Synthesis of DNA and RNA Oligonucleotides Containing a Dual-Purpose Selenium-Modified Fluorescent Nucleoside Probe.

Development of efficient tools that would enable direct correlation of nucleic acid structure and recognition in solution and in solid state at atomic resolution is highly desired. In this context, we recently developed dual-purpose nucleoside probes made of a 5-selenophene-modified uracil core, which serves both as a conformation-sensitive fluorophore and as an X-ray crystallography phasing agent. In this article, we provide a detailed synthetic procedure to synthesize the phosphoramidites of 5-selenophene-modified 2'-deoxyuridine and 5-selenophene-modified uridine analogs. We also describe their site-specific incorporation into therapeutically relevant DNA and RNA oligonucleotide motifs by an automated solid support synthesis protocol. The dual-purpose and minimally invasive nature of the probes enables efficient analysis of the conformation and ligand binding abilities of bacterial decoding site RNA (A-site) and G-quadruplex structures of the human telomeric overhang in real time by fluorescence and in 3D by X-ray crystallography. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 5-selenophene-2'-deoxyuridine 2 and its phosphoramidite 5 Support Protocol 1: Synthesis of 2-(tri-n-butylstannyl) selenophene Support Protocol 2: Synthesis of 5'-O-DMT-protected 5-iodo-2'-deoxyuridine 3 Basic Protocol 2: Synthesis of 5-selenophene-modified uridine 7 and its phosphoramidite 11 Basic Protocol 3: Synthesis of DNA oligonucleotides containing 5-selenophene-modified 2'-deoxyuridine 2 Basic Protocol 4: Synthesis of an RNA oligonucleotide containing 5-selenophene-modified uridine 7.

Duplex-forming Oligonucleotide of Triazole-linked RNA.

An oligonucleotide of triazole-linked RNA (TL RNA) was synthesized by performing consecutive copper-catalyzed azide-alkyne cycloaddition reactions for elongation. The reaction conditions that had been optimized for the synthesis of 3-mer TL RNA were found to be inappropriate for longer oligonucleotides, and the conditions were reoptimized for the solid-phase synthesis of an 11-mer TL RNA oligonucleotide. Duplex formation of the 11-mer TL RNA oligonucleotide was examined with the complementary oligonucleotide of natural RNA to reveal the effects of the 2'-OH groups on the duplex stability.

Phosphodiester photo-tethers for the (multi-)cyclic conformational caging of oligonucleotides.

The ability to address the function of oligonucleotides with light is highly desirable since they are often used experimentally in the regulation of biological processes that need to be controlled in time, space and activation level. Here we present an extension of our initial approach of using photo-tethers that force single strands of nucleic acids into a circle, thus making them unable to form a duplex with a complementary DNA- or RNA-strand. Due to the persistence length a single strand can form a circle of, for example, 30 nucleotides, but a duplex cannot. We show that these new photo-tethers can also be easily installed on the phosphodiester backbone. This simplifies the approach considerably and leads to temporarily inhibited oligonucleotides that can only form a duplex after linearization by photoactivation.

Discovery of the first small-molecule CsrA-RNA interaction inhibitors using biophysical screening technologies.

AIM: CsrA is a global post-transcriptional regulator protein affecting mRNA translation and/or stability. Widespread among bacteria, it is essential for their full virulence and thus represents a promising anti-infective drug target. Therefore, we aimed at the discovery of CsrA-RNA interaction inhibitors. Results & methodology: We followed two strategies: a screening of small molecules (A) and an RNA ligand-based approach (B). Using surface plasmon resonance-based binding and fluorescence polarization-based competition assays, (A) yielded seven small-molecule inhibitors, among them MM14 (IC50 of 4 µM). (B) resulted in RNA-based inhibitor GGARNA (IC50 of 113 µM). CONCLUSION: The first small-molecule inhibitors of the CsrA-RNA interaction were discovered exhibiting micromolar affinities. These hits represent tools to investigate the effects of CsrA-RNA interaction inhibition on bacterial virulence.

N-Branched acyclic nucleoside phosphonates as monomers for the synthesis of modified oligonucleotides.

Protected N-branched nucleoside phosphonates containing adenine and thymine bases were prepared as the monomers for the introduction of aza-acyclic nucleotide units into modified oligonucleotides. The phosphotriester and phosphoramidite methods were used for the incorporation of modified and natural units, respectively. The solid phase synthesis of a series of nonamers containing one central modified unit was successfully performed in both 3'→5' and 5'→3' directions. Hybridization properties of the prepared oligoribonucleotides and oligodeoxyribonucleotides were evaluated. The measurement of thermal characteristics of the complexes of modified nonamers with the complementary strand revealed a considerable destabilizing effect of the introduced units. We also examined the substrate/inhibitory properties of aza-acyclic nucleoside phosphono-diphosphate derivatives (analogues of nucleoside triphosphates) but neither inhibition of human and bacterial DNA polymerases nor polymerase-mediated incorporation of these triphosphate analogues into short DNA was observed.

