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- Aaldering, L. J., et al.
(författare)
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Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule
- 2017
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Ingår i: ChemBioChem. - : Wiley-VCH Verlag. - 1439-4227 .- 1439-7633. ; 18:8, s. 755-763
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Tidskriftsartikel (refereegranskat)abstract
- The thrombin-binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G-quadruplex-forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three-carbon spacer (spacer-C3), unlocked nucleic acid (UNA) and 3′-amino-modified UNA (amino-UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G-quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer-C3 introduction at the T7 loop position (TBA-SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA-SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.
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| 2. |
- Bestas, Burcu, et al.
(författare)
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Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model
- 2014
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Ingår i: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 124:9, s. 4067-4081
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Tidskriftsartikel (refereegranskat)abstract
- X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTKtranscripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.
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| 3. |
- Geny, Sylvain, et al.
(författare)
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Next-generation bis-locked nucleic acids with stacking linker and 2 '-glycylamino-LNA show enhanced DNA invasion into supercoiled duplexes
- 2016
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 44:5, s. 2007-2019
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Tidskriftsartikel (refereegranskat)abstract
- Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.
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| 11. |
- Hernandez, Frank J, et al.
(författare)
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Aptamers as a model for functional evaluation of LNA and 2′-amino LNA
- 2009
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Ingår i: Bioorganic & Medicinal Chemistry Letters. - : Pergamon Press. - 0960-894X .- 1464-3405. ; 19:23, s. 6585-6587
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Tidskriftsartikel (refereegranskat)abstract
- The affinity change upon incorporation of LNA and 2′-amino-LNA monomers into an avidin binding DNA aptamer is described. The kinetic profile of selected modified-aptamer was obtained by surface plasmon resonance experiments and compared with the profile of the parent unmodified DNA aptamer. We report significant improvement of avidin binding affinity by the incorporation of single LNA modifications into the aptamer, and successful incorporation of 2′-amino LNA as a novel monomer in aptamers with potential function as carrier unit for additional molecular entities. © 2009 Elsevier Ltd. All rights reserved.
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| 15. |
- Pabon-Martinez, YV, et al.
(författare)
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LNA effects on DNA binding and conformation: from single strand to duplex and triplex structures
- 2017
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Ingår i: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1, s. 11043-
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Tidskriftsartikel (refereegranskat)abstract
- The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked nucleic acid (LNA) substitutions in the ON when targeting a c-MYC or FXN (Frataxin) sequence. We found that LNA-containing single strand TFOs are conformationally pre-organized for major groove binding. Reduced content of LNA at consecutive positions at the 3′-end of a TFO destabilizes the triplex structure, whereas the presence of Twisted Intercalating Nucleic Acid (TINA) at the 3′-end of the TFO increases the rate and extent of triplex formation. A triplex-specific intercalating benzoquinoquinoxaline (BQQ) compound highly stabilizes LNA-containing triplex structures. Moreover, LNA-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.
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| 17. |
- Pasternak, Anna, et al.
(författare)
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Improved thrombin binding aptamer by incorporation of a single unlocked nucleic acid monomer
- 2011
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 39:3, s. 1155-1164
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Tidskriftsartikel (refereegranskat)abstract
- A 15-mer DNA aptamer (named TBA) adopts a G-quadruplex structure that strongly inhibits fibrin-clot formation by binding to thrombin. We have performed thermodynamic analysis, binding affinity and biological activity studies of TBA variants modified by unlocked nucleic acid (UNA) monomers. UNA-U placed in position U3, U7 or U12 increases the thermodynamic stability of TBA by 0.15-0.50kcal/mol. In contrast, modification of any position within the two G-quartet structural elements is unfavorable for quadruplex formation. The intramolecular folding of the quadruplexes is confirmed by Tm versus ln c analysis. Moreover, circular dichroism and thermal difference spectra of the modified TBAs displaying high thermodynamic stability show bands that are characteristic for antiparallel quadruplex formation. Surface plasmon resonance studies of the binding of the UNA-modified TBAs to thrombin show that a UNA monomer is allowed in many positions of the aptamer without significantly changing the thrombin-binding properties. The biological effect of a selection of the modified aptamers was tested by a thrombin time assay and showed that most of the UNA-modified TBAs possess anticoagulant properties, and that the construct with a UNA-U monomer in position 7 is a highly potent inhibitor of fibrin-clot formation. © 2010 The Author(s).
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| 22. |
- Umek, T., et al.
(författare)
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Oligonucleotides Targeting DNA Repeats Downregulate Huntingtin Gene Expression in Huntington's Patient-Derived Neural Model System
- 2021
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Ingår i: Nucleic Acid Therapeutics. - : Mary Ann Liebert Inc. - 2159-3337 .- 2159-3345. ; 31:6, s. 443-456
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Tidskriftsartikel (refereegranskat)abstract
- Huntington's disease (HD) is one of the most common, dominantly inherited neurodegenerative disorders. It affects the striatum, cerebral cortex, and other subcortical structures leading to involuntary movement abnormalities, emotional disturbances, and cognitive impairments. HD is caused by a CAG center dot CTG trinucleotide-repeat expansion in exon 1 of the huntingtin (HTT) gene leading to the formation of mutant HTT (mtHTT) protein aggregates. Besides the toxicity of the mutated protein, there is also evidence that mtHTT transcripts contribute to the disease. Thus, the reduction of both mutated mRNA and protein would be most beneficial as a treatment. Previously, we designed a novel anti-gene oligonucleotide (AGO)-based strategy directly targeting the HTT trinucleotide-repeats in DNA and reported downregulation of mRNA and protein in HD patient fibroblasts. In this study, we differentiate HD patient-derived induced pluripotent stem cells to investigate the efficacy of the AGO, a DNA/Locked Nucleic Acid mixmer with phosphorothioate backbone, to modulate HTT transcription during neural in vitro development. For the first time, we demonstrate downregulation of HTT mRNA following both naked and magnetofected delivery into neural stem cells (NSCs) and show that neither emergence of neural rosette structures nor self-renewal of NSCs is compromised. Furthermore, the inhibition potency of both HTT mRNA and protein without off-target effects is confirmed in neurons. These results further validate an anti-gene approach for the treatment of HD.
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