smFISH for Plants.

Single-molecule fluorescence in situ hybridization (smFISH) is a powerful method for the visualization and quantification of individual RNA molecules within intact cells. With its ability to probe gene expression at the single cell and single-molecule level, the technique offers valuable insights into cellular processes and cell-to-cell heterogeneity. Although widely used in the animal field, its use in plants has been limited. Here, we present an experimental smFISH workflow that allows researchers to overcome hybridization and imaging challenges in plants, including sample preparation, probe hybridization, and signal detection. Overall, this protocol holds great promise for unraveling the intricacies of gene expression regulation and RNA dynamics at the single-molecule level in whole plants.

Single-Molecule Sandwich Aptasensing on Nanoarrays by Tethered Particle Motion Analysis.

High-throughput single-molecule techniques are expected to challenge the demand for rapid, simple, and sensitive detection methods in health and environmental fields. Based on a single-DNA-molecule biochip for the parallelization of tethered particle motion analyses by videomicroscopy coupled to image analysis and its smart combination with aptamers, we successfully developed an aptasensor enabling the detection of single target molecules by a sandwich assay. One aptamer is grafted to the nanoparticles tethered to the surface by a long DNA molecule bearing the second aptamer in its middle. The detection and quantification of the target are direct. The recognition of the target by a pair of aptamers leads to a looped configuration of the DNA-particle complex associated with a restricted motion of the particles, which is monitored in real time. An analytical range extending over 3 orders of magnitude of target concentration with a limit of detection in the picomolar range was obtained for thrombin.

Convertible and Constrained Nucleotides: The 2'-Deoxyribose 5'-C-Functionalization Approach, a French Touch.

Many strategies have been developed to modulate the biological or biotechnical properties of oligonucleotides by introducing new chemical functionalities or by enhancing their affinity and specificity while restricting their conformational space. Among them, we review our approach consisting of modifications of the 5'-C-position of the nucleoside sugar. This allows the introduction of an additional chemical handle at any position on the nucleotide chain without disturbing the Watson-Crick base-pairing. We show that 5'-C bromo or propargyl convertible nucleotides (CvN) are accessible in pure diastereoisomeric form, either for nucleophilic displacement or for CuAAC conjugation. Alternatively, the 5'-carbon can be connected in a stereo-controlled manner to the phosphate moiety of the nucleotide chain to generate conformationally constrained nucleotides (CNA). These allow the precise control of the sugar/phosphate backbone torsional angles. The consequent modulation of the nucleic acid shape induces outstanding stabilization properties of duplex or hairpin structures in accordance with the preorganization concept. Some biological applications of these distorted oligonucleotides are also described. Effectively, the convertible and the constrained approaches have been merged to create constrained and convertible nucleotides (C2NA) providing unique tools to functionalize and stabilize nucleic acids.

Convertible and conformationally constrained nucleic acids (C2NAs).

We introduce the concept of Convertible and Constrained Nucleic Acids (C2NAs). By means of the synthesis of a stereocontrolled N-propargyl dioxo-1,3,2-oxaza-phosphorinane as an internucleotidic linkage, the torsional angles α and ß can adopt either the canonical (g-, t) set of values able to increase DNA duplex stability or the non-canonical (g+, t) set that stabilized the hairpin structure when installed within the loop moiety. With an appended propargyl function on the nitrogen atom of the six-membered ring, the copper catalysed Huisgen's cycloaddition (CuAAC click chemistry) allows for the introduction of new functionalities at any location on the nucleic acid chain while maintaining the properties brought by the geometrical constraint and the neutral internucleotidic linkage.

DNA Three Way Junction Core Decorated with Amino Acids-Like Residues-Synthesis and Characterization.