A fluorescence turn-on detection of copper(II) based on the template-dependent click ligation of oligonucleotides.

In this work, a fluorescence turn-on method for copper(II) detection is reported. A molecular beacon (MB) was designed as a template. Cu(2+) was reduced to Cu(+) in the presence of a reductant (ascorbic acid). Two short single-stranded oligonucleotides one was labeled with a 5'-alkyne and the other with 3'-azide group, proceeded a template-dependent chemical ligation through the Cu(I)-catalyzed azide-alkyne cycloaddition. The newly generated click-ligated long chain oligonucleotide, which was complementary to the MB, opened the MB hairpin structure and resulted in a turn on fluorescence. The increase in fluorescence intensity is directly proportional to the amount of Cu(2+) added to the assay solution. The present assay is quite sensitive and allows the detection of 2 nM Cu(2+). The described assay also exhibits high selectivity over other metal ions.

First synthesis of fully modified 4'-selenoRNA and 2'-OMe-4'-selenoRNA based on the mechanistic considerations of an unexpected strand break.

This study investigated oligonucleotide (ON) synthesis containing 4'-selenoribonucleoside(s) under standard phosphoramidite conditions. Careful operation using a manual ON synthetic system revealed that an unexpected strand break occurred to afford a C2-symmetric homodimer as a byproduct. In addition, this side reaction occurred during I2 oxidation. On the basis of these findings, the first synthesis of fully modified 4'-selenoRNA and 2'-OMe-4'-selenoRNA was achieved using tert-butyl hydroperoxide (TBHP) as the alternative oxidant.

Solid-phase synthesis of P-boronated oligonucleotides by the H-boranophosphonate method.

Recently, P-boronated oligonucleotides have been attracting much attention as potential therapeutic oligonucleotides. In this study, we developed H-boranophosphonate oligonucleotide bearing a borano group and hydrogen atom on the internucleotidic phosphorus and demonstrated that this novel P-boronated oligonucleotide is a versatile precursor to various P-boronated oligonucleotides such as boranophosphate, boranophosphorothioate, and boranophosphoramidate. The method was also applicable to the synthesis of a locked nucleic acid-modified boranophosphate oligonucleotide, which exhibited a dramatically enhanced affinity to complementary oligonucleotides.

Aminosilane functionalizations of mesoporous oxidized silicon for oligonucleotide synthesis and detection.

Direct solid phase synthesis of peptides and oligonucleotides (ONs) requires high chemical stability of the support material. In this work, we have investigated the passivation ability of porous oxidized silicon multilayered structures by two aminosilane compounds, 3-aminopropyltriethoxysilane and 3-aminopropyldimethylethoxysilane (APDMES), for optical label-free ON biosensor fabrication. We have also studied by spectroscopic reflectometry the hybridization between a 13 bases ON, directly grown on the aminosilane modified porous oxidized silicon by in situ synthesis, and its complementary sequence. Even if the results show that both devices are stable to the chemicals (carbonate/methanol) used, the porous silica structure passivated by APDMES reveals higher functionalization degree due to less steric hindrance of pores.

Synthesis of selenomethylene-locked nucleic acid (SeLNA)-modified oligonucleotides by polymerases.

Enzymatic recognition of SeLNA nucleotides was investigated. KOD XL DNA polymerase was found to be an efficient enzyme in primer extension reactions. Polymerase chain reaction (PCR) amplification of SeLNA-modified DNA templates was also efficiently achieved by Phusion and KOD XL DNA polymerases.

Hollow spherical nucleic acids for intracellular gene regulation based upon biocompatible silica shells.

Cellular transfection of nucleic acids is necessary for regulating gene expression through antisense or RNAi pathways. The development of spherical nucleic acids (SNAs, originally gold nanoparticles functionalized with synthetic oligonucleotides) has resulted in a powerful set of constructs that are able to efficiently transfect cells and regulate gene expression without the use of auxiliary cationic cocarriers. The gold core in such structures is primarily used as a template to arrange the nucleic acids into a densely packed and highly oriented form. In this work, we have developed methodology for coating the gold particle with a shell of silica, modifying the silica with a layer of oligonucleotides, and subsequently oxidatively dissolving the gold core with I(2). The resulting hollow silica-based SNAs exhibit cooperative binding behavior with respect to complementary oligonucleotides and cellular uptake properties comparable to their gold-core SNA counterparts. Importantly, they exhibit no cytotoxicity and have been used to effectively silence the eGFP gene in mouse endothelial cells through an antisense approach.

Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2'-C-piperazino-UNA monomer.

Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2'-C-piperazino-UNA residue and UNA residues incorporated in several positions on thermodynamics, kinetics and biological properties of the aptamer. 2'-C-Piperazino-UNA is characterized by more efficient stabilization of quadruplex structure in comparison to regular UNA and increases thermodynamic stability of TBA by 0.28-0.44 kcal/mol in a position depending manner with retained quadruplex topology and molecularity. The presence of UNA-U in positions U3, U7, and U12 results in the highest stabilization of G-quadruplex structure (ΔΔG(37)(°)=-1.03kcal/mol). On the contrary, the largest destabilization mounting to 1.79 kcal/mol was observed when UNA residues were placed in positions U7, G8, and U9. Kinetic studies indicate no strict correlation between thermodynamic stability of modified variants and their binding affinity to thrombin. Most of the studied variants bind thrombin, albeit with decreased affinity in reference to unmodified TBA. Thrombin time assay studies indicate three variants as being as potent as TBA in fibrin clotting inhibition.

Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms.

Oligonucleotides, in which one of the two nonbridging oxygen atoms of internucleotidic phosphates is replaced by a different type of atom or a substituent, are useful as therapeutic agents and probes to elucidate mechanisms of enzymatic reactions. The internucleotidic phosphorus atoms of these oligonucleotides are chiral, and the properties of these oligonucleotides are affected by the absolute configuration of the chiral phosphorus atoms. In order to address the issue of chirality, various methods have been developed to synthesize these P-chiral oligonucleotide analogs in a stereocontrolled manner. This critical review focuses on the recent progress in this field (123 references).

Autonomously pairing cysteinyl-linked nucleotide analogues with a unique architecture.

We report the efficient pairing in water of the first representative of oligonucleotide analogues in which the backbone is replaced by linking elements between the nucleobases. The architecture of the new analogue demonstrates that the structural differentiation of oligonucleotides into a contiguous backbone and nucleobases, as embodied by the natural nucleic acids and all nucleotide analogues analyzed to date, is not a prerequisite for pairing. UV and circular dichroism analyses of self-complementary and non-self-complementary octanucleotide analogues strongly suggest the fully reversible, sequence-specific association of our new analogues to form a left-handed double helix with an antiparallel strand orientation that is characterized by melting temperatures and free enthalpies higher than those of natural RNA and DNA of the same sequence. The linking element incorporates an L-cysteine moiety that allows a short and efficient synthesis of the monomeric building blocks and, through the choice of either L- or D-cysteine, gives access to either one of the enantiomeric oligomers and thus to left- or right-handed helices.

Discovery of novel anti-HIV active G-quadruplex-forming oligonucleotides.

A series of d((5')TGGGAG(3')) sequences, 5'-conjugated with a variety of aromatic groups through phosphodiester linkages, were synthesized, showing CD spectra diagnostic of parallel-stranded, tetramolecular G-quadruplex structures. When tested for anti-HIV-1 and HIV-2 activity, potent inhibition of HIV-1 infection in CEM cell cultures was found, associated with high selectivity index values. Surface Plasmon Resonance assays revealed specific binding to HIV-1 gp120 and gp41.

Simple and rapid enzymatic method for the synthesis of single-strand oligonucleotides containing trifluorothymidine.

To investigate the mechanism of trifluorothymidine (TFT)-induced DNA damage, we developed an enzymatic method for the synthesis of single-strand oligonucleotides containing TFT-monophosphate residues. Sixteen-mer oligonucleotides and 14-mer 5'-phosphorylated oligonucleotides were annealed to the template of 25-mer, so as to empty one nucleotide site. TFT-triphosphate was incorporated into the site by DNA polymerase and then ligated to 5'-phosphorylated oligonucleotides by DNA ligase. The synthesized 31-mer oligonucleotides containing TFT residues were isolated from the 25-mer complementary template by denaturing polyacrylamide electrophoresis. Using these single-strand oligonucleotides containing TFT residues, the cleavage of TFT residues from DNA, using mismatch uracil-DNA glycosylase (MUG) of E.coli origin, was compared with that of 5-fluorouracil (5FU) and 5-bromodeoxyuridine (BrdU). The TFT/A pair was not cleaved by MUG, while the other pairs, namely, 5FU/A, 5FU/G, BrdU/A, BrdU/G, and TFT/G, were easily cleaved from each synthesized DNA. Thus, this method is useful for obtaining some site-specifically modified oligonucleotides.

