Unlocked nucleic acids: implications of increased conformational flexibility for RNA/DNA triplex formation

2014 ◽  
Vol 464 (2) ◽  
pp. 203-211 ◽  
Author(s):  
Weronika Kotkowiak ◽  
Michał Kotkowiak ◽  
Ryszard Kierzek ◽  
Anna Pasternak
Keyword(s):  
Nucleic Acids ◽  
Conformational Flexibility ◽  
Hairpin Structure ◽  
Triplex Formation ◽  
Dna Triplex

UNA moieties within the TFO strongly destabilize triplexes. Introduction of UNA into specific positions in the hairpin structure is energetically favourable for triplex formation. UNA increases the resistance of the oligonucleotides to serum nucleases when incorporated at specific hairpin positions.

scholarly journals RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 3020 ◽  
Author(s):  
Alan Ann Lerk Ong ◽  
Jiazi Tan ◽  
Malini Bhadra ◽  
Clément Dezanet ◽  
Kiran M. Patil ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Nucleic Acids ◽  
Splice Site ◽  
Peptide Nucleic Acids ◽  
Amino Sugar ◽  
Hairpin Structure ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Antisense Peptide ◽  
Exon 10

Alternative splicing of tau pre-mRNA is regulated by a 5′ splice site (5′ss) hairpin present at the exon 10–intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5′ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10–15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3′ arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5′ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5′ arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5′ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.

scholarly journals Inhibition of Interleukin-4- and CD40-induced IgE Germline Gene Promoter Activity by 2′-Aminoethoxy-modified Triplex-forming Oligonucleotides

2001 ◽  
Vol 276 (15) ◽  
pp. 11759-11765 ◽  
Author(s):  
Adrian M. Stütz ◽  
Jutta Hoeck ◽  
Francois Natt ◽  
Bernard Cuenoud ◽  
Maximilian Woisetschläger
Keyword(s):  
B Cells ◽  
Critical Role ◽  
Gene Promoter ◽  
Gene Induction ◽  
Modified Oligonucleotides ◽  
Germline Gene ◽  
Triplex Formation ◽  
Human B Cells ◽  
Dna Triplex

Elevated levels of IgE are intimately associated with a number of allergic diseases, such as allergic rhinitis or asthma. Therefore, prevention of IgE production in human B-cells represents an attractive therapeutic target. IL-4-induced IgE germline gene transcription represents a crucial early step during IgE isotype switch differentiation. Gene induction is orchestrated by the coordinated action of the transcription factors STAT6 (signal transducer and activator of transcription), NF-κB, PU.1, and C/EBP. This study shows that 2′-aminoethoxy-modified oligonucleotides, which partially overlap with the STAT6 and the adjacent PU.1/NF-κB binding site, inhibit DNA binding of all three proteins with high affinity in a dose- and time-dependent fashionin vitro. Loss of protein binding correlated strongly with increasing DNA triplex formation. Importantly, the oligomers also effectively displaced pre-bound recombinant NF-κB p50 from double-stranded DNAin vitro. Functionally, the oligonucleotides led to a selective inhibition of IL-4-induced reporter gene activity from a construct driven by the IgE germline gene promoter in human B-cells. These data confirm the critical role of this cytokine-responsive regulatory region in IgE germline gene induction and further support the concept of specific modulation of gene expression by DNA triplex formation induced with chemically modified oligonucleotides.

A Molecular Beacon Strategy for the Thermodynamic Characterization of Triplex DNA:  Triplex Formation at the Promoter Region of Cyclin D1†

Biochemistry ◽  
2001 ◽  
Vol 40 (31) ◽  
pp. 9387-9395 ◽  
Author(s):  
Thomas Antony ◽  
Thresia Thomas ◽  
Leonard H. Sigal ◽  
Akira Shirahata ◽  
T. J. Thomas
Keyword(s):  
Cyclin D1 ◽  
Promoter Region ◽  
Molecular Beacon ◽  
Triplex Dna ◽  
Thermodynamic Characterization ◽  
Triplex Formation ◽  
Dna Triplex

Polyamines favor DNA triplex formation at neutral pH

Biochemistry ◽  
1991 ◽  
Vol 30 (18) ◽  
pp. 4455-4459 ◽  
Author(s):  
Ken J. Hampel ◽  
Paul Crosson ◽  
Jeremy S. Lee
Keyword(s):  
Triplex Formation ◽  
Neutral Ph ◽  
Dna Triplex

Thermodynamic and kinetic studies of DNA triplex formation of an oligohomopyrimidine and a matched duplex by filter binding assay

Biochemistry ◽  
1993 ◽  
Vol 32 (34) ◽  
pp. 8963-8969 ◽  
Author(s):  
Heisaburo Shindo ◽  
Hidetaka Torigoe ◽  
Akinori Sarai
Keyword(s):  
Binding Assay ◽  
Kinetic Studies ◽  
Triplex Formation ◽  
Dna Triplex
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