ABSTRACT
Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation of HTT gene-associated RNA-polymerase II is demonstrated, suggestive of reduced transcription downstream the ON-targeted repeat. Different backbone chemistries were found to have a strong impact on the ON efficiency. We also successfully use different delivery vehicles as well as naked uptake of the ONs, demonstrating versatility and possibly providing insights for in vivo applications.
Subject(s)
Down-Regulation/drug effects , Huntingtin Protein/genetics , Phosphorothioate Oligonucleotides/pharmacology , Trinucleotide Repeat Expansion/genetics , Alleles , DNA/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Knockdown Techniques , Humans , Huntingtin Protein/metabolism , Nucleic Acid Denaturation/drug effects , Peptides/metabolism , Phosphorylation/drug effects , Phosphoserine/metabolism , RNA Polymerase II/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Restriction Mapping , Ultraviolet RaysABSTRACT
Forty modified versions of a streptavidin-binding aptamer each containing single or multiple LNA or α-l-LNA-substitutions were synthesized and their dissociation constants determined by surface plasmon resonance experiments. Both full-length and truncated versions of the aptamer were studied and compared with the unmodified DNA aptamers. A â¼two-fold improvement in binding affinity was achieved by incorporation of LNA nucleotides in the 3'-part of the stems of the streptavidin-binding aptamer whereas LNA- and α-l-LNA-substitutions in the terminal stem increased the serum stability.
Subject(s)
Aptamers, Nucleotide/chemistry , Oligonucleotides/chemistry , Streptavidin/chemistry , Aptamers, Nucleotide/metabolism , Base Sequence , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides/metabolism , Streptavidin/metabolism , Surface Plasmon ResonanceABSTRACT
Herein we describe fluorescent oligonucleotides prepared by click chemistry between novel alkyne-modified locked nucleic acid (LNA) strands and a series of fluorescent azides for homogeneous (all-in-solution) detection of nucleic acids and autoimmune antibodies.
Subject(s)
Autoantibodies/analysis , Nucleic Acids/analysis , Oligonucleotides/chemical synthesis , Alkynes/chemistry , Antibodies, Monoclonal/analysis , Azides/chemistry , Click Chemistry , DNA/immunology , DNA Probes , Spectrometry, FluorescenceABSTRACT
Synthesis of three pyrene-modified nucleosides was accomplished using the CuAAC reaction. Hereby, pyrene is attached either to the 5'-position of thymidine or to the 2'-position of 2'-deoxyuridine through triazolemethylene linkers, or to the 2'-position of 2'-deoxyuridine through a more rigid triazole linker. The three nucleosides were incorporated into oligonucleotides, and these were combined in different duplexes and other secondary structures, which were analyzed by thermal stability and fluorescence studies. The three monomers were found to have different impacts on the nucleic acid complexes. Hence, pyrene attached to the 5'-position shows a tendency for intercalation into the duplex as indicated by a general decrease in fluorescence intensity followed by an increase in duplex thermal stability. Pyrene attached to the 2'-position through a rigid triazole linker also shows a tendency for intercalation but with decrease in duplex stability, whereas the pyrene attached to the 2'-position through a triazolemethylene linker is primarily situated in the minor groove as indicated by an increase in fluorescence but here in most cases leading to increase in duplex stability. All three pyrene nucleotides lead to thermal stabilization of bulged duplexes and three-way junctions. In some cases when two pyrenes were introduced into the core of these complexes, the formation or disappearance of a fluorescence excimer band can be used to indicate the hybridization process. Hereby these oligonucleotides can act as specific recognition probes.
Subject(s)
Nucleosides/chemical synthesis , Nucleotides/chemical synthesis , Oligonucleotides/chemistry , Oligonucleotides/chemical synthesis , Pyrenes/chemistry , Pyrenes/chemical synthesis , Base Pairing , Molecular Sequence Data , Nucleic Acid Hybridization , Nucleosides/chemistry , Nucleotides/chemistryABSTRACT
Four double-headed nucleosides were prepared by the CuAAC reaction. Hereby, a triazole-containing linker connects an additional thymine or adenine to the 2'-position of 2'-deoxyuridine, a thymine to the 5'-position of thymidine and a thymine to the 6'-position of an LNA-thymidine monomer. Whereas no conclusive recognition effects of the additional thymines were found when introduced in LNA or at the 5'-position, both thymine and adenine in the 2'-position were found to stabilise three-way junctions in both dsDNA and DNA : RNA contexts and to give cross-strand interactions in a DNA-duplex, when specifically introduced in a so-called (+1)-zipper motif.
