Structure Tuning of Cationic Oligospermine-siRNA Conjugates for Carrier-Free Gene Silencing.

Oligospermine-siRNA conjugates are able to induce efficient luciferase gene silencing upon carrier-free transfection. These conjugates are readily accessible by a versatile automated chemistry that we developed using a DMT-spermine phosphoramidite reagent. In this article, we used this chemistry to study a wide range of structural modifications of the oligospermine-siRNA conjugates, i.e., variation of conjugate positions and introduction of chemical modifications to increase nuclease resistance. At first we examined gene silencing activity of a series of siRNA-tris(spermine) conjugates with and without chemical modifications in standard carrier assisted conditions. The three spermine units attached at one of the two ends of the sense strand or at the 3'-end of the antisense strand are compatible with gene silencing activity whereas attachment of spermine units at the 5'-end of the antisense strand abolished the activity. 2'-O-Methylated nucleotides introduced in the sense strand are compatible while not in the antisense strand. Thiophosphate links could be used without activity loss at the 3'-end of both strands and at the 5'-end of the sense strand to conjugate oligospermine. Consequently a series of oligospermine-siRNA conjugates containing 15 to 45 spermines units in various configurations were chosen, prepared, and examined in carrier-free conditions. Attachment of 30 spermine units singly at the 5'-end of the sense strand provides the most potent carrier-free siRNA. Longevity of luciferase gene silencing was studied using oligospermine-siRNA conjugates. Five day long efficiency with more than 80% gene expression knockdown was observed upon transfection without vector. Oligospermine-siRNA conjugates targeting cell-constitutive natural lamin A/C gene were prepared. Efficient gene silencing was observed upon carrier-free transfection of siRNA conjugates containing 20 or 30 spermine residues grafted at the 5'-end of the sense strand.

MMT, Npeoc-protected spermine, a valuable synthon for the solid phase synthesis of oligonucleotide oligospermine conjugates via guanidine linkers.

Solid phase spermine oligomerization via guanidine linkers was achieved using activated thiourea coupling reaction with primary amino group. Disymmetric spermine synthon was efficiently synthesised in eight steps from spermine. MMT group was used as coupling monitor and resulting oligomeric spermines were conjugated to oligonucleotides.

Chemical synthesis, DNA incorporation and biological study of a new photocleavable 2'-deoxyadenosine mimic.

The phototriggered cleavage of chemical bonds has found numerous applications in biology, particularly in the field of gene sequencing through photoinduced DNA strand scission. However, only a small number of modified nucleosides that are able to cleave DNA at selected positions have been reported in the literature. Herein, we show that a new photoactivable deoxyadenosine analogue, 3-nitro-3-deaza-2'-deoxyadenosine (d(3-NiA)), was able to induce DNA backbone breakage upon irradiation (lambda > 320 nm). The d(3-NiA) nucleoside was chemically incorporated at desired positions into 40-mer oligonucleotides as a phosphoramidite monomer and subsequent hybridization studies confirmed that the resulting modified duplexes display a behaviour that is close to that of the related natural sequence. Enzymatic action of the Klenow fragment exonuclease free revealed the preferential incorporation of dAMP opposite the 3-NiA base. On the other hand, incorporation of the analogous 3-NiA triphosphate to a primer revealed high enzyme efficiency and selectivity for insertion opposite thymine. Furthermore, only the enzymatically synthesized base pair 3-NiA:T was a substrate for further extension by the enzyme. All the hybridization and enzymatic data indicate that this new photoactivable 3-NiA triphosphate can be considered as a photochemically cleavable dATP analogue.

Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription.

Most nucleic acid-based technologies rely upon sequence recognition between an oligonucleotide and its nucleic acid target. With the aim of improving hybridization by decreasing electrostatic repulsions between the negatively charged strands, novel modified oligonucleotides named Zip nucleic acids (ZNAs) were recently developed. ZNAs are oligonucleotide-oligocation conjugates whose global charge is modulated by the number of cationic spermine moieties grafted on the oligonucleotide. It was demonstrated that the melting temperature of a hybridized ZNA is easily predictable and increases linearly with the length of the oligocation. Furthermore, ZNAs retain the ability to discriminate between a perfect match and a single base-pair-mismatched complementary sequence. Using quantitative PCR, we show here that ZNAs are specific and efficient primers displaying an outstanding affinity toward their genomic target. ZNAs are particularly efficient at low magnesium concentration, low primer concentrations and high annealing temperatures, allowing to improve the amplification in AT-rich sequences and potentially multiplex PCR applications. In reverse transcription experiments, ZNA gene-specific primers improve the yield of cDNA synthesis, thus increasing the accuracy of detection, especially for genes expressed at low levels. Our data suggest that ZNAs exhibit faster binding kinetics than standard and locked nucleic acid-containing primers, which could explain why their target recognition is better for rare targets.

