide, showing a considerable destabilizing effect produced by the structure at the point of branching. The data obtained with looped oligonucleotides demonstrated considerable stabilization of the hybrid (double/triple helix) complexes with the complement. The data reported may be useful in attempting to design new antisense or antigene oligonucleotides capable of forming selective and stable bimolecular hybrid complexes with nucleic acids.     

Selective binding of looped oligonucleotides to a single-stranded DNA and its influence on replication in vitro.

Complexing of looped and circular oligonucleotides, composed of either 2'-deoxyribo- or 2'-O-methylribonucleoside units, with completely matching or partially mismatching complementary DNA sequences was studied. Melting experiments revealed considerable differences among the stabilities of these hybrid complexes. Maximum stability and selectivity was displayed by oligomers 2 and 5. It was concluded that a linear stretch, attached to 1'-O- of 3'-deoxypsicothymidine unit (Z) increases the selectivity of hybridisation and stability of the complex as a whole. This allows one to aim the target DNA very precisely at its polyadenine part as well as at adjacent sequence simultaneously. Experiments on termination of primer extension catalysed by different DNA-polymerases--Sequenase, Klenow fragment and Tth--have demonstrated that looped oligomer 5, composed of 2'-O-methylribonucleosides appears to be a highly selective and potent inhibitor of replication in vitro. Features of looped oligonucleotides, composed of 2'-O-methylribonucleosides seem to be useful for design of highly specific antigene oligonucleotides.     

Synthesis and hybridization property of sugar and phosphate linkage modified oligonucleotides.

1-[3-Deoxy-5-O-(4,4'-dimethoxytriphenylmethyl)-3-C-hydroxymethyl-2 -O-(2-methoxyethoxymethyl)-beta-D-erythro-pentofuranosyl]thymine (13) was synthesized from 1,2-isopropylidene-D-xylose (1) as a building block of modified oligonucleotides. Three types of novel oligonucleotides were synthesized from 13 and their T(m)s were compared with those of the corresponding natural oligonucleotides. It was found that our synthesized oligomers had lower affinity to DNA and RNA than the natural oligomers.     

Interaction of the ADP-ribosylating enzyme from the hyperthermophilic archaeon S. solfataricus with DNA and ss-oligo deoxy ribonucleotides

The DNA-binding ability of the poly-ADPribose polymerase-like enzyme from the extremely thermophilic archaeon Sulfolobus solfataricus was determined in the presence of genomic DNA or single stranded oligodeoxyribonucleotides. The thermozyme protected homologous DNA against thermal denaturation by lowering the amount of melted DNA and increasing melting temperature. The archaeal protein induced structural changes of the nucleic acid by modifying the dichroic spectra towards a shape typical of condensing DNA. However, enzyme activity was slightly increased by DNA. Competition assays demonstrated that the protein interacted also with heterologous DNA. In order to characterize further the DNA binding properties of the archaeal enzyme, various ss-oligodeoxyribonucleotides of different base composition, lengths (12-mer to 24-mer) and structure (linear and circular) were used for fluorescence titration measurements. Intrinsic fluorescence of the archaeal protein due to tryptophan (excitation at 295 nm) was measured in the presence of each oligomer at 60 degrees C. Changes of tryptophan fluorescence were induced by all compounds in the same range of base number per enzyme molecule, but independently from the structural features of oligonucleotides, although the protein exhibited a slight preference for those adenine-rich and circular. The binding affinities were comparable for all oligomers, with intrinsic association constants of the same order of magnitude (K=10(6) M(-1)) in 0.01 M Na-phosphate buffer, pH 8.0, and accounted for a "non-specific" binding protein. Circular dichroism analysis showed that at 60 degrees C the native protein was better organized in a secondary structure than at 20 degrees C. Upon addition of oligonucleotides, enzyme structure was further stabilized and changed towards a beta-conformation. This effect was more marked with the circular oligomer. The analysed oligodeoxyribonucleotides slightly enhanced enzyme activity with the maximal increase of 50% as compared to the control. No activation was observed with the circular oligomer.     

