Spin-Labeled Riboswitch Synthesized from a Protected TPA Phosphoramidite Building Block.

The nitroxide TPA (2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene) is an excellent spin label for EPR studies of RNA. Previous synthetic methods, however, are complicated and require special equipment. Herein, we describe a uridine derived phosphoramidite with a photocaged TPA unit attached. The light sensitive 2-nitrobenzyloxymethyl group can be removed in high yield by short irradiation at 365 nm. Based on this approach, a doubly spin-labeled 27mer neomycin sensing riboswitch was synthesized and studied by PELDOR. The overall thermal stability of the fold is not much reduced by TPA. In-line probing nevertheless detected changes in local mobility.

Exploring the Druggability of Conserved RNA Regulatory Elements in the SARS-CoV-2 Genome.

SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.

Redirection of miRNA-Argonaute Complexes to Specific Target Sites by Synthetic Adaptor Molecules.

Dysregulation of miRNAs is connected with a multitude of diseases for which antagomirs and miRNA replacement are discussed as therapeutic options. Here, we suggest an alternative concept based on the redirection of RISCs to non-native target sites. Metabolically stable DNA-LNA mixmers are used to mediate the binding of RISCs to mRNAs without any direct base complementarity to the presented guide RNA strand. Physical redirection of a dye-labeled miRNA model and of specific miRNA-programmed RISC fractions present in HeLa extracts is demonstrated by pull-down experiments with biotinylated capture oligonucleotides.

Site-Specific Cleavage of RNAs Derived from the PIM1 3'-UTR by a Metal-Free Artificial Ribonuclease.

Oligonucleotide conjugates of tris(2-aminobenzimidazole) have been reported previously to cleave complementary RNA strands with high levels of sequence and site specificity. The RNA substrates used in these studies were oligonucleotides not longer than 29-mers. Here we show that ~150⁻400-mer model transcripts derived from the 3'-untranslated region of the PIM1 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates. The replacement of DNA by DNA/LNA mixmers further increased the cleavage rate. Tris(2-aminobenzimidazoles) were designed to interact with phosphates and phosphate esters. A cell, however, contains large amounts of phosphorylated species that may cause competitive inhibition of RNA cleavage. It is thus important to note that no loss in reaction rates was observed in phosphate buffer. This opens the way to in-cell applications for this type of artificial nuclease. Furthermore, we disclose a new synthetic method giving access to tris(2-aminobenzimidazoles) in multigram amounts.

A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full-Length TAR RNA and Investigation by In-Line Probing and EPR Spectroscopy.

EPR studies on RNA are complicated by three major obstacles related to the chemical nature of nitroxide spin labels: Decomposition while oligonucleotides are chemically synthesized, further decay during enzymatic strand ligation, and undetected changes in conformational equilibria due to the steric demand of the label. Herein possible solutions for all three problems are presented: A 2-nitrobenzyloxymethyl protective group for nitroxides that is stable under all conditions of chemical RNA synthesis and can be removed photochemically. By careful selection of ligation sites and splint oligonucleotides, high yields were achieved in the assembly of a full-length HIV-1 TAR RNA labeled with two protected nitroxide groups. PELDOR measurements on spin-labeled TAR in the absence and presence of arginine amide indicated arrest of interhelical motions on ligand binding. Finally, even minor changes in conformation due to the presence of spin labels are detected with high sensitivity by in-line probing.

Phosphoramidite building blocks with protected nitroxides for the synthesis of spin-labeled DNA and RNA.

TEMPO spin labels protected with 2-nitrobenzyloxymethyl groups were attached to the amino residues of three different nucleosides: deoxycytidine, deoxyadenosine, and adenosine. The corresponding phosphoramidites could be incorporated by unmodified standard procedures into four different self-complementary DNA and two RNA oligonucleotides. After photochemical removal of the protective group, elimination of formic aldehyde and spontaneous air oxidation, the nitroxide radicals were regenerated in high yield. The resulting spin-labeled palindromic duplexes could be directly investigated by PELDOR spectroscopy without further purification steps. Spin-spin distances measured by PELDOR correspond well to the values obtained from molecular models.