Cationic siRNAs provide carrier-free gene silencing in animal cells.

siRNA-mediated gene silencing requires intracellular delivery of the nucleic acid. We have developed a carrierless molecular approach that follows the same cell entry route as cationic supramolecular complexes, yet should avoid the extracellular barriers encountered by nanoparticles. Cationic oligospermine-oligonucleotide conjugates (ZNAs, for Zip Nucleic Acids) were synthesized stepwise on an oligonucleotide synthesizer using a DMT-spermine phosphoramidite derivative. They were shown to enter cells and have access to the cytoplasm, provided their formal charge ratio N/P was >1.5. Cationic siRNAs that fulfilled this condition were shown to achieve selective inhibition of luciferase gene expression in the submicromolar concentration range in constitutively luciferase-expressing cells.

Oligonucleotide-oligospermine conjugates (zip nucleic acids): a convenient means of finely tuning hybridization temperatures.

Synthesis of oligonucleotide probes and control of their hybridization temperature are key aspects of polymerase chain reaction (PCR)-based detection of genetic sequences. A straightforward means to approach the last goal is to decrease the repulsion between the polyanionic probe and target strands. To this end, we have developed a versatile automated synthesis of oligonucleotide-oligospermine derivatives that gave fast access to a large variety of compounds. Plots of their hybridization temperatures T(m) vs overall charge provided a measure of the impact of interstrand phosphate repulsion (and of spermine-mediated attraction) on the main driving force of duplex formation, i.e., base pairing. It showed that stabilization brought about by excess cationic charges can be of larger absolute magnitude than interstrand repulsion, even in high salt media. Base sequence and conjugation site (3' or 5') hardly influenced the effect of spermine on T(m). In typical PCR probe conditions, the T(m) increased linearly with the number of grafted spermines (e.g., 6.2 degrees C per spermine for a decanucleotide probe). The large data set of T(m) vs number of spermines and oligonucleotide length allowed us to empirically derive a simple mathematical relation that is accurately predicting the T(m) of any oligonucleotide-oligospermine derivative. Zip nucleic acids (ZNA) are thus providing an interesting alternative to locked nucleic acids (LNA) or minor groove binders (MGB) for raising the stability of 8-12-mer oligonucleotides up to ca. 70 degrees C, the level required for quantitative PCR experiments.

Cationic Oligospermine-Oligonucleotide Conjugates Provide Carrier-free Splice Switching in Monolayer Cells and Spheroids.

We report the evaluation of 18-mer 2'-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5' end to the oligospermine units (Sn-: n = 5, 15, 20, 25, and 30 [number of spermine units]) as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein-labeled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in three-dimensional (3D) cell culture (multicellular tumor spheroids [MCTSs]) that mimics living tissues. Whereas vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared with the assay in monolayer culture, an efficient exon skipping without significant toxicity was observed with oligospermine-grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labeled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS, whereas the vector-complexed SSO penetrated only the cells of the spheroid's outer layer.

Hybridization properties and enzymatic replication of oligonucleotides containing the photocleavable 7-nitroindole base analog.

Universal DNA base analogs having photocleavable properties would be of great interest for development of new nucleic acid fragmentation tools. The photocleavable 7-nitroindole 2'-deoxyribonucleoside d(7-Ni) was previously shown to furnish a highly efficient approach to photochemically trigger DNA backbone cleavage at preselected position when inserted in a DNA fragment. In the present report, we examine its potential use as universal DNA nucleoside, by analogy with the 5-nitroindole analog that is generally considered as universal base. The d(7-Ni) phosphoramidite was incorporated into oligonucleotides. Hybridization properties of resulting 11mer duplexes indicated a behavior close to that of the 5-nitroindole analog. Enzymatic recognition by Klenow fragment exonuclease-free using 40mers containing the unnatural bases as templates indicated notably a decrease of the polymerase activity with preferential incorporation of dAMP opposite both the 7-Ni and 5-Ni bases. Incorporation of the d(7-Ni) triphosphate was also studied indicating absence of significant differences between the incorporation kinetics opposite each natural base in the template. All the hybridization and enzymatic data indicate that 7-nitroindole can be considered as a cleavable base analog, although not strictly fulfilling, like the 5-nitro isomer, all properties required for a universal base.

