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.     

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.     

How are Nucleic Acids Released in Cells from Cationic Lipid-Nucleic Acid Complexes?

Abstract

Cationic lipid-nucleic acid complexes are widely used to deliver oligonucleotides, RNA and DNA into cells. Although much has been learned about the structure and forces that hold the complex together, an understanding of the mechanism of release of the nucleic acids from the complex into cells has been lacking. Recent studies have shown that anionic liposomes with compositions similar to the cytoplasmic face of the endosomal membrane are potent agents for inducing the rapid release of oligonucleotides and DNA from cationic lipid-nucleic acid complexes. Based upon these results, we propose that after the cationic lipid/nucleic complex is internalized by endocytosis it destabilizes the endosomal membrane. This destabilization induces flip-flop of anionic lipids from the cytoplasmic facing monolayer, which laterally diffuse into the complex and form a charge neutral ion-pair with the cationic lipids. This results in displacement of the nucleic acid from the cationic lipid and subsequent release of the nucleic acid into cytoplasm of the cell. We review the data that show the proposed mechanism accounts for a variety of observations on cationic lipid/nucleic acid complex-cell interactions.     

1,5-Anhydro-2-Deoxy-D-Altritol Oligonucleotides as Conformationally Restricted Analogues of Rna

Abstract

As part of a project concerning the investigation of new hexitol nucleic acids (HNA), the 1,5-anhydro-2-deoxy-D-altritol nucleoside building blocks with a uracil, cytosine, adenine and guanine base moiety were synthesized. The uracil analogue was used for the automated synthesis of corresponding oligonucleotides. Hybridization capabilities of these altritol nucleic acids (ANA) are illustrated by the Tm values obtained for the (a ^hU)₁₃/(dA)₁₃ duplex.     

Enhanced Fluorescence in the Binding of Oligonucleotides With a Pyrene Group in the Sugar Fragment to Complementary Polynucleotides

Abstract

The fluorescence intensity and lifetime of oligonucleotides with a pyrenylmethyl group at the specific sugar residue were increased upon binding to their complementary polynucleotide in aqueous solution. The present oligonucleotide-pyrene conjugates provide new fluorescent probes for detection of specific nucleic acids.     

Visible Light Activatable Binary System of Oligonucleotide Conjugates for Nucleic Acids Modification

Abstract

A binary system of oligonucleotides conjugated to perfluoroarylazide and perylene for sequence-specific photomodification of nucleic acids has been developed. The system can be activated by visible light (450-580 nm), reacts 300000 times faster than azide in the absence of perylene and provides highly efficient (up to 99%) photomodification of target ssDNA.     

Divers Stereoisomers of N-Acetylhydroxyprolinol as Sugar Substitute in Oligonucleotides

Abstract

A detailed comparison of the hybridisation characteristics of oligonucleotides with 4-hydroxy-N-acetylprolinol or 3-hydroxy-N-acetylprolinol as sugar substitute, reveals dramatic differences. The 4-HO oligonucleotides are able to form stable complexes which are in general duplexes when natural nucleic acids are used as the complement, and the system has a strong preference for isochiral interaction. For the 3-HO oligonucleotides on the other hand complexes are generally weak, triple stranded, and often isochiral and heterochiral hybrids have a similar stability.     

Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells

To modulate gene expression in research studies or in potential clinical therapies, transfection of exogenous nucleic acids including plasmid DNA and small interference RNA (siRNA) are generally performed. However, the cellular processing and the fate of these nucleic acids remain elusive. By investigating the cellular behavior of transfected nucleic acids using confocal imaging, here we show that when siRNA was co-transfected into cultured cells with other nucleic acids, including single-stranded RNA oligonucleotides, single and double-stranded DNA oligonucleotides, as well as long double-stranded plasmid DNA, they all aggregate in the same cytoplasmic granules. Interestingly, the amount of siRNA aggregating in granules was found not to correlate with the gene silencing activity, suggesting that assembly of cytoplasmic granules triggered by siRNA transfection may be separable from the siRNA silencing event. Our results argue against the claim that the siRNA-aggregating granules are the functional site of RNA interference (RNAi). Taken together, our studies suggest that, independent of their types or forms, extraneously transfected nucleic acids are processed through a common cytoplasmic pathway and trigger the formation of a new type of cytoplasmic granules “transfection granules”.     