Effect of thiazole orange doubly labeled thymidine on DNA duplex formation

Nucleic acid oligonucleotides are widely used in hybridization experiments for specific detection of complementary nucleic acid sequences. For design and application of oligonucleotides, an understanding of their thermodynamic properties is essential. Recently, exciton-controlled hybridization-sensitive fluorescent oligonucleotides (ECHOs) were developed as uniquely labeled DNA oligomers containing commonly one thymidine having two covalently linked thiazole orange dye moieties. The fluorescent signal of an ECHO is strictly hybridization-controlled, where the dye moieties have to intercalate into double-stranded DNA for signal generation. Here we analyzed the hybridization thermodynamics of ECHO/DNA duplexes, and thermodynamic parameters were obtained from melting curves of 64 ECHO/DNA duplexes measured by ultraviolet absorbance and fluorescence. Both methods demonstrated a substantial increase in duplex stability (ΔΔG°(37) ～ -2.6 ± 0.7 kcal mol(-1)) compared to that of DNA/DNA duplexes of the same sequence. With the exception of T·G mismatches, this increased stability was mostly unaffected by other mismatches in the position opposite the labeled nucleotide. A nearest neighbor model was constructed for predicting thermodynamic parameters for duplex stability. Evaluation of the nearest neighbor parameters by cross validation tests showed higher predictive reliability for the fluorescence-based than the absorbance-based parameters. Using our experimental data, a tool for predicting the thermodynamics of formation of ECHO/DNA duplexes was developed that is freely available at http://genome.gsc.riken.jp/echo/thermodynamics/ . It provides reliable thermodynamic data for using the unique features of ECHOs in fluorescence-based experiments.     

Cell binding, uptake and cytosolic partition of HIV anti-gag phosphodiester oligonucleotides 3'-linked to cholesterol derivatives in macrophages

The purpose of this study is to evaluate the cell interactions of a new class of compounds composed of phosphodiester oligonucleotides linked to the cholesterol group at position 3, 7, or 22 of the steroid structure. The resulting conjugates were assessed for their capacity to bind, penetrate and partition in the cytoplasmic compartment of murine macrophages. The results showed that lipophilic conjugates bind to cells much faster (t(1/2) < or = 10 min) than do underivatized oligomers. Oligomers tethered to the cholesterol at positions 3 and 7 (PO-GEM-3-Chol and PO-GEM-7-Chol) interacted more efficiently with cell membranes and were better internalized than oligomers attached to the cholesterol moiety at position 22 (PO-GEM-22-Chol). The cytosolic fraction of internalized oligomers was studied by a digitonin-based membrane permeabilization method. The recovered fraction of oligomers that can freely diffuse from the cytosol was comparable for GEM-91, a phosphorothioate congener, and for PO-GEM-7-Chol (50-60% of the internalized oligomers), while that of PO-GEM-3-Chol was less (30% of the internalized oligomers) indicating a higher membrane affinity of the latter derivative as compared to the other investigated compounds. Membrane binding and cell internalization correlated well with the hydrophobicity of the conjugates as characterized by their partition coefficients in a water-octanol system. Due to their capacity of rapid binding and cytosolic partition in cells, cholesterol-derivatized oligonucleotides at position 3 or 7 of the steroid molecule appeared as good candidates for systemic delivery of anti-HIV antisense compounds.     

Synthesis and triplex-forming properties of cyclic oligonucleotides with (G,A)-antiparallel strands