Construction and physico-chemical behavior of DNA three way junction (3WJ) functionalized by protein-like residues (imidazole, alcohol and carboxylic acid) at unpaired positions at the core is described. One 5'-C(S)-propargyl-thymidine nucleotide was specifically incorporated on each strand to react through a post synthetic CuACC reaction with either protected imidazolyl-, hydroxyl- or carboxyl-azide. Structural impacts of 5'-C(S)-functionalization were investigated to evaluate how 3WJ flexibility/stability is affected.

Differential effects on allele selective silencing of mutant huntingtin by two stereoisomers of α,ß-constrained nucleic acid.

We describe the effects of introducing two epimers of neutral backbone α,ß-constrained nucleic acid (CNA) on the activity and allele selectivity profile of RNase H active antisense oligonucleotides (ASOs) targeting a single nucleotide polymorphism (SNP) for the treatment of Huntington's disease (HD). ASOs modified with both isomers of α,ß-CNA in the gap region showed good activity versus the mutant allele, but one isomer showed improved selectivity versus the wild-type allele. Analysis of the human RNase H cleavage patterns of α,ß-CNA modified ASOs versus matched and mismatched RNA revealed that both isomers support RNase H cleavage on the RNA strand across from the site of incorporation in the ASO--an unusual observation for a neutral linkage oligonucleotide modification. Interestingly, ASOs modified with (R)- and (S)-5'-hydroxyethyl DNA (RHE and SHE respectively) formed by partial hydrolysis of the dioxaphosphorinane ring system in α,ß-CNA also showed good activity versus the mutant allele but an improved selectivity profile was observed for the RHE modified ASO. Our observations further support the profiling of neutral and 5'-modified nucleic acid analogs as tools for gene silencing applications.

Pd(0)-Catalyzed methyl transfer on nucleosides and oligonucleotides, envisaged as a PET tracer.

The methyl transfer reaction from activated monomethyltin, via a modified Stille coupling reaction, was studied under "ligandless" conditions on fully deprotected 5'-modified nucleosides and one dinucleotide. The reaction was optimized to proceed in a few minutes and quantitative yield, even under dilute conditions, thus affording a rapid and efficient new method for oligonucleotide labelling with carbon-11.

Oligonucleotide labelling using a fluorogenic "click" reaction with a hemicarboxonium salt.

Two fluorescent streptocyanine labelled oligonucleotides have been synthesized by a simple "click" reaction between a non-fluorescent hemicarboxonium salt and aminoalkyl functionalized thymidines within the oligonucleotide and their spectrophotometric properties have been studied.

Dioxaphosphorinane-constrained nucleic Acid dinucleotides as tools for structural tuning of nucleic acids.

We describe a rational approach devoted to modulate the sugar-phosphate backbone geometry of nucleic acids. Constraints were generated by connecting one oxygen of the phosphate group to a carbon of the sugar moiety. The so-called dioxaphosphorinane rings were introduced at key positions along the sugar-phosphate backbone allowing the control of the six-torsion angles α to ζ defining the polymer structure. The syntheses of all the members of the D-CNA family are described, and we emphasize the effect on secondary structure stabilization of a couple of diastereoisomers of α,ß-D-CNA exhibiting wether B-type canonical values or not.

α,ß-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction.

(S(C5'), R(P)) α,ß-D- Constrained Nucleic Acids (CNA) are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.

Silicon-based chemistry: an original and efficient one-step approach to [18F]-nucleosides and [18F]-oligonucleotides for PET imaging.

Take it eaSi! Nucleosides, dinucleotides, and one oligonucleotide, all modified by click chemistry, have for the first time been directly and very efficiently labeled with (18)F by using a silicon-based, one-step approach that opens the way for the development of a new class of positron emission tomography (PET) tracers (see graphic).

Alpha,beta-D-CNA preorganization of unpaired loop moiety stabilizes DNA hairpin.

DNA hairpin structures can be stabilized by introduction of constraint on the sugar phosphate backbone within the unpaired loop moiety by means of dinucleotides locked with an alpha torsion angle in gauche(+) configuration.

