Biophysical and Biological Characterization of Hairpin and Molecular Beacon RNase H Active Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) are single stranded, backbone modified nucleic acids, which mediate cleavage of complementary RNA by directing RNase H cleavage in cell culture and in animals. It has generally been accepted that the single stranded state in conjunction with the phosphorothioate modified backbone is necessary for cellular uptake and transport to the active compartment. Herein, we examine the effect of using hairpin structured ASOs to (1) determine if an ASO agent requires a single stranded conformation for efficient RNA knock down, (2) use a fluorophore-quencher labeled ASO to evaluate which moieties the ASO interacts with in cells and examine if cellular distribution can be determined with such probes, and (3) evaluate if self-structured ASOs can improve allele selective silencing between closely related huntingtin alleles. We show that hairpin shaped ASOs can efficiently down-regulate RNA <i>in vitro</i>, but potency correlates strongly negatively with increasing stability of the hairpin structure. Furthermore, self-structured ASOs can efficiently reduce huntingtin mRNA in the central nervous system of mice

A Constrained Tricyclic Nucleic Acid Analogue of α‑l‑LNA: Investigating the Effects of Dual Conformational Restriction on Duplex Thermal Stability

A constrained tricyclic analogue of α-l-LNA (<b>2</b>), which contains dual modes of conformational restriction about the ribose sugar moiety, has been synthesized and characterized by X-ray crystallography. Thermal denaturation experiments of oligonucleotide sequences containing this tricyclic α-l-LNA analogue (α-l-TriNA 2, <b>5</b>) indicate that this modification is moderately stabilizing when paired with complementary DNA and RNA, but less stabilizing than both α-l-LNA (<b>2</b>) and α-l-TriNA 1 (<b>4</b>)

A Constrained Tricyclic Nucleic Acid Analogue of α‑l‑LNA: Investigating the Effects of Dual Conformational Restriction on Duplex Thermal Stability

A constrained tricyclic analogue of α-l-LNA (<b>2</b>), which contains dual modes of conformational restriction about the ribose sugar moiety, has been synthesized and characterized by X-ray crystallography. Thermal denaturation experiments of oligonucleotide sequences containing this tricyclic α-l-LNA analogue (α-l-TriNA 2, <b>5</b>) indicate that this modification is moderately stabilizing when paired with complementary DNA and RNA, but less stabilizing than both α-l-LNA (<b>2</b>) and α-l-TriNA 1 (<b>4</b>)

A Constrained Tricyclic Nucleic Acid Analogue of α‑l‑LNA: Investigating the Effects of Dual Conformational Restriction on Duplex Thermal Stability

A constrained tricyclic analogue of α-l-LNA (<b>2</b>), which contains dual modes of conformational restriction about the ribose sugar moiety, has been synthesized and characterized by X-ray crystallography. Thermal denaturation experiments of oligonucleotide sequences containing this tricyclic α-l-LNA analogue (α-l-TriNA 2, <b>5</b>) indicate that this modification is moderately stabilizing when paired with complementary DNA and RNA, but less stabilizing than both α-l-LNA (<b>2</b>) and α-l-TriNA 1 (<b>4</b>)

C5-Alkynyl-Functionalized α‑L‑LNA: Synthesis, Thermal Denaturation Experiments and Enzymatic Stability

Major efforts are currently being devoted to improving the binding affinity, target specificity, and enzymatic stability of oligonucleotides used for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. One of the most popular strategies toward this end has been to introduce additional modifications to the sugar ring of affinity-inducing conformationally restricted nucleotide building blocks such as locked nucleic acid (LNA). In the preceding article in this issue, we introduced a different strategy toward this end, i.e., C5-functionalization of LNA uridines. In the present article, we extend this strategy to α-L-LNA: i.e., one of the most interesting diastereomers of LNA. α-L-LNA uridine monomers that are conjugated to small C5-alkynyl substituents induce significant improvements in target affinity, binding specificity, and enzymatic stability relative to conventional α-L-LNA. The results from the back-to-back articles therefore suggest that C5-functionalization of pyrimidines is a general and synthetically straightforward approach to modulate biophysical properties of oligonucleotides modified with LNA or other conformationally restricted monomers

Ground of sufficient level of gold-value backlogs of the state

У статті розглядаються основні цілі і завдання управління золотиовалютними резервами держави. Визначено основні методи оцінки ефективності системи управління та достатності золотовалютних резервів.Primary purposes and tasks of management of the state золотиовалютними backlogs are examined in the article. The basic methods of estimation of efficiency of control system and sufficientness of gold-value backlogs are certain