Cyclic oligonucleotides carrying an oligopurine Watson-Crick sequence linked to the corresponding (G,A)- and (G,T)-antiparallel strands were prepared by nonenzymatic template-assisted cyclization of phosphorylated precursors. Cyclization was attempted using 3'-phosphate and 5'-phosphate linear precursors with carbodiimide or BrCN activation. The best results were obtained with the 5'-phosphorylated precursors and carbodiimide activation. Cyclic oligonucleotides bind polypyrimidine target sequence by formation of antiparallel triplexes. We have used UV and circular dichroism (CD) spectroscopy to analyze triplexes formed by cyclic oligonucleotides carrying G and A in the reverse-Hoogsteen strand. The relative stability of the triplexes formed by cyclic and linear oligonucleotides with a common polypyrimidine target was determined by melting experiments. The most-stable triplexes were formed by the cyclic oligonucleotide, followed by the unphosphorylated and phosphorylated oligonucleotide precursors, and, finally, the corresponding hairpin. Although the differences in binding affinity between cyclic oligonucleotides and their corresponding linear precursors are small, the use of cyclic oligonucleotides offers a clear advantage over conventional duplex recognition.     

Herpes simplex virus type 2 growth and latency reactivation by cocultivation are inhibited with antisense oligonucleotides complementary to the translation initiation site of the large subunit of ribonucleotide reductase (RR1)

Antisense oligonucleotides complementary to the translation initiation site of the herpes simplex virus type 2 (HSV-2) large subunit of ribonucleotide reductase (RR1) were studied for their ability to inhibit RR1 expression, HSV-2 growth, and its reactivation from latently infected ganglia. The oligomers caused a significant decrease (90%-97% inhibition) in HSV-2 RR1 expression and inhibited HSV-2 growth, with IC50 and IC90 values of 0.11 and 1.0 microM, respectively. The titers of HSV-2 mutants that are respectively deleted in the PK (ICP10deltaPK) or RR (ICP10deltaRR) domains of RR1 were also significantly (500-20,000-fold) decreased, indicating that the antisense oligomers interfere with the independent contributions of the two RR1 functions (PK and RR) toward virus growth. Inhibition was sequence specific, as evidenced by the failure of a two-base mutant (RR1TImu) to inhibit protein expression and HSV-2 growth. Furthermore, the antisense oligomers inhibited HSV-2 reactivation by cocultivation of latently infected ganglia (0/8). Virus was reactivated from ganglia cultured without oligomers, in the presence of unrelated oligomers (6/8), or in the presence of the two-base mutant RR1TImu (5/8) (p < 0.007 by two-tailed Fisher exact test). HSV-2 growth was not inhibited by antisense oligonucleotides complementary to the splice junction of HSV-2 immediate-early (IE) pre-mRNA 4 and 5 (IE4,5SA) or the translation initiation site of IE mRNA 4 (IE4TI), although the respective HSV-1-specific oligomers inhibit HSV-1 growth.     

2'-Pyrene modified oligonucleotide provides a highly sensitive fluorescent probe of RNA.

Oligonucleotide 9mers containing 2'-O-(1-pyrenylmethyl)uridine [U(pyr)] at the center position were synthesized by using a protected U(pyr) phosphoramidite. The UV melting behaviors indicate that the pyrene-modified oligonucleotides can bind to both their complementary DNA and RNA in aqueous solution. When compared with the unmodified oligonucleotides, the pyrene-modified oligonucleotides showed higher affinity for DNA while exhibiting lower affinity for RNA. The pyrene-modified oligonucleotides in diluted solution exhibited fluorescence typical of pyrene monomer emission [lambdamax 378 (band I) and 391 nm (band III)]. When these oligomers bound to DNA, the fluorescence intensity ratio of band III/band I was increased. With this fluorescence change, a new broad emission (lambdamax 450 nm) due to exciplex between the pyrene and an adjacent nucleobase appeared. In contrast, addition of RNA to the pyrene oligonucleotides resulted in enhancement of the pyrene monomer emission with decrease in the fluorescence band ratio. The extent of the emission enhancement was found to be highly dependent on the nucleobase adjacent to the U(pyr) in the pyrene oligomers. The pyrene oligonucleotide containing dC at the 3'-site of the modification showed remarkable increase (approximately 250 times) in fluorescence (375 nm) upon binding to complementary RNA. The present findings would open the way to the design of a highly sensitive fluorescent probe of RNA.     

