New Benchmark in DNA-Based Asymmetric Catalysis: Prevalence of Modified DNA/RNA Hybrid Systems.

By harnessing the chirality of the DNA double helix, chemists have been able to obtain new, reliable, selective, and environmentally friendly biohybrid catalytic systems with tailor-made functions. Nonetheless, despite all the advances made throughout the years in the field of DNA-based asymmetric catalysis, many challenges still remain to be faced, in particular when it comes to designing a "universal" catalyst with broad reactivity and unprecedented selectivity. Rational design and rounds of selection have allowed us to approach this goal.

Pegylation of RNA Spiegelmers by a Novel Widely Applicable Two-Step Process for the Conjugation of Carboxylic Acids to Amino-Modified Oligonucleotides.

The reaction between N-hydroxy succinimide (NHS) ester-activated carboxylic acids and amino-modified nucleic acids is commonly used for the post-synthetic modification of oligonucleotides. Here, we report a two-step variation of the method in which the NHS ester is replaced by the corresponding parent carboxylic acid. In the first step, the carboxylic acid is activated with a standard peptide coupling reagent like HBTU in an anhydrous water-miscible aprotic organic solvent.

Novel Tumor Pretargeting System Based on Complementary l-Configured Oligonucleotides.

Unnatural mirror image l-configured oligonucleotides (L-ONs) are a convenient substance class for the application as complementary in vivo recognition system between a tumor specific antibody and a smaller radiolabeled effector molecule in pretargeting approaches. The high hybridization velocity and defined melting conditions are excellent preconditions of the L-ON based methodology. Their high metabolic stability and negligible unspecific binding to endogenous targets are superior characteristics in comparison to their d-configured analogs.

Expanding biohybrid-mediated asymmetric catalysis into the realm of RNA.

The recent development of biohybrid catalytic systems has allowed synthetic chemists to reach high levels of selectivity on a wide variety of valuable synthetic transformations. In this context, DNA-based catalysts have emerged as particularly appealing tools. Interestingly, while long RNA sequences (ribozymes) are known to catalyse specific biochemical reactions with remarkable efficiencies, RNA-based catalysts involving a catalytically active metal complex interacting in a non-covalent fashion with short sequences have never been evaluated to date.

SDF-1 inhibition targets the bone marrow niche for cancer therapy.

Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l-oligonucleotide (olaptesed-pegol).

DNA vs. mirror-image DNA: a universal approach to tune the absolute configuration in DNA-based asymmetric catalysis.

Mirror mirror on the wall: By taking advantage of the unique structural features of L-DNA, the first examples of left-helical enantioselective induction in the field of DNA-based asymmetric catalysis were realized. Most importantly, this approach is the only one that allows a reliable and predictable access to both enantiomers for any given reaction.

Methods for L-ribooligonucleotide sequence determination using LCMS.

The ability to verify the sequence of a nucleic acid-based therapeutic is an essential step in the drug development process. The challenge associated with sequence identification increases with the length and nuclease resistance of the nucleic acid molecule, the latter being an important attribute of therapeutic oligonucleotides. We describe methods for the sequence determination of Spiegelmers, which are enantiomers of naturally occurring RNA with high resistance to enzymatic degradation.

RNA aptamers and spiegelmers: synthesis, purification, and post-synthetic PEG conjugation.

This unit describes the solid-phase synthesis and downstream processing for RNA oligonucleotides with a length of up to 40 to 50 nucleotides on a 1- to 4-mmol scale with subsequent conjugation to PEG using the L-RNA spiegelmer NOX-E36 as an example. Following synthesis and two-step deprotection, the crude oligonucleotide is purified by preparative reversed-phase HPLC and desalted by tangential flow ultrafiltration. The resulting intermediate amino-modified oligonucleotide is reacted with NHS-ester-activated PEG, and the oligonucleotide-PEG conjugate is obtained after preparative AX-HPLC purification, followed by ultrafiltration and lyophilization.

Polyetheylenimine-polyplexes of Spiegelmer NOX-A50 directed against intracellular high mobility group protein A1 (HMGA1) reduce tumor growth in vivo.

High mobility group A1 (HMGA1) proteins belong to a group of architectural transcription factors that are overexpressed in a range of human malignancies, including pancreatic adenocarcinoma. They promote anchorage-independent growth and epithelial-mesenchymal transition and are therefore suggested as potential therapeutic targets. Employing in vitro selection techniques against a chosen fragment of HMGA1, we have generated biostable l-RNA oligonucleotides, so-called Spiegelmers, that specifically bind HMGA1b with low nanomolar affinity.

Radiosynthesis of new [90Y]-DOTA-based maleimide reagents suitable for the prelabeling of thiol-bearing L-oligonucleotides and peptides.

We describe the radiosynthesis of two new [(90)Y]-DOTA-based maleimide reagents, suitable for the mild radiolabeling of L-RNAs and peptides modified with thiol-bearing linkers. The synthesis procedure of both maleimide-bearing (90)Y complexes, [{(2S)-2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl}tetraacetato][(90)Y]yttrate(1-)([(90)Y]3) and [{(2S)-2-(4-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}benzyl)-1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetrayl]tetraacetato}[(90)Y]yttrate(1-)([(90)Y]4), was optimized in terms of an easy purification method via solid-phase extraction (SPE). Application as well as reactivity of both maleimide reagents were initially evaluated by the prelabeling of glutathione (GSH) and a thiol-modified 12mer L-RNA as model substances.

