Synthesis and properties of small interfering RNA duplexes carrying 5-ethyluridine residues

Oligoribonucleotides carrying 5-ethyluridine units were prepared using solid-phase phosphoramidite chemistry. The introduction of the tert-butyldimethylsilyl group at the 2'-OH position proceeded in good yield and very high 2'-regioselectivity. RNA duplexes carrying 5-ethyluridine either at the sense or the guide strands display RNAi activity comparable to or slightly better than that of unmodified RNA duplexes. Gene suppression experiments using luciferase targets in SH-SY5Y cells show that the ethyl group is generally well accepted at all positions although a small decrease in RNA interference activity is observed when one 5-ethylU residue is incorporated in the 3' overhangs

Modified siRNAs for the study of the PAZ domain

Chemical modifications aimed at stabilizing the interaction between the 3'-end of siRNAs and the PAZ domain of RISC have been tested for their effect on RNAi activity. Such modifications contribute positively to the stability of siRNAs in human serum

Stepwise synthesis of oligonucleotide-peptide conjugates containing guanidinium and lipophilic groups in their 3'-termini

Two different series of oligonucleotide-peptide conjugates have been efficiently synthesized by stepwise solid-phase synthesis. First, oligonucleotides and oligonucleotide phosphorothioates containing polar groups at the 3′-termini, such as amine and guanidinium groups were prepared. ODNs conjugates carrying several lysine residues were obtained directly from Fmoc deprotection whereas ODN conjugates with guanidinium groups were obtained by post-synthetic guanidinylation. The second family contains different urea moieties that were achieved by standard protocols. All products were fully characterized by reversed phase HPLC and MALDI-TOF mass spectrometry yielding satisfactory results. Oligonucleotide-phosphorothioate conjugates were evaluated as potential antisense oligonucleotides in the inhibition of the luciferase gene

Influence of pH and a porphyrin ligand on the stability of a G-quadruplex structure within a duplex segment near the promoter region of the SMARCA4 gene

In a previous work, the formation of G-quadruplex structures in a 44-nucleotide long sequence found near the promoter region of the SMARCA4 gene was reported. The central 25 nucleotides were able to fold into an antiparallel G-quadruplex structure, the stability of which was pH-dependent. In the present work, the effect of the presence of lateral nucleotides and the complementary cytosine-rich strand on the stability of this G-quadruplex has been characterized. Moreover, the role of the model ligand TMPyP4 has been studied. Spectroscopic and separation techniques, as well as multivariate data analysis methods, have been used with these purposes. The results have shown that stability of the G-quadruplex as a function of pH or temperature is greatly reduced in the presence of the lateral nucleotides. The influence of the complementary strand does not prevent the formation of the G-quadruplex. Moreover, attempts to modulate the equilibria by an external ligand led us to determine the influence of the TMPyP4 porphyrin on these complex equilibria. This study could eventually help to understand the regulation of SMARCA4 expression

Tuning G-quadruplex nanostructures with lipids. Towards designing hybrid scaffolds for oligonucleotide delivery

Two G-quadruplex forming oligonucleotides [d(TG4T)4 and d(TG6T)4] were selected as two tetramolecular quadruplex nanostructures because of their demonstrated ability to be modified with hydrophobic molecules. This allowed us to synthesize two series of G-quadruplex conjugates that differed in the number of G-tetrads, as well as in the terminal position of the lipid modification. Both solution and solid-phase syntheses were carried out to yield the corresponding lipid oligonucleotide conjugates modified at their 3′- and 5′-termini, respectively. Biophysical studies confirmed that the presence of saturated alkyl chains with different lengths did not affect the G-quadruplex integrity, but increased the stability. Next, the G-quadruplex domain was added to an 18-mer antisense oligonucleotide. Gene silencing studies confirmed the ability of such G-rich oligonucleotides to facilitate the inhibition of target Renilla luciferase without showing signs of toxicity in tumor cell lines

Parallel Clamps and Polypurine Hairpins (PPRH) for Gene Silencing and Triplex-Affinity Capture: Design, Synthesis, and Use

Nucleic acid triplexes are formed when a DNA or RNA oligonucleotide binds to a polypurine-polypyrimidine-rich sequence. Triplexes have wide therapeutic applications such as gene silencing or site-specific mutagenesis. In addition, protocols based on triplex-affinity capture have been used for detecting nucleic acids in biosensing platforms. In this article, the design, synthesis, and use of parallel clamps and polypurine-reversed hairpins (PPRH) to bind to target polypyrimidine targets are described. The combination of the polypurine Watson-Crick strand with the triplex-forming strand in a single molecule produces highly stable triplexes allowing targeting of single- and double-stranded nucleic acid sequences. On the other hand, PPRHs are easily prepared and work at nanomolar range, like siRNAs, and at a lower concentration than that needed for antisense ODNs or TFOs. However, the stability of PPRHs is higher than that of siRNAs. In addition, PPRHs circumvent off-target effects and are non-immunogenic