DSC deconvolution of the structural complexity of c-MYC P1 promoter G-quadruplexes

We completed a biophysical characterization of the c-MYC proto-oncogene P1 promoter quadruplex and its interaction with a cationic porphyrin, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin (TMPyP4), using differential scanning calorimetry, isothermal titration calorimetry, and circular dichroism spectroscopy. We examined three different 24-mer oligonucleotides, including the wild-type (WT) sequence found in the c-MYC P₁ promoter and two mutant G→T sequences that are known to fold into single 1:2:1 and 1:6:1 loop isomer quadruplexes. Biophysical experiments were performed on all three oligonucleotide sequences at two different ionic strengths (30 mM [K⁺] and 130 mM [K⁺]). Differential scanning calorimetry experiments demonstrated that the WT quadruplex consists of a mixture of at least two different folded conformers at both ionic strengths, whereas both mutant sequences exhibit a single two-state melting transition at both ionic strengths. Isothermal titration calorimetry experiments demonstrated that both mutant sequences bind 4 mols of TMPyP4 to 1 mol of DNA, in similarity to the WT sequence. The circular dichroism spectroscopy signatures for all three oligonucleotides at both ionic strengths are consistent with an intramolecular parallel stranded G-quadruplex structure, and no change in quadruplex structure is observed upon addition of saturating amounts of TMPyP4 (i.e., 4:1 TMPyP4/DNA).     

Synthesis and Some Properties of Modified Oligonucleotides. II. Oligonucleotides Containing 2′-Deoxy-2′-fluoro-β-D-arabinofuranosyl Pyrimidine Nucleosides

Abstract

In order to find the effects of unnatural nucleosides on the stability of duplex, several oligonucleotides containing 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-uracil(FAU),-cytosine (FAC) and -thymine (FMAU) were synthesized by two alternative approaches: phosphoramidite method on an ABI 392 synthesizer and H-phosphonate procedure on our GeneSyn I universal module synthesizer. It was shown from the melting profiles that the presence of FMAU has a large stabilizing effect on the duplex. Replacement of thymidine with FAU, or deoxycytidine with FAC resulted in the formation of less stable duplexes. Temperature-dependent CD spectroscopy demonstrated that the structures of the fluorine containing oligomers are very similar to those of unmodified oligomers.     

Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs

The antisense activity of oligomers with 2'-O-methyl (2'-O-Me) phosphorothioate, 2'-O-methoxyethyl (2'-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2'-O-Me and 2'-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of L-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.     

A new synthetic protocol for labeled oligonucleotides, using a chemically cleavable universal linker

A two-step general method for labeling of synthetic oligonucleotides is described. The protocol employs a cleavable universal linker, 5'-O-(4,4'-dimethoxytrityl)-3'-O-benzoyl-2'-O-(2-cyanoethyl-N,N-diisopropyl)-uridine phosphoramidite, to effect coupling to polymer-bound oligonucleotide chains. Sequentially, coupling with commercially available phosphoramidite reagent of an appropriate label (Biotin, HEX etc.) in an automated DNA synthesizer is carried out. The labeled oligomers, obtained after cleavage and deprotection reactions, are analyzed on RP-HPLC. A distinctive feature of this protocol is the recovery of free oligomers from their labeled analogs under mild conditions. The oligomers obtained are comparable to the corresponding standard oligonucleotides (HPLC).     

Carboranyl oligonucleotides: 4. synthesis and physicochemical studies of oligonucleotides containing 2'-O-(o-carboran-1-yl)methyl group