A Bis(guanidinium)alcohol Attached to a Hairpin Polyamide: Synthesis, DNA Binding, and Plasmid Cleavage.

Bis(guanidinium)alcohols have been designed to react with phosphodiester substrates in a fast transphosphorylation step, a quasi-intramolecular process taking place in contact ion pairs. Here the attachment of such compounds to Dervan-type hairpin polyamides is described. The resulting conjugate 1 binds to AT-rich DNA duplexes with affinity similar to that of the parent polyamide as shown by UV melting experiments and CD titrations. Conjugate 1 nicks plasmid DNA at concentrations ranging from micromolar to high nanomolar.

Fragment based search for small molecule inhibitors of HIV-1 Tat-TAR.

Basic molecular building blocks such as benzene rings, amidines, guanidines, and amino groups have been combined in a systematic way to generate ligand candidates for HIV-1 TAR RNA. Ranking of the resulting compounds was achieved in a fluorimetric Tat-TAR competition assay. Although simple molecules such as phenylguanidine are inactive, few iteration steps led to a set of ligands with IC50 values ranging from 40 to 150 µM. 1,7-Diaminoisoquinoline 17 and 2,4,6-triaminoquinazoline 22 have been further characterized by NMR titrations with TAR RNA. Compound 22 is bound to TAR at two high affinity sites and shows slow exchange between the free ligand and the RNA complex. These results encourage investigations of dimeric ligands built from two copies of compound 22 or related heterocycles.

Cleavage of phosphodiesters and of DNA by a bis(guanidinium)naphthol acting as a metal-free anion receptor.

Phosphoric acid diesters form anions at neutral pH. As a result of charge repulsion they are notoriously resistant to hydrolysis. Nucleophilic attack, however, can be promoted by different types of electrophilic catalysts that bind to the anions and reduce their negative charge density. Although in most cases phosphodiester-cleaving enzymes and synthetic catalysts rely on Lewis acidic metal ions, some exploit the guanidinium residues of arginine as metal-free electrophiles. Here we report that a combination of two guanidines and a hydroxy group yields highly reactive receptor molecules that can attack a broad range of phosphodiester substrates by nucleophilic displacement at phosphorus in a single-turnover mode. Some stable O-phosphates were isolated and characterized further by NMR spectroscopy. The bis(guanidinium)naphthols also cleave plasmid DNA, presumably by a transphosphorylation mechanism.

Binding sites of the viral RNA element TAR and of TAR mutants for various peptide ligands, probed with LILBID: a new laser mass spectrometry.

A new laser-based mass spectrometry method, called laser induced liquid bead ion desorption (LILBID), was applied to investigate RNA:ligand interactions. As model system the HIV Tat:TAR transactivation complex and its binding behavior were analyzed. TARwt of HIV Type 1 and Type 2 and different artificial mutants were compared regarding their binding to Tat and different peptide ligands. Specific and nonspecific association to TAR was deduced, with the bulge being the well known specific binding site of TAR. In the case of triple arginine (RRR) as a nonspecific ligand, multiple electrostatic binding to TAR was found at higher concentration of RRR. This contrasted with the formation of only ternary complexes in competitive binding studies with TAR, Tat, and potential inhibitors. The fact that the stoichiometries of the complexes can be determined is a major advantage of MS methods over the widely applied fluorimetric methods. A quantitative evaluation of the spectra by a numeric model for ternary complex formation allows conclusions about the role and strength of the binding sites of the RNAs, the specificity and affinity of different ligands, the determination of apparent IC50 and KD values, and a comparison of the binding efficiencies of potential inhibitors.

Fluorescence-based on-line detection as an analytical tool in RNA electrophoresis.

