We report the effect of introducing a single incorporation of 2-thio-deoxythymidine (2S-dT) or C5-Triazolylphenyl-deoxythymidine (5-TrPh-dT) at four positions within the gap region of RNase H gapmer ...antisense oligonucleotides (ASOs) for reducing wild-type and mutant huntingtin mRNA in human patient fibroblasts. We show that these modifications can modulate processing of the ASO/RNA heteroduplexes by recombinant human RNase H1 in a position-dependent manner. We also created a structural model of the catalytic domain of human RNase H bound to ASO/RNA heteroduplexes to rationalize the activity and selectivity observations in cells and in the biochemical assays. Our results highlight the ability of chemical modifications in the gap region to produce profound changes in ASO behavior.
A constrained tricyclic analogue of α-l-LNA (2), 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, 5) indicate that this modification is moderately stabilizing when paired with complementary DNA and RNA, but less stabilizing than both α-l-LNA (2) and α-l-TriNA 1 (4).
The design, synthesis and biophysical evaluation of two highly-constrained tricyclic analogues of locked nucleic acid (LNA), which restrict rotation around the C4'-C5'-exocyclic bond (torsion angle ...γ) and enhance hydrophobicity in the minor groove and along the major groove, are reported. A structural model that provides insights into the sugar-phosphate backbone conformations required for efficient hybridization to complementary nucleic acids is also presented.
We describe the effects of introducing two epimers of neutral backbone α,β-constrained nucleic acid (CNA) on the activity and allele selectivity profile of RNase H active antisense oligonucleotides ...(ASOs) targeting a single nucleotide polymorphism (SNP) for the treatment of Huntington’s disease (HD). ASOs modified with both isomers of α,β-CNA in the gap region showed good activity versus the mutant allele, but one isomer showed improved selectivity versus the wild-type allele. Analysis of the human RNase H cleavage patterns of α,β-CNA modified ASOs versus matched and mismatched RNA revealed that both isomers support RNase H cleavage on the RNA strand across from the site of incorporation in the ASOan unusual observation for a neutral linkage oligonucleotide modification. Interestingly, ASOs modified with (R)- and (S)-5′-hydroxyethyl DNA (RHE and SHE respectively) formed by partial hydrolysis of the dioxaphosphorinane ring system in α,β-CNA also showed good activity versus the mutant allele but an improved selectivity profile was observed for the RHE modified ASO. Our observations further support the profiling of neutral and 5′-modified nucleic acid analogs as tools for gene silencing applications.
Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (locked nucleic acid) and its diastereomer α-L-LNA are two promising examples ...thereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization, and molecular modeling of N2′-functionalized 2′-amino-α-L-LNA is described. Chemoselective N2′-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2′-N-(pyren-1-yl)carbonyl-2′-amino-α-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 °C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small nonaromatic N2′-functionalities such as 2′-N-acetyl-2′-amino-α-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as −16.5 °C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases in circular dichroism signal intensity, and molecular modeling studies suggest that pyrene-functionalized 2′-amino-α-L-LNA monomers W−Y having short linkers between the bicyclic skeleton and the pyrene moiety allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development of probes for emerging therapeutic and diagnostic applications focusing on DNA targeting.
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 in vitro, 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.
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.
Single nucleotide polymorphisms (SNPs) are important markers in disease genetics and pharmacogenomic studies. Oligodeoxyribonucleotides (ONs) modified with ...5‐3‐(1‐pyrenecarboxamido)propynyl‐2′‐deoxyuridine monomer X enable detection of SNPs at non‐stringent conditions due to differential fluorescence emission of matched versus mismatched nucleic acid duplexes. Herein, the thermal denaturation and optical spectroscopic characteristics of monomer X are compared to the corresponding locked nucleic acid (LNA) and α‐L‐LNA monomers Y and Z. ONs modified with monomers Y or Z result in a) larger increases in fluorescence intensity upon hybridization to complementary DNA, b) formation of more brightly fluorescent duplexes due to markedly larger fluorescence emission quantum yields (ΦF=0.44–0.80) and pyrene extinction coefficients, and c) improved optical discrimination of SNPs in DNA targets. Optical spectroscopy studies suggest that the nucleobase moieties of monomers X–Z adopt anti and syn conformations upon hybridization with matched and mismatched targets, respectively. The polarity‐sensitive 1‐pyrenecarboxamido fluorophore is, thereby, either positioned in the polar major groove or in the hydrophobic duplex core close to quenching nucleobases. Calculations suggest that the bicyclic skeletons of LNA and α‐L‐LNA monomers Y and Z influence the glycosidic torsional angle profile leading to altered positional control and photophysical properties of the C5‐fluorophore.
SNP discrimination with LNA: Single‐nucleotide polymorphisms (SNPs) are discriminated by using DNA monomer X or the corresponding C5‐functionalized locked nucleic acid (LNA) and α‐L‐LNA monomers Y and Z. Improved positional control of the polarity‐sensitive pyrene label results in larger hybridization‐induced increases in fluorescence intensity, formation of brightly fluorescent duplexes upon hybridization to complementary nucleic acids, and efficient SNP discrimination.
In a platform trial involving patients hospitalized with Covid-19, among 314 patients who were also being treated with remdesivir, those who received the monoclonal antibody LY-CoV555 did not have ...better pulmonary function at day 5 than those who received placebo. The trial was stopped for futility.
The synthesis and biophysical evaluation of R and S-5′-Me-α-l-LNA nucleoside phosphoramidites and modified oligo-2′-deoxyribonucleotides is reported. Synthesis of the nucleoside phosphoramidites was ...accomplished in multi-gram quantities starting from diacetone glucose. The 5′-methyl group in the S configuration was introduced by reacting the sugar 5′-aldehyde with MeMgBr. Synthesis of the R-5′-Me isomer was accomplished from the S-5′-Me nucleoside by a late stage inversion using Mitsunobu conditions. Evaluation of the modified oligonucleotides in thermal denaturation experiments revealed that R-5′-Me-α-l-LNA showed similar RNA affinity as α-l-LNA while the S-5′-Me analog was less stabilizing. This result is in contrast to the β-d-series where the S-5′-Me isomer showed LNA-like affinity for RNA while the R-5′-Me group completely reversed the stabilization effect on duplex thermostability.
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