Linear polyethylenimine as a tool for comparative studies of antisense and short double-stranded RNA oligonucleotides

Despite the recently enlarged field of available RNA knock-down technologies, e.g., antisense oligonucleotides (ASOs) and duplexes of synthetic 21 nucleotides RNAs (siRNAs), no versatile transfection reagent has been reported to deliver different nucleic acids formats at high rates of efficiency. We have evaluated the versatility and efficacy of linear PEI in transfecting and properly delivering a broad panel of nucleic acids such as short oligonucleotides and double-stranded RNA into cells in culture.     

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.     

Use of C5-Diamino-Substituted-Pyridine in Triple Helix Forming Oligonucleotides

Abstract

Triple helical structures can be observed between double-stranded nucleic acids and a third strand through the formation of Hoogsteen hydrogen bonds. We report here the synthesis and the preliminary evaluation of oligonucleotides incorporating. 5[(N-2-aminoethyl)-3-aminopropynyl]-2′-deoxyuridine 1 as well as its reduced analogue 2. Synthesis of two phosphoramidites 8 and 9 and the first melting temperature measurements are described.     

A new oxygen modification cyclooctaoxygen binds to nucleic acids as sodium crown complex

BackgroundOxygen exists in two gaseous and six solid allotropic modifications. An additional allotropic modification of oxygen, the cyclooctaoxygen, was predicted to exist in 1990.MethodsCyclooctaoxygen sodium was synthesized in vitro from atmospheric oxygen, or catalase effect-generated oxygen, under catalysis of cytosine nucleosides and either ninhydrin or eukaryotic low-molecular weight RNA. Thin-layer chromatographic mobility shift assays were applied on specific nucleic acids and the cyclooctaoxygen sodium complex.ResultsWe report the first synthesis and characterization of cyclooctaoxygen as its sodium crown complex, isolated in the form of three cytosine nucleoside hydrochloride complexes. The cationic cyclooctaoxygen sodium complex is shown to bind to nucleic acids (RNA and DNA), to associate with single-stranded DNA and spermine phosphate, and to be essentially non-toxic to cultured mammalian cells at 0.1–1.0 mM concentration.ConclusionsWe postulate that cyclooctaoxygen is formed in most eukaryotic cells in vivo from dihydrogen peroxide in a catalase reaction catalyzed by cytidine and RNA. A molecular biological model is deduced for a first epigenetic shell of eukaryotic in vivo DNA. This model incorporates an epigenetic explanation for the interactions of the essential micronutrient selenium (as selenite) with eukaryotic in vivo DNA.General significanceSince the sperminium phosphate/cyclooctaoxygen sodium complex is calculated to cover the active regions (2.6%) of bovine lymphocyte interphase genome, and 12.4% of murine enterocyte mitotic chromatin, we propose that the sperminium phosphate/cyclooctaoxygen sodium complex coverage of nucleic acids is essential to eukaryotic gene regulation and promoted proto-eukaryotic evolution.     

Locked Nucleic Acids: Promising Nucleic Acid Analogs for Therapeutic Applications

Locked Nucleic Acid (LNA) is a unique nucleic‐acid modification possessing very high binding affinity and excellent specificity toward complementary RNA or DNA oligonucleotides. The remarkable properties exhibited by LNA oligonucleotides have been employed in different nucleic acid‐based therapeutic strategies both in vitro and in vivo. Herein, we highlight the applications of LNA nucleotides for controlling gene expression.     