PRO-051, an antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy.

PRO-051 (GSK-2402968), being developed by GlaxoSmithKline plc, under license from Leiden University Medical Center and Prosensa Therapeutics BV, is a 2'-O-methyl phosphorothioate antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy (DMD). The PRO-051 oligonucleotide sequence induces skipping of exon 51 of the dystrophin gene by binding to a sequence within the dystrophin pre-mRNA and masking the exon inclusion signals that are used for splicing. Removal of exon 51 from an exon 45 to 50, 47 to 50, 48 to 50, 49 to 50, 50, 52 or 52 to 63 deleted transcript allows restoration of the open reading frame and synthesis of an internally truncated, semi-functional dystrophin protein. By targeting exon 51, approximately 13% of patients with DMD could be treated, the largest proportion of patients that could benefit from targeting a single dystrophin exon. A proof-of-concept clinical trial of PRO-051 in patients with DMD demonstrated that a single intramuscular administration of PRO-051 induced exon skipping within muscle fibers adjacent to the injection site, while biopsies revealed dystrophin expression in treated but not control muscle fibers. At the time of publication, a phase I/IIa trial to evaluate subcutaneous delivery of PRO-051 had been completed, although full results were yet to be published.

Synthesis and crystallographic analysis of 5-Se-thymidine DNAs.

We investigated the possibility of the interaction of 5-CH(3) of thymidine and its 5'-phosphate backbone (C-H...O(-)-PO(3) interaction) in DNA via the insertion of the atomic probe (a selenium atom) into the exo-5-position of thymidine (5-Se-T). 5-Se-T was synthesized for the first time, via Mn(OAc)(3) assisted electrophilic addition of CH(3)SeSeCH(3) to 3',5'-di-O-benzoyl-2'-deoxyuridine. The 5-Se-T phosphoramidite was subsequently synthesized and incorporated into DNA in over 99% coupling yield. Biophysical and structural investigations of the 5-Se-T DNAs revealed that the Se-modified and nonmodified DNAs are virtually identical. In addition, the crystallographic analysis of a 5-Se-T DNA strongly suggests a hydrogen-bond formation between the 5-CH(3) and 5'-phosphate groups (CH(3)...PO(4)(-) interaction).

Selectivity of arginine chromatography in promoting different interactions using synthetic oligonucleotides as model.

Arginine has been effectively used in several chromatography methodologies to improve recovery, resolution, and to suppress aggregation. Recently, arginine chromatography was used to fully separate supercoiled and open circular plasmid DNA isoforms. The specific recognition of supercoiled plasmid isoform by arginine was hypothesised to be due to the ability of arginine matrix to be involved in complex interactions that are partly dependent on the conformation of the DNA molecule. In light of these considerations a study was conducted to understand the several interactions that a DNA molecule can promote with the arginine support, in accordance with the chromatographic conditions established. Consequently, knowing the ideal conditions to promote the specific interactions, it could be possible to perform a more targeted and efficient purification. This work describes the chromatography of oligonucleotides with sizes up to 30 bases on the arginine-agarose gel. The effect of several conditions like hydrophobic character of the individual bases, molecular mass of the oligonucleotides, presence of secondary structures, temperature and elution buffer composition (salt and arginine supplemented buffer) was investigated. According to previous atomic data referent to possible interactions between amino acids and DNA nucleotides, arginine can preferentially interact with guanine by hydrogen bond, but other interactions (ionic interactions, van der Waals contacts, water mediated bonds) may also be present and become dominant depending on the conditions used. The results also revealed that the application of arginine in the elution buffer led to an effective elution of oligonucleotides from the arginine chromatographic support by a competition strategy. In general, it was suggested that the affinity interaction promoted by the arginine support is responsible for the specific recognition of particular oligonucleotide bases, involving multiple interactions.

Membrane-anchored DNA assembly for energy and electron transfer.

In this work we examine the trapping and conversion of visible light energy into chemical energy using a supramolecular assembly. The assembly consists of a light-absorbing antenna and a porphyrin redox center, which are covalently attached to two complementary 14-mer DNA strands, hybridized to form a double helix and anchored to a lipid membrane. The excitation energy is finally trapped in the lipid phase of the membrane as a benzoquinone radical anion that could potentially be used in subsequent chemical reactions. In addition, in this model complex, the hydrophobic porphyrin moiety acts as an anchor into the liposome positioning the DNA construct on the lipid membrane surface. The results show the suitability of our system as a prototype for DNA-based light-harvesting devices, in which energy transfer from the aqueous phase to the interior of the lipid membrane is followed by charge separation.