Universal labeling chemistry for nucleic acid detection on DNA chips.

An efficient strategy for nucleic acid labeling and analysis on deoxyribonucleic acid (DNA) chips has been developed. This approach, which combines the fragmentation and the labeling steps, is based on the reactivity of the phosphates of DNA and ribonucleic acid (RNA) fragments and is using reporter molecules bearing a bromomethyl- or aryldiazomethane-reactive group. In this chapter, we describe the preparation of the reactive label and protocols for efficient labeling of any nucleic acid sequence, DNA or RNA, prior to their hybridization, detection, and analysis on DNA chips.

Synthesis of Monodisperse Sequence-Coded Polymers with Chain Lengths above DP100.

Non-natural, sequence-encoded polyphosphates were prepared using the phosphoramidite approach on a DNA synthesizer. Two phosphoramidite monomers, namely, 2-cyanoethyl (3-dimethoxytrityloxy-propyl) diisopropylphosphoramidite (0) and 2-cyanoethyl (3-dimethoxytrityloxy-2,2-dimethyl-propyl) diisopropylphosphoramidite (1), were used in this approach to form binary-coded sequences. Using 1000 Å controlled pore glass as a support and a large excess of monomers at each step, it was possible to synthesize homopolymers and sequence-coded copolymers of high chain-length. For instance, monodisperse polymers containing 16, 24, 56, and 104 coded monomer units were synthesized and characterized in this work. These results indicate that highly efficient phosphoramidite steps are suitable for the synthesis of long non-natural information-containing macromolecules.

Deoxyribonolactone lesion in DNA: synthesis of fluorinated analogues.

Mono- and difluorinated derivatives of 2-deoxyribonolactone were synthesized using diastereoselective Reformatski reaction as a key step.

Aryldiazomethane derivatives as reagents for site specific labeling of nucleic acids at phosphate.

An efficient and direct labeling method based on direct alkylation of nucleic acids at phosphates by aryldiazomethane derivatives is described.

Cell-penetrating cationic siRNA and lipophilic derivatives efficient at nanomolar concentrations in the presence of serum and albumin.

Despite its considerable interest in human therapy, in vivo siRNA delivery is still suffering from hurdles of vectorization. We have shown recently efficient gene silencing by non-vectorized cationic siRNA. Here, we describe the synthesis and in vitro evaluation of new amphiphilic cationic siRNA. C12-, (C12)2- and cholesteryl-spermine(x)-siRNA were capable of luciferase knockdown at nanomolar concentrations without vectorization (i.e. one to two orders of magnitude more potent than commercially available cholesteryl siRNA). Moreover, incubation in the presence of serum did not impair their efficiency. Finally, amphiphilic cationic siRNA was pre-loaded on albumin. In A549Luc cells in the presence of serum, these siRNA conjugates were highly effective and had low toxicity.

New substituted aryldiazomethyl labels for high density DNA chip-based nucleic acid testing.

DNA and RNA labeling and detection are key steps in nucleic acid testing, particularly for molecular diagnostics applications. Here, we report a new class of aryldiazomethyl labels that include in their structure a biotin moiety as a detectable unit and a nitro substituted phenyl diazomethyl as a reactive group. The greatest reactivity towards phosphates of nucleic acids, the water solubility and the stability of these new molecules were demonstrated. These very important properties, which are the main requirements for nucleic acid labeling in aqueous conditions using automated protocols within integrated diagnostic devices, make them the perfect labeling tools for hybridization-based analysis, especially for high-density DNA chips.

Versatile synthesis of oligodeoxyribonucleotide-oligospermine conjugates.

A protocol for the rapid, automated synthesis of oligospermine-oligonucleotide sequences is described. To this end, a protected spermine phosphoramidite derivative was synthesized in six steps from spermine and used as the fifth synthon in an oligonucleotide synthesizer. Parameters were optimized to reach greater than 95% coupling yields. Cationic oligonucleotides show enhanced hybridization and strand invasion properties, and hence are an alternative to conventional oligonucleotides for molecular biology, diagnostic and potential therapeutic applications. A multi-gram-scale synthesis of the spermine phosphoramidite allowing several hundred coupling steps takes 2-3 weeks. Oligonucleotide synthesis and purification takes approximately 3 d.