In this chapter, we present methods for denaturing and native gel electrophoresis of nucleic acids based on fluorescently labeled probes and automatic signal detection by a deoxyribonucleic acid sequencer. Specific examples are given for the determination of ribonucleic acid (RNA) fragmentation patterns, of (deoxy)ribozyme kinetics, and of direct or competitive gel-shift assays. These methods can replace widely used radioisotope-based protocols, for example, for secondary structure mapping of RNA and for the characterization of nucleic acid ligand interactions.

The concept of template-based de novo design from drug-derived molecular fragments and its application to TAR RNA.

Principles of fragment-based molecular design are presented and discussed in the context of de novo drug design. The underlying idea is to dissect known drug molecules in fragments by straightforward pseudo-retro-synthesis. The resulting building blocks are then used for automated assembly of new molecules. A particular question has been whether this approach is actually able to perform scaffold-hopping. A prospective case study illustrates the usefulness of fragment-based de novo design for finding new scaffolds. We were able to identify a novel ligand disrupting the interaction between the Tat peptide and TAR RNA, which is part of the human immunodeficiency virus (HIV-1) mRNA. Using a single template structure (acetylpromazine) as reference molecule and a topological pharmacophore descriptor (CATS), new chemotypes were automatically generated by our de novo design software Flux. Flux features an evolutionary algorithm for fragment-based compound assembly and optimization. Pharmacophore superimposition and docking into the target RNA suggest perfect matching between the template molecule and the designed compound. Chemical synthesis was straightforward, and bioactivity of the designed molecule was confirmed in a FRET assay. This study demonstrates the practicability of de novo design to generating RNA ligands containing novel molecular scaffolds.

Fluorescence correlation spectroscopy at single molecule level on the Tat-TAR complex and its inhibitors.

The TAR element of HIV and the viral protein Tat form a molecular switch regulating transcriptional efficiency in HIV. We show that fluorescence correlation spectroscopy at the single molecule level is a powerful method to study the association between a Tat-derived peptide and TAR fragments. We also investigated the inhibition of the peptide-RNA complex by different ligands. Utilizing cross correlation measurements, the dissociation constants (K(D)) were determined. To demonstrate the important role of the bulge for the binding of Tat, we compared wt-TAR with three RNA mutants, mainly differing in the bulge region. For the TAR mutants studied at equimolar concentration of RNA and peptide (25 nM), the K(D) values are 15-35 times larger than that of wt-TAR. This gives evidence that the bulge region is the most crucial part of the TAR RNA for specific Tat binding. The IC(50) values for different inhibitors of the Tat/TAR complex both with wt-TAR and mutants have been determined. Neamine conjugate proved to be the best inhibitor of the complex formation. Our results are in agreement with earlier published data on this system using alternative biophysical and biochemical methods, respectively.

Tripeptides from synthetic amino acids block the Tat-TAR association and slow down HIV spread in cell cultures.

Non-natural amino acids with aromatic or heteroaromatic side chains were incorporated into tripeptides of the general structure Arg-X-Arg and tested as ligands of the HIV RNA element TAR. Some of these compounds could compete efficiently with the association of TAR and Tat and downregulated a TAR-controlled reporter gene in HeLa cells. Peptide 7, which contains a 2-pyrimidinyl-alkyl chain, also inhibited the spread of HIV-1 in cell cultures. NMR studies of 7 bound to HIV-2-TAR gave evidence for contacts in the bulge region.

Site-specific cleavage of RNA by a metal-free artificial nuclease attached to antisense oligonucleotides.

RNA cleaving tris(2-aminobenzimidazoles) have been attached to DNA oligonucleotides via disulfide or amide bonds. The resulting conjugates are effective organocatalytic nucleases showing substrate and site selectivity as well as saturation kinetics. The benzimidazole conjugates also degrade enantiomeric RNA. This observation rules out contamination effects as an alternative explanation of RNA degradation. The pH dependency shows that the catalyst is most active in the deprotonated state. Typical half-lifes of RNA substrates are in the range of 12-17 h. Thus, conjugates of tris(2-aminobenzimidazoles) can compete with the majority of metal-dependent artificial nucleases.

