RNA functionalization strategies and their application to RNA folding dynamics and experimental RNomics

The oversimplified notion of RNA being a mere carrier of sequence information from gene to protein has been repeatedly undermined over the decades by yet another newly discovered function performed by certain RNA species. These new species include in particular RNAs which regulate gene expression in response to a metabolite sensing event. These RNAs — known as riboswitches — elegantly couple metabolite recognition with gene regulation in the apparent absence of protein helpers. Here we first sought to investigate the folding dynamics of the S-adenosyl-L-methionine responsive riboswitch by FRET spectroscopy. This requires the synthesis of full-length riboswitch constructs site-specifically modified with multiple fluorophores. For this challenging task we have established a 5-way splinted-ligation strategy to prepare dual-fluorophore labelled full-length riboswitch constructs in an unprecedented overall yield of 10 %. These constructs have further been subjected to bulk and single molecule FRET spectroscopy for ligand induced folding analysis. We confirmed similar folding dynamics for the aptamer of the complete riboswitch (aptamer + expression platform) as reported earlier for constructs containing the aptamer alone. However, we also observed a few other folding phenomena induced by a chemically slightly different, yet non-cognate metabolite, which cannot be explained by any facts known about this riboswitch to date and require further experiments to reach a final conclusion. During the course of the aforesaid work, we realized the limitations of existing nucleic acid functionalization strategies. Therefore we decided to use bioorthogonal click reactions as part of our labelling strategy. Among various different click reactions, we first had to find the one which best suits our purpose and to optimize its conditions. Having the optimized click reaction conditions at hand, we developed enzymatic strategies to site-specifically functionalize long RNAs with clickable residues. In our nucleic acid labelling strategy a diverse array of different chemical functionalities can be introduced exploiting the modular nature of click chemistry. This does not demand either de novo synthesis or optimizations of enzymatic reaction conditions for each new single compound. Furthermore, we developed a chemical approach using two different mutually orthogonal click reactions for concurrent, site-specific labelling of DNA molecules with multiple fluorophores. Moreover, we sought to extend this strategy of enzymatic, site-specific transfer of clickable residues to long RNAs towards photochemical transfer of clickable moieties to a target RNA in a mixture of many unrelated sequences. This technique, which we call Affinity-based Chemical RNomics is a chemical approach in experimental RNomics whereby RNA sequences which bind to a given small-molecule metabolite are to be isolated from a total RNA isolate of any organism just by the virtue of its tight binding to its cognate metabolite and without any prior knowledge of its sequence. This method would therefore allow for the discovery of previously unknown riboswitches, currently the only known kind of natural RNA that binds small-molecule metabolites. Since all currently known riboswitches have been discovered by rational approaches, this will considerably extend the chances of discovering new riboswitches

Exploring the energy landscape of a SAM-I riboswitch

SAM-I riboswitches regulate gene expression through transcription termination upon binding a S-adenosyl-L-methionine (SAM) ligand. In previous work, we characterized the conformational energy landscape of the full-length Bacillus subtilis yitJ SAM-I riboswitch as a function of Mg$^{2+}$ and SAM ligand concentrations. Here, we have extended this work with measurements on a structurally similar ligand, S-adenosyl-L-homocysteine (SAH), which has, however, a much lower binding affinity. Using single-molecule Förster resonance energy transfer (smFRET) microscopy and hidden Markov modeling (HMM) analysis, we identified major conformations and determined their fractional populations and dynamics. At high Mg$^{2+}$ concentration, FRET analysis yielded four distinct conformations, which we assigned to two terminator and two antiterminator states. In the same solvent, but with SAM added at saturating concentrations, four states persisted, although their populations, lifetimes and interconversion dynamics changed. In the presence of SAH instead of SAM, HMM revealed again four well-populated states and, in addition, a weakly populated ‘hub’ state that appears to mediate conformational transitions between three of the other states. Our data show pronounced and specific effects of the SAM and SAH ligands on the RNA conformational energy landscape. Interestingly, both SAM and SAH shifted the fractional populations toward terminator folds, but only gradually, so the effect cannot explain the switching action. Instead, we propose that the noticeably accelerated dynamics of interconversion between terminator and antiterminator states upon SAM binding may be essential for control of transcription

Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 3′-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2′-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2′-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules

MULTIPLE ORTHOGONAL LABELLING OF OLGONUCLEOTIDES

In Summary, the present invention concerns a method for multiple orthogonal labelling of oligonucleotides, preferably RNA or DNA, by simultaneously performing the inverse Diels-Alder reaction (DAinv) and the copper-catalyzed click reaction (CuAAC), wherein the method is employed in a single step by just adding the different reaction components together and incubating the aqueous reaction mixture prefer ably for one hour at room temperature. In detail, the reaction components are one or more N-modified labels, a copper compound, a stabilizing ligand, a reducing agent and one or more electron-deficient label-modified dienes that are added together with an at least double-modified oligonucleotide having one more nucleotides containing one or more N3-re active groups and one or more electron-rich dienophiles, wherein a terminal alkyne moiety is preferably used as N3-re active group(s) and afrans-cyclooctene moiety or norbornene is preferably used as electron-rich dienophile(s), more pref erably frans-cyclooctene. Therefore, the present invention provides a one-pot method for post-synthetic multiple orthogonal labeling of oligonucleotides, which allows the site-specific introduction of more than one label, preferably of at least two labels into oligonucleotides after solid-phase synthesis, wherein the DAinv takes place on the dienophile modification only and the CuAAC selectively takes place on the N3-reactive group modification

MULTIPLE ORTHOGONAL LABELLING OF OLGONUCLEOTIDES

In Summary, the present invention concerns a method for multiple orthogonal labelling of oligonucleotides, preferably RNA or DNA, by simultaneously performing the inverse Diels-Alder reaction (DAinv) and the copper-catalyzed click reaction (CuAAC), wherein the method is employed in a single step by just adding the different reaction components together and incubating the aqueous reaction mixture prefer ably for one hour at room temperature. In detail, the reaction components are one or more N-modified labels, a copper compound, a stabilizing ligand, a reducing agent and one or more electron-deficient label-modified dienes that are added together with an at least double-modified oligonucleotide having one more nucleotides containing one or more N3-re active groups and one or more electron-rich dienophiles, wherein a terminal alkyne moiety is preferably used as N3-re active group(s) and afrans-cyclooctene moiety or norbornene is preferably used as electron-rich dienophile(s), more pref erably frans-cyclooctene. Therefore, the present invention provides a one-pot method for post-synthetic multiple orthogonal labeling of oligonucleotides, which allows the site-specific introduction of more than one label, preferably of at least two labels into oligonucleotides after solid-phase synthesis, wherein the DAinv takes place on the dienophile modification only and the CuAAC selectively takes place on the N3-reactive group modification

Hydroxyapatite particle shape affects screw attachment in cancellous bone when augmented with hydroxyapatite-containing hydrogels

Screw-bone construct failures are a true challenge in orthopaedic implant fixation, particularly in poor quality bone. Whilst augmentation with polymeric or ceramic bone cement can improve the primary stability of the screws, the cement may block the flow of blood and nutrients and hamper bone remodelling. In this study, soft, non-setting biomaterials based on Hyalectin gels and hydroxyapatite (HA) particles with different morphological parameters were evaluated as potential augmentation materials, using a lapine ex vivo bone model. The pull-out force, stiffness, and work to fracture were considered in evaluating screw attachment. The pull-out force of constructs reinforced with Hyalectin containing irregularly shaped nano-HA and spherically shaped micro-HA particles were found to be significantly higher than the control group (no augmentation material). The pull-out stiffness increased for the micro-HA particles and the work to fracture increased for the irregular nano-HA particles. However, there were no significant augmentation effect found for the spherical shaped nano-HA particles. In conclusion, injectable Hyalectin gel loaded with hydroxyapatite particles was found to have a potentially positive effect on the primary stability of screws in trabecular bone, depending on the HA particle shape and size