Synthesis of 3-deaza-3-nitro-2'-deoxyadenosine.

Photoactivable deoxyadenosine mimic, 3-deaza-3-nitro-2'-deoxyadenosine (2), was prepared using two different synthetic routes. The first route involved base catalyzed glycosylation of 3-deaza-3-nitroadenine, which was prepared by regioselective nitration of 3-deazaadenine. In the second route, the convertible nucleoside 6-O-(2,4,6-trimethylphenyl)-3-deaza-2'-deoxyadenosine (28) was used to introduce 6-NH2 group in the last step.

Pyrenyldiazomethane, a versatile reagent for nucleotide phosphate alkylation.

Pyrenyldiazomethane was shown to react quantitatively and selectively at phosphate with 2'-, 3'-, and 5'-nucleotide phosphates incorporating the different nucleic bases.

Biotin-phenyldiazomethane conjugates as labeling reagents at phosphate in mono and polynucleotides.

Molecules 2-5 that include in their structure a biotin moiety as detectable unit and differently substituted phenyl diazo functions as reactive group were prepared as reagents for labeling the phosphate group in mono and polynucleotides. These molecules were shown to react selectively and quantitatively with the model nucleotide 3'-UMP. They were used successfully in the labeling step of DNA and RNA analysis using high-density DNA-chips (or microarrays) technology.

Human DNA glycosylases involved in the repair of oxidatively damaged DNA.

Reactive oxygen species from endogenous and environmental sources induce oxidative damage to DNA, and hence pose an enormous threat to the genetic integrity of cells. Such oxidative DNA damage is restored by the base excision repair (BER) pathway that is conserved from bacteria to humans and is initiated by DNA glycosylases, which simply remove the aberrant base from the DNA backbone by hydrolyzing the N-glycosidic bond (monofunctional DNA glycosylase), or further catalyze the incision of a resulting abasic site (bifunctional DNA glycosylase). In human cells, oxidative pyrimidine lesions are generally removed by hNTH1, hNEIL1, or hNEIL2, whereas oxidative purine lesions are removed by hOGG1. hSMUG1 excises a subset of oxidative base damage that is poorly recognized by the above enzymes. Unlike these enzymes, hMYH removes intact A misincorporated opposite template 8-oxoguanine during DNA replication. Although hNTH1, hOGG1, and hMYH account for major cellular glycosylase activity for inherent substrate lesions, mouse models deficient in the enzymes exhibit no overt phenotypes such as the development of cancer, implying backup mechanisms. Contrary to the mouse model, hMYH mutations have been shown to lead to a multiple colorectal adenoma syndrome and high colorectal cancer risk. For cleavage of the N-glycosidic bond, bifunctional DNA glycosylases (hNTH1, hNEIL1, hNEIL2, and hOGG1) use Lys or Pro for direct attack on sugar C1', whereas monofunctional DNA glycosylases (hSMUG1 and hMYH) use an activated water molecule. DNA glycosylases for oxidative damage, if not all, are covalently trapped by DNA containing 2-deoxyribonolactone or oxanine. Thus, the depletion of functional DNA glycosylases using covalent trapping may reduce the BER capacity of cancer cells, hence potentiating the efficacy of anticancer drugs or radiation therapy.

Chemistry of the 2-deoxyribonolactone lesion in oligonucleotides: cleavage kinetics and products analysis.

Deoxyribonolactone in DNA is an oxidized abasic site damage that is produced by a variety of physical and chemical agents such as gamma-irradiation and ene-diyne antibiotics. The extent and biological significance of the lesion are poorly documented due to the high lability of the damaged DNA. The chemistry of degradation of deoxyribonolactone-containing DNA was investigated using oligonucleotides of different length (5-, 11-, 23-, 34-mers) in which the lactone was photochemically generated, as already reported, from oligonucleotide precursors containing a photoactive nitroindole residue. The procedure was successfully extended to double-strand synthesis by irradiation of the preformed duplex in which one strand contained the nitroindole residue. The degradation kinetics were investigated as a function of pH, temperature, length, and ionic strength. The cleavage fragments resulting from beta- and delta-eliminations were isolated and identified by (1)H NMR. It was found that the lesion is extremely sensitive to pH and temperature while slightly dependent upon ionic strength, length, and sequence. The cleavage rates for the beta- and delta-elimination steps are of the same order of magnitude. The deoxyribonolactone site leads to greater instability of DNA than the "regular" deoxyribose abasic site.