Physicochemical stability of NOX-E36, a 40mer L-RNA (Spiegelmer) for therapeutic applications.

Spiegelmers are structured mirror-image oligonucleotides that are designed to bind and inhibit pharmacologically relevant target molecules. The synthesis and purification of mirror-image oligonucleotides is comparable to the manufacturing of standard oligonucleotides that consist of naturally configured nucleotides. Due to the use of the non-natural L-nucleotides in Spiegelmers, these oligonucleotides show an exceptional biostability.

An 86Y-labeled mirror-image oligonucleotide: influence of Y-DOTA isomers on the biodistribution in rats.

A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide (86)Y (t(1/2) = 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5'-aminohexyl UGA CUG ACU GAC-3'; MW 3975) was performed using (S)-p-SCN-Bn-DOTA.

Comparison of d-g3139 and its enantiomer L-g3139 in melanoma cells demonstrates minimal in vitro but dramatic in vivo chiral dependency.

G3139 (Genasense), an 18mer phosphorothioate antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 messenger RNA (mRNA), downregulates Bcl-2 protein and mRNA expression in many cell lines. However, both the in vitro and in vivo mechanisms of action of G3139 are still uncertain. The isosequential L-deoxyribose enantiomer L-G3139, which does not downregulate Bcl-2 expression, was synthesized to study the role of the Bcl-2 protein in melanoma cells.

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.

Mapping the melatonin receptor. 7. Subtype selective ligands based on beta-substituted N-acyl-5-methoxytryptamines and beta-substituted N-acyl-5-methoxy-1-methyltryptamines.

A series of beta-substituted and beta,beta-disubstituted N-acyl 5-methoxy-1-methyltryptamines and 5-methoxytryptamines have been prepared as melatonin analogues to investigate the nature of the binding site of the melatonin receptor. The affinity of analogues was determined in a radioligand binding assay using cloned human MT(1) and MT(2) receptor subtypes expressed in NIH 3T3 cells. Agonist and antagonist potency of all analogues was measured using the pigment aggregation response of a clonal line of Xenopus laevis melanophores.

An L-RNA-based aquaretic agent that inhibits vasopressin in vivo.

A class of diuretic/aquaretic agents based on mirror-image oligonucleotides (so-called Spiegelmers) has been identified. These molecules directly bind and inhibit the neuropeptide vasopressin (AVP). AVP is the major regulatory component of body fluid homeostasis mediated through binding to the renal V(2) receptor.

In vivo biodistribution and pharmacokinetics of 18F-labelled Spiegelmers: a new class of oligonucleotidic radiopharmaceuticals.

Purpose: Single-stranded mirror-image oligonucleotides (Spiegelmers) are highly resistant to nuclease degradation and are capable of tightly and specifically binding to protein targets. Here we explored the potential of Spiegelmers as in vivo imaging probes for positron emission tomography (PET).

Methods: We investigated the biodistribution and pharmacokinetics of [18F]-L-DNA and [18F]-L-RNA Spiegelmers by dynamic quantitative whole-body PET imaging after intravenous administration in non-human primates.

Inhibition of ghrelin action in vitro and in vivo by an RNA-Spiegelmer.

Employing in vitro selection techniques, we have generated biostable RNA-based compounds, so-called Spiegelmers, that specifically bind n-octanoyl ghrelin, the recently discovered endogenous ligand for the type 1a growth hormone secretagogue (GHS) receptor. Ghrelin is a potent stimulant of growth hormone release, food intake, and adiposity. We demonstrate that our lead compound, L-NOX-B11, binds ghrelin with low-nanomolar affinity and inhibits ghrelin-mediated GHS-receptor activation in cell culture with an IC(50) of 5 nM.

Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ.

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers are L-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared to D-oligonucleotides.

Aminomodified nucleobases: functionalized nucleoside triphosphates applicable for SELEX.

5-Aminoallyl-2'-fluoro-dUTP, 5-aminoallyl-UTP, and N(6)-([6-aminohexyl]carbamoylmethyl)-ATP were systematically tested for their suitability for the systematic evolution of ligands by exponential enrichment (SELEX) process with the aim of introducing additional functionalities to RNA libraries. All three aminomodified nucleoside triphosphates proved to be compatible with the enzymatic steps required for SELEX and maintained strict Watson-Crick basepairing. Complementary RNA molecules modified with the two uridine analogues show a significantly increased melting temperature, whereas the introduction of N(6)-([6-aminohexyl]carbamoylmethyl)-ATP leads to a decreased T(m) and thus less stable basepairing.

Internal 32P-labeling of L-deoxyoligonucleotides.

A general two step procedure for the internal labeling of L-deoxyoligonucleotides, Spiegelmers, has been developed. Through radioactive labeling oligonucleotides can easily be detected and monitored in biological samples. T4 polynucleotide kinase is shown to efficiently phosphorylate strands of L-nucleic acids which allows the labeling with phosphorous isotopes such as (32)P.