Thrombin is a serine protease that plays a crucial role in hemostasis, fibrinolysis, cell proliferation, and migration. Thrombin binding aptamer (TBA) is able to inhibit the activity of thrombin molecule via binding to its exosite I. This 15-nt DNA oligonucleotide forms an intramolecular, antiparallel G-quadruplex structure with a chair-like conformation. In this paper, we report on our investigations on the influence of certain modified nucleotide residues on thermodynamic stability, folding topology, and biological properties of TBA variants. In particular, the effect of single incorporation of a novel 4-thiouracil derivative of unlocked nucleic acid (UNA), as well as single incorporation of 4-thiouridine and all four canonical UNAs, was evaluated. The studies presented herein have shown that 4-thiouridine in RNA and UNA series, as well as all four canonical UNAs, can efficiently modulate G-quadruplex thermodynamic and biological stability, and that the effect is strongly position dependent. Interestingly, TBA variants containing the modified nucleotide residues are characterized by unchanged folding topology. Thrombin time assay revealed that incorporation of certain UNA residues may improve G-quadruplex anticoagulant properties. Noteworthy, some TBA variants, characterized by decreased ability to inhibit thrombin activity, possess significant antiproliferative properties reducing the viability of the HeLa cell line even by 95% at 10 μM concentration.
Alternative
splicing of MAPT cassette exon 10
produces tau isoforms with four microtubule-binding repeat domains
(4R) upon exon inclusion or three repeats (3R) upon exon skipping.
In human neurons, deviations from the ∼1:1 physiological 4R:3R
ratio lead to frontotemporal dementia with Parkinsonism linked to
chromosome 17 (FTDP-17). Certain FTDP-17-associated mutations affect
a regulatory hairpin that sequesters the exon 10 5′ splice
site (5′ss, located at the exon 10–intron 10 junction).
These mutations tend to increase the 4R:3R ratio by destabilizing
the hairpin, thereby improving 5′ss recognition by U1 snRNP.
Interestingly, a single C-to-G mutation at the 19th nucleotide in
intron 10 (C19G or +19G) decreases the level of exon 10 inclusion
significantly from 56% to 1%, despite the disruption of a G-C base
pair in the bottom stem of the hairpin. Here, we show by biophysical
characterization, including thermal melting, fluorescence, and single-molecule
mechanical unfolding using optical tweezers, that the +19G mutation
alters the structure of the bottom stem, resulting in the formation
of a new bottom stem with enhanced stability. The cell culture alternative
splicing patterns of a series of minigenes reveal that the splicing
activities of the mutants with destabilizing mutations on the top
stem can be compensated in a position-dependent manner by the +19G
mutation in the bottom stem. We observed an excellent correlation
between the level of exon 10 inclusion and the rate of mechanical
unfolding at 10 pN, indicating that the unfolding of the splice site
hairpins (to facilitate subsequent binding of U1 snRNA) may be aided
by helicases or other proteins.
This study focused on determining design rules for gapmer-type antisense oligonucleotides (ASOs), that can differentiate cleavability of two SNP variants of RNA in the presence of ribonuclease H based on the mismatch type and position in the heteroduplex. We describe the influence of structural motifs formed by several arrangements of multiple mismatches (various types of mismatches and their position within the ASO/target RNA duplex) on RNase H cleavage selectivity of five different SNP types. The targets were mRNA fragments of APP, SCA3, SNCA and SOD1 genes, carrying C-to-G, G-to-C, G-to-A, A-to-G and C-to-U substitutions. The results show that certain arrangements of mismatches enhance discrimination between wild type and mutant SNP alleles of RNA in vitro as well as in HeLa cells. Among the over 120 gapmers tested, we found two gapmers that caused preferential degradation of the mutant allele APP 692 G and one that led to preferential cleavage of the mutant SNCA 53 A allele, both in vitro and in cells. However, several gapmers promoted selective cleavage of mRNA mutant alleles in in vitro experiments only.
Off-target effects remain as ignificant challengei n the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the yearsv arious modificationsh ave been synthesizeda nd incorporated into AONs,h owever,p recise control of RNase H-induced cleavage and target sequences electivity has yet to be realized.H erein, the synthesis of the uracil and cytosine derivatives of anovel class of 2'-deoxy-2'fluoro-3'-C-hydroxymethyl-b-d-lyxo-configured nucleotides has been accomplisheda nd the targetm oleculesh ave been incorporated into AONs. Experiments on exonucleased egradation showed improvedn ucleolytic stability relative to the unmodified control.U pon the introduction of one or two of the novel 2'-fluoro-3'-C-hydroxymethyl nucleotides as modificationsi nt he gapr egion of ag apmer AON was associated with efficient RNase H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably,atailored single cleavage event could be engineered depending on the positioning of as ingle modification. The effect of single mismatched base pairs was scanned along the full gap regiond emonstrating that the modificatione nables ar emarkable specificity of RNase Hc leavage. Ac ell-basedm odel systemw as used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.
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