Porphyrin binding mechanism is altered by protonation at the loops in G-quadruplex DNA formed near the transcriptional activation site of the human c-kit gene

Background G-quadruplex DNA structures are hypothesized to be involved in the regulation of gene expression and telomere homeostasis. The development of small molecules that modulate the stability of G-quadruplex structures has a potential therapeutic interest in cancer treatment and prevention of aging. Methods Molecular absorption and circular dichroism spectra were used to monitor thermal denaturation, acid base titration and mole ratio experiments. The resulting data were analyzed by multivariate data analysis methods. Surface plasmon resonance was also used to probe the kinetics and affinity of the DNA-drug interactions. Results We investigated the interaction between a G-quadruplex-forming sequence in the human c-kit proto-oncogene and the water soluble porphyrin TMPyP4. The role of cytosine and adenine residues at the loops of G-quadruplex was studied by substitution of these residues by thymidines. Conclusions Here, we show the existence of two binding modes between TMPyP4 and the considered G-quadruplex. The stronger binding mode (formation constant around 107) involves end-stacking, while the weaker binding mode (formation constant around 106) is probably due to external loop binding. Evidence for the release of TMPyP4 upon protonation of bases at the loops has been observed. General significance The results may be used for the design of porphyrin-based anti-cancer molecules with a higher affinity to G-quadruplex structures which may have anticancer properties. Graphical abstract Protonation pushes away TMPyP4 molecules from the loops in G-quadruplex structures. The interaction of TMPyP4 porphyrin with the G-quadruplex structure formed by a guanine-rich sequence in the promoter region of c-kit gene was studied. Up to three ligand molecules may be bound to the G-quadruplex structure. Protonation at the loops induces the release of one TMPyP4 molecule

Understanding the effect of the nature of the nucleobase in the loops on the stability of the i-motif structure

The nature and length of loops connecting cytosine tracts in i-motif structures may affect their stability. In this work, the influence of the nature of the nucleobases located in two of the loops of an intramolecular i-motif is studied using spectroscopy, separation techniques, and multivariate data analysis. Insertion of bases other than thymine induces an additional acid-base equilibrium with pKa~4.5. The presence of two guanine bases in the loops, placed opposite to each other, decreases the thermal stability of the structure. In contrast, thymine and cytosine bases in these positions stabilize the structur

DNA-origami-aided lithography for sub-10 nanometer pattern printing

We report the first DNA-based origami technique that can print addressable patterns on surfaces with sub-10 nm resolution. Specifically, we have used a two-dimensional DNA origami as a template (DNA origami stamp) to transfer DNA with pre-programmed patterns (DNA ink) on gold surfaces. The DNA ink is composed of thiol-modified staple strands incorporated at specific positions of the DNA origami stamp to create patterns upon thiol-gold bond formation on the surface (DNA ink). The DNA pattern formed is composed of unique oligonucleotide sequences, each of which is individually addressable. As a proof-of-concept, we created a linear pattern of oligonucleotide-modified gold nanoparticles complementary to the DNA ink pattern. We have developed an in silico model to identify key elements in the formation of our DNA origami-driven lithography and nanoparticle patterning as well as simulate more complex nanoparticle patterns on surfaces

Ethylcellulose nanoparticles as a new "in vitro" transfection tool for antisense oligonucleotide delivery

Oil-in-water nano-emulsions have been obtained in the HEPES 20 mM buffer solution / [Alkylamidoammonium:Kolliphor EL = 1:1] / [6 wt% ethylcellulose in ethyl acetate] system over a wide oil-to-surfactant range and above 35 wt% aqueous component at 25 °C. The nano-emulsion with an oil-to-surfactant ratio of 70/30 and 95 wt% aqueous component was used for nanoparticles preparation. These nanoparticles (mean diameter around 90 nm and zeta potential of +22 mV) were non-toxic to HeLa cells up to a concentration of 3 mM of cationic species. Successful complexation with an antisense phosphorothioate oligonucleotide targeting Renilla luciferase mRNA was achieved at cationic/anionic charge ratios above 16, as confirmed by zeta potential measurements and an electrophoretic mobility shift assay, provided that no Fetal Bovine Serum is present in the cell culture medium. Importantly, Renilla luciferase gene inhibition shows an optimum efficiency (40%) for the cationic/anionic ratio 28, which makes these complexes promising for "in vitro" cell transfection