Synthesis and luminescence properties of a trinucleotide-europium(III) complex conjugate.

Two trinucleotide conjugates of the macrocyclic ligand 1,4,7-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane are prepared. One contains only DNA (1) and the second is a chimeric RNA/DNA conjugate (2). The synthetic methodology used to prepare the trinucleotide macrocyclic ligand conjugates is based on the introduction of a convertible nucleoside which has an electrophilic function to facilitate the attachment of any nucleophilic ligand to the 5-position of the 3-nucleoside unit. The convertible nucleoside is first treated with the macrocyclic ligand, 1,4,7,10-tetraazacyclododecane, followed by alkylation of the three remaining amine groups to give a conjugated macrocyclic ligand with three pendent amide groups. Addition of an equivalent of EuCl3 to trinucleotide (1) or (2) yields the complexes Eu(1) and Eu(2), respectively. Studies using time-resolved and steady state direct excitation luminescence spectroscopy show that Eu(III) binds to the macrocyclic moiety in 1 and in 2. The excitation peak frequency for the 7Fo5Do transition and the unexpectedly low number of water ligands in Eu(1) and Eu(2) are consistent with additional interactions of the Eu(III) macrocycle with one of the phosphate diester groups. Studies show that Eu(2) undergoes cleavage at the uridine nucleotide. The unique point of attachment of the macrocyclic complex will enable the preparation of new lanthanide nucleic acid conjugates with useful properties.

Alpha,beta-D-CNA induced rigidity within oligonucleotides.

Introduction of alpha,beta-D-CNA featuring canonical values of the torsional angles alpha and beta within oligonucleotides leads to an overall stabilization and improved rigidity of the duplex DNA as demonstrated by UV experiments, circular dichroism and corroborated by molecular dynamics simulations.

Constrained nucleic acids (CNA). Part 2. Synthesis of conformationally restricted dinucleotide units featuring noncanonical alpha/beta/gamma or delta/epsilon/zeta torsion angle combinations.

Four dinucleotide building units of nucleic acids in which three out of six backbone torsion angles are included in a dioxaphosphorinane ring structure (D-CNA family) have been synthesized: two diastereoisomeric alpha,beta,gamma-D-CNA {cis and trans} and two diastereoisomeric delta,epsilon,zeta-D-CNA {(S(C4'),R(P)) and (S(C4'),S(P))}. The structural analysis of these conformationally restricted dinucleotides, using NMR spectroscopy and X-ray crystallography, shows that these D-CNA structural elements allow the stabilization of torsion angle combinations, alpha/beta/gamma and delta/epsilon/zeta, that are significantly different from those typically observed in canonical A- or B-form duplexes.

Diastereoselective synthesis of a conformationally restricted dinucleotide with predefined alpha and beta torsional angles for the construction of alpha,beta-constrained nucleic acids (alpha,beta-CNA).

[reaction: see text] The synthesis of a diastereopure 1,3,2-dioxaphosphorinane linkage in which two, alpha and beta, out of six torsion angles of the natural phosphodiester backbone are constrained with predefined values of ca. +60 degrees (g(+)) and 180 degrees (t), respectively, is described. The stable and unstrained six-membered cyclic phosphotriester structure represents the smallest possible ring allowing the conformational locking of the alpha torsion angle at a significant positive value that is typical of many bulged regions of nucleic acids.

Total Synthesis of a Thymidine 2-Deoxypolyoxin C Analogue.

The synthesis of the thymidine 2-deoxypolyoxin C analogue 10 from a noncarbohydrate precursor was achieved in 10 steps and 9% yield starting from a chiral gamma,delta-epoxy-beta-hydroxy ester 11 readily available from cis-2-butene-1,4-diol. The main steps concern the stereo- and regioselective opening of the epoxide ring by an azide anion, the stereoselective introduction of the thymine base, and the transformation of the primary alcohol to the acid functionality of the final product. Two other approaches have also been investigated.