Metal-free catalysts for the hydrolysis of RNA derived from guanidines, 2-aminopyridines, and 2-aminobenzimidazoles.

2-aminopyridine and 2-aminobenzimidazole were chosen as structural analogues to substitute guanidinium groups in receptor molecules designed as phosphoryl transfer catalysts. Shifting the pKa of the guanidinium analogues toward 7 was expected to raise catalytic activities in aqueous buffer. Although the pKa's of both heterocycles are similar (6.2 and 7.0), only 2-aminobenzimidazole led to active RNA cleavers. All cleavage assays were run with fluorescently labeled substrates and a DNA sequencer. RNase contaminations would degrade RNA enantioselectively. In contrast, achiral catalysts such as 9b and 10b necessarily induce identical cleavage patterns in RNA and its mirror image. This principle allowed us to safely rule out contamination effects in this study. The most active catalysts, tris(2-aminobenzimidazoles) 9b and 10b, were shown by fluorescence correlation spectroscopy (FCS) to aggregate with oligonucleotides. However, at very low concentrations the compounds are still active in the nonaggregated state. Conjugates of 10b with antisense oligonucleotides or RNA binding peptides, therefore, will be promising candidates as site specific artificial ribonucleases.

New inhibitors of the Tat-TAR RNA interaction found with a "fuzzy" pharmacophore model.

TAR RNA is a potential target for AIDS therapy. Ligand-based virtual screening was performed to retrieve novel scaffolds for RNA-binding molecules capable of inhibiting the Tat-TAR interaction, which is essential for HIV replication. We used a "fuzzy" pharmacophore approach (SQUID) and an alignment-free pharmacophore method (CATS3D) to carry out virtual screening of a vendor database of small molecules and to perform "scaffold-hopping". A small subset of 19 candidate molecules were experimentally tested for TAR RNA binding in a fluorescence resonance energy transfer (FRET) assay. Both methods retrieved molecules that exhibited activities comparable to those of the reference molecules acetylpromazine and chlorpromazine, with the best molecule showing ten times better binding behavior (IC50 = 46 microM). The hits had molecular scaffolds different from those of the reference molecules.

A peptide template as an allosteric supramolecular catalyst for the cleavage of phosphate esters.

The heptapeptide H-Iva-Api-Iva-ATANP-Iva-Api-Iva-NHCH(3) (P1a), where Iva is (S)-isovaline, Api is 4-amino-4-carboxypiperidine, and ATANP is (S)-2-amino-3-[1-(1,4,7-triazacyclononane)]propanoic acid, has been synthesized. Its conformation in aqueous solution is essentially that of a 3(10)-helix. By connecting three copies of P1a to a functionalized Tris(2-aminoethyl)amine (Tren) platform a new peptide template, [T(P1)(3)], was obtained. This molecule is able to bind up to four metal ions (Cu(II) or Zn(II)): one in the Tren subsite and three in the azacyclononane subunits. The binding of the metals to the Tren platform induces a change from an open to a closed conformation in which the three short, helical peptides are aligned in a parallel manner with the azacyclonane units pointing inward within the pseudocavity they define. T(P1)(3) shows a peculiar behavior in the transphosphorylation of phosphate esters; the tetrazinc complex is a catalyst of the cleavage of 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP), whereas the free ligand is a catalyst of the cleavage of an oligomeric RNA sequence with selectivity for pyrimidine bases. In the case of HPNP, Zn(II) acts as a positive allosteric effector by enhancing the catalytic efficiency of the system. In the case of the polyanionic RNA substrate, Zn(II) switches off the activity, thus behaving as a negative allosteric regulator. It is suggested that the opposite behavior of the catalyst induced by Zn(II) is associated with the change of conformation of the Tren platform, and consequently of the relative spatial disposition of the three linked peptides, that occurs after binding of the metal ion.