Efficient Cleavage of pUC19 DNA by Tetraaminonaphthols.

In an attempt to create models of phosphodiesterases, we previously investigated bis(guanidinium) naphthols. Such metal-free anion receptors cleaved aryl phosphates and also plasmid DNA. Observed reaction rates, however, could not compete with those of highly reactive metal complexes.

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.

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.

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.

Site-Specific Cleavage of RNAs Derived from the 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 mRNA reacted with rates and specificities comparable to those of short oligonucleotide substrates.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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".

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.

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.

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.