Synthesis and Antisense Properties of Fluoro Cyclohexenyl Nucleic Acid (F-CeNA), a Nuclease Stable Mimic of 2′-Fluoro RNA

We report the design and synthesis of 2′-fluoro cyclohexenyl nucleic acid (F-CeNA) pyrimidine phosphoramidites and the synthesis and biophysical, structural, and biological evaluation of modified oligonucleotides. The synthesis of the nucleoside phosphoramidites was accomplished in multigram quantities starting from commercially available methyl-d-mannose pyranoside. Installation of the fluorine atom was accomplished using nonafluorobutanesulfonyl fluoride, and the cyclohexenyl ring system was assembled by means of a palladium-catalyzed Ferrier rearrangement. Installation of the nucleobase was carried out under Mitsunobu conditions followed by standard protecting group manipulations to provide the desired pyrimidine phosphoramidites. Biophysical evaluation indicated that F-CeNA shows behavior similar to that of a 2′-modified nucleotide, and duplexes with RNA showed slightly lower duplex thermostability as compared to that of the more rigid 3′-fluoro hexitol nucleic acid (FHNA). However, F-CeNA modified oligonucleotides were significantly more stable against digestion by snake venom phosphodiesterases (SVPD) as compared to unmodified DNA, 2′-fluoro RNA (FRNA), 2′-methoxyethyl RNA (MOE), and FHNA modified oligonucleotides. Examination of crystal structures of a modified DNA heptamer duplex d­(GCG)-T*-d­(GCG):d­(CGCACGC) by X-ray crystallography indicated that the cyclohexenyl ring system exhibits both the ³H₂ and ²H₃ conformations, similar to the C3′-endo/C2′-endo conformation equilibrium seen in natural furanose nucleosides. In the ²H₃ conformation, the equatorial fluorine engages in a relatively close contact with C8 (2.94 Å) of the 3′-adjacent dG nucleotide that may represent a pseudo hydrogen bond. In contrast, the cyclohexenyl ring of F-CeNA was found to exist exclusively in the ³H₂ (C3′-endo like) conformation in the crystal structure of the modified A-form DNA decamer duplex [d­(GCGTA)-T*-d­(ACGC)]_2. In an animal experiment, a 16-mer F-CeNA gapmer ASO showed similar RNA affinity but significantly improved activity compared to that of a sequence matched MOE ASO, thus establishing F-CeNA as a useful modification for antisense applications

Synthesis and Biophysical Properties of C5-Functionalized LNA (Locked Nucleic Acid)

Oligonucleotides modified with conformationally restricted nucleotides such as locked nucleic acid (LNA) monomers are used extensively in molecular biology and medicinal chemistry to modulate gene expression at the RNA level. Major efforts have been devoted to the design of LNA derivatives that induce even higher binding affinity and specificity, greater enzymatic stability, and more desirable pharmacokinetic profiles. Most of this work has focused on modifications of LNA’s oxymethylene bridge. Here, we describe an alternative approach for modulation of the properties of LNA: i.e., through functionalization of LNA nucleobases. Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites were synthesized and incorporated into oligodeoxyribonucleotides (ONs), which were then characterized with respect to thermal denaturation, enzymatic stability, and fluorescence properties. ONs modified with monomers that are conjugated to small alkynes display significantly improved target affinity, binding specificity, and protection against 3′-exonucleases relative to regular LNA. In contrast, ONs modified with monomers that are conjugated to bulky hydrophobic alkynes display lower target affinity yet much greater 3′-exonuclease resistance. ONs modified with C5-fluorophore-functionalized LNA-U monomers enable fluorescent discrimination of targets with single nucleotide polymorphisms (SNPs). In concert, these properties render C5-functionalized LNA as a promising class of building blocks for RNA-targeting applications and nucleic acid diagnostics

Efficient Synthesis and Biological Evaluation of 5′-GalNAc Conjugated Antisense Oligonucleotides

Conjugation of triantennary <i>N</i>-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5′-hexylamino modified antisense oligonucleotides (5′-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3′-end of an ASO to the 5′-end results in improved potency in cells and animals

Allele-Specific Suppression of Mutant Huntingtin Using Antisense Oligonucleotides: Providing a Therapeutic Option for All Huntington Disease Patients

<div><p>Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.</p></div