Boronated oligonucleotides are potential candidates for antisense oligonucleotide technology (AOT), boron neutron capture therapy (BNCT), and as tools in molecular biology. A method was developed for the solid phase synthesis of oligonucleotides containing 2'-O-(o-carboran-1-yl-methyl) (2'-CBM) group. Synthesis was performed using a standard beta-cyanoethyl cycle and automated DNA synthesizer. Manual steps were performed for the insertion of a modified monomer bearing the 2'-CBM group. Several tetradecanucleotides complementary to DNA-HCMV, and bearing 2'-CBM modification near the 3'-end or 5'-end or in the middle of the oligonucleotide chain were synthesized. The resulting oligomers were characterized by polyacrylamide gel electrophoresis (PAGE), reverse phase high-performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and ultraviolet spectroscopy (UV), circular dichroism (CD), and melting temperature (Tm) measurements. Tm of duplexes formed between 2'-CBM-modified tetradecanucleotides and complementary DNA and RNA template were compared with those formed by the unmodified oligonucleotide and complementary sequence. The stability of 2'-CBM oligonucleotides in the presence of phosphodiesterase I from Crotalus atrox venom and in human serum was studied. Oligonucleotides bearing the 2'-CBM group are characterized by increased resistance to enzymatic digestion, increased lipophilicity, and the ability to form stable duplexes with complementary templates.     

Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") and origins of their nuclease resistance properties.

The tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") are known to significantly enhance the binding affinity of oligonucleotides to their complementary target DNA or RNA strands. To investigate their effect on the nuclease resistance, they were incorporated into model oligomers with a natural phosphodiester backbone, and enzymatic degradation was monitored in an in vitro assay with snake venom phosphodiesterase as the hydrolytic enzyme. In both cases, a single incorporation at the 3'-terminus completely protected the oligonucleotides against 3'-exonuclease attack. Further investigations indicate that the observed high nuclease resistance is not due to the lack of binding affinity to the enzyme's active site, since these modified oligonucleotides were able to inhibit degradation of a natural DNA fragment by bovine intestinal mucosal phosphodiesterase in a dose-dependent manner.     

Systemically delivered antisense oligomers upregulate gene expression in mouse tissues

Systemically injected 2'-O-methoxyethyl (2'-O-MOE)-phosphorothioate and PNA-4K oligomers (peptide nucleic acid with four lysines linked at the C terminus) exhibited sequence-specific antisense activity in a number of mouse organs. Morpholino oligomers were less effective, whereas PNA oligomers with only one lysine (PNA-1K) were completely inactive. The latter result indicates that the four-lysine tail is essential for the antisense activity of PNA oligomers in vivo. These results were obtained in a transgenic mouse model designed as a positive readout test for activity, delivery, and distribution of antisense oligomers. In this model, the expressed gene (EGFP-654) encoding enhanced green fluorescence protein (EGFP) is interrupted by an aberrantly spliced mutated intron of the human beta-globin gene. Aberrant splicing of this intron prevented expression of EGFP-654 in all tissues, whereas in tissues and organs that took up a splice site-targeted antisense oligomer, correct splicing was restored and EGFP-654 expression upregulated. The sequence-specific ability of PNA-4K and the 2'-O-MOE oligomers to upregulate EGFP-654 provides strong evidence that systemically delivered, chemically modified oligonucleotides affect gene expression by sequence-specific true antisense activity, validating their application as potential therapeutics.     

Synthesis and Hybridization Properties of Modified Oligonucleotides with PNA-DNA Dimer Blocks

Abstract

Different modified PNA-DNA dimer-analogous synthons (I and II) were synthesized as phosphoramidites. These dimer units were assembled by a 5′-modified deoxythymidine and a modified PNA monomer. These synthons were used in the routine coupling procedure for oligonucleotides. Therefore no PNA coupling chemistry is necessary to synthesize PNA-DNA chimeric oligonucleotides. Various deoxyoligonucleotides were synthesized introducing the dimer blocks I and II at different positions in the sequences. Melting temperatures of the modified oligonucleotides with their complementary DNA analogues were determined.

Backbone modifications of oligonucleotides are required in the antisense strategy for protection against endonucleolytic cleavage in biological environment. Peptide nucleic acids (PNA fragments) are known to be nuclease resistant analogues, which show stable and discriminating hybridization. For this reason we prepared chimeric PNA-DNA oligomers by incorporation of two different modified PNA-DNA dimer blocks (Scheme A) into oligonucleotides. Melting temperatures of the modified oligonucleotides with their complementary DNA were determined.