Influence of the incorporation of a cyclohexenyl nucleic acid (CeNA) residue onto the sequence d(CGCGAATTCGCG)

Cyclohexene nucleic acids (CeNA), which are characterized by the presence of a cyclohexene moiety instead of a natural (deoxy)ribose sugar, are known to increase the thermal and enzymatic stability when incorporated in RNA oligonucleotides. As it has been demonstrated that even a single cyclohexenyl nucleoside, when incorporated in an oligonucleotide, can have a profound effect on the biological activity of the oligonucleotide, further research is warranted to study the complex of such oligonucleotides with target proteins. In order to analyse the influence of CeNA residues onto the helix conformation and hydration of natural nucleic acid structures, a cyclohexenyl-adenine building block (xAr) was incorporated into the Dickerson sequence CGCGA(xAr)TTCGCG. The crystal structure of this sequence determined to a resolution of 1.90 Å. The global helix belongs to the B-type family and shows a water spine, which is partially broken up by the apolar cyclohexene residue. The cyclohexene ring adopts the ²E-conformation allowing a better incorporation of the residue in the dodecamer sequence. The crystal packing is stabilized by cobalt hexamine residues and belongs to space group P222₁, never before reported for nucleic acids.     

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract

Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety¹ has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation^2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oligonucleotides with techniques such as capillary gel electrophoresis (CGE) and mass spectrometry help to determine the purity as well as the in vivo fate of these complex molecules. Large-scale synthesis is becoming a tangible reality for antisense oligonucleotides. Nucleic acid chemists and biologists alike are beginning to understand the structure-biological activity in terms of basic physical-organic parameters such as the gauche effect, the charge effect and conformational constraints. Synthesis of chimeric designer oligonucleotides bringing the attractive features of different modifications to a given antisense oligonucleotide sequence to generate synergistic interactions is forthcoming³⁰. These advances along with the potential availability of complete human genome sequence information promise a bright future for the widespread use of nucleic acid based therapeutics.     

Les aptamères : du concept à l’outil

Aptamers are oligonucleotides, generally DNA or RNA but occasionnally chemically modified nucleic acids, which are identified within randomly synthesized libraries containing up to 10¹⁵ different candidates. They are obtained following a process of in vitro selection termed systematic evolution of ligands by exponantial enrichment (SELEX) that makes use of iterative steps of selection and amplification. Aptamers were successfully raised against a wide range of targets: amino acids, antibiotics, dyes, peptides, proteins, nucleic acids, intact viruses or live cells. They generally display high affinity (Kd in the nanomolar range or lower are frequent for proteins) and high specificity. They are easily obtained by chemical synthesis and can be converted in tools of interest for diagnostic purposes, by conjugation to various pendant groups. They rival antibodies and can be used in vivo in human beings.     

Quantitative detection of siRNA and single-stranded oligonucleotides: relationship between uptake and biological activity of siRNA

The quantitative detection of oligomeric nucleic acids including short double-stranded RNA in cells and tissues becomes increasingly important. Here, we describe a method for the detection of siRNA in extracts prepared from mammalian cells, which is based on liquid hybridization with a ³²P-labelled probe followed by a nuclease protection step. The limit of detection of absolute amounts of siRNA is in the order of 10–100 amol. This methodology is suited to quantitatively follow the spontaneous uptake of siRNA by mammalian cells, i.e. without the use of carrier substances. This protocol may also be used to detect extremely low amounts of other kinds of short nucleic acids, including antisense oligonucleotides.     

Anti-Influenza Virus Activities of Nicked and Circular Dumbbell RNA/DNA Chimeric Oligonucleotides

Abstract

We have designed a new type of antisense oligonucleotide, containing two hairpin loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem (nicked and circular dumbbell DNA/RNA chimeric oligonucleotides). The reaction of the nicked and circular dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding anti-DNA together with the sense RNA cleavage products. These oligonucleotides were more resistant to exonuclease attack. We also describe the anti-Fluv activities of nicked and circular dumbbell DNMA chimeric oligonucleotides.     

Synthesis of 1,1,2-trisubstituted cyclopropane nucleosides in enantiomerically pure forms

Abstract

Due to the unique rigid and small steric feature of cyclopropane, cyclopropane nucleosides (CPNs) in which the ribose (deoxyribose) of nucleosides are replaced by a hydroxy-substituted cyclopropane, are of great biological interest. Novel 1,1,2-trisubstituted cyclopropane nucleosides were synthesized in enantiomerically pure forms as potential antiviral agents. In the synthesis, two cyclopropane tosylates, which were prepared from chiral cyclopropane lactones previously reported by us, were used effectively as common intermediates for the CPNs. These CPNs are also potentially useful as nucleoside units to incorporate into oligonucleotides in nucleic acids chemotherapy studies.