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1.
JACS Au ; 2(8): 1910-1917, 2022 Aug 22.
Article in English | MEDLINE | ID: mdl-36032523

ABSTRACT

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. We report here the development of a DNA/RNA hybrid catalytic system featuring a covalently attached bipyridine ligand, which exhibits unmatched levels of selectivity throughout the current DNA toolbox and opens new avenues in asymmetric catalysis.

2.
Curr Protoc Nucleic Acid Chem ; 81(1): e109, 2020 06.
Article in English | MEDLINE | ID: mdl-32438494

ABSTRACT

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. In the second step, the solution of the activated carboxylic acid is added to the amino-modified oligonucleotide in water. The method is demonstrated using 40-kDa polyethylene glycol (PEG) carboxylic acid and biotin as examples. Recycling of the carboxylic acid, which is typically used in molar excess over the nucleic acid, is shown for the conjugation with 40-kDa PEG carboxylic acid. This conjugation method is generally applicable to the conjugation of carboxylic acids to amino-modified oligonucleotides, thus enabling the attachment of small to large molecular entities such as dyes, tags, peptides, and other macromolecules. © 2020 Wiley Periodicals LLC. Basic Protocol 1: General protocol for the conjugation of an amino-modified oligonucleotide with a carboxylic acid, exemplified for 40-kDa PEG carboxylic acid Basic Protocol 2: Biotinylation of an amino-modified oligonucleotide using the general conjugation protocol Basic Protocol 3: Recycling of the carboxylic acid component from the conjugation reaction, demonstrated for 40-kDa PEG carboxylic acid using ultrafiltration Support Protocol 1: Analytical AEX-HPLC method used as in-process control method to monitor the conjugation reaction with 40-kDa PEG carboxylic acid Support Protocol 2: Analytical AEX-HPLC method used as in-process control method to monitor the conjugation reaction with biotin Support Protocol 3: Analytical IP-RP-HPLC method used as in-process control method to monitor the conjugation reaction Alternate Protocol: Separation of 40-kDa PEG carboxylic acid from unreacted and conjugated oligonucleotide by preparative AEX-HPLC.


Subject(s)
Carboxylic Acids/chemistry , Oligonucleotides/chemistry , RNA/chemistry , Chromatography, High Pressure Liquid , Spectrophotometry, Ultraviolet
3.
Bioconjug Chem ; 28(4): 1176-1188, 2017 04 19.
Article in English | MEDLINE | ID: mdl-28222590

ABSTRACT

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. In this study, a radiopharmacological evaluation of a new l-ONs based pretargeting system using the epidermal growth factor receptor (EGFR) specific antibody cetuximab (C225) as target-seeking component is presented. An optimized PEGylated 17mer-L-DNA was conjugated with p-SCN-Bn-NOTA (NOTA') to permit radiolabeling with the radionuclide 64Cu. C225 was modified with the complementary 17mer-L-DNA (c-L-DNA) strand as well as with NOTA' for radiolabeling and use for positron emission tomography (PET). Two C225 conjugates were coupled with 1.5 and 5.0 c-L-DNA molecules, respectively. In vitro characterization was done with respect to hybridization studies, competition and saturation binding assays in EGFR expressing squamous cell carcinoma cell lines A431 and FaDu. The modified C225 derivatives exhibited high binding affinities in the low nanomolar range to the EGFR. PET and biodistribution experiments on FaDu tumor bearing mice with directly 64Cu-labeled NOTA'3-C225-(c-L-DNA)1.5 conjugate revealed that a pretargeting interval of 24 h might be a good compromise between tumor accumulation, internalization, blood background, and liver uptake of the antibody. Despite internalization of the antibody in vivo pretargeting experiments showed an adequate hybridization of 64Cu-radiolabeled NOTA'-L-DNA to the tumor located antibody and a good tumor-to-muscle ratio of about 11 resulting in a clearly visible image of the tumor after 24 h up to 72 h. Furthermore, low accumulation of radioactivity in organs responsible for metabolism and excretion was determined. The presented results indicate a high potential of complementary L-ONs for the pretargeting approach which can also be applied to therapeutic radionuclides such as 177Lu, 90Y, 186Re, or 188Re.


Subject(s)
Cetuximab/therapeutic use , Immunoconjugates/chemistry , Oligonucleotides/chemistry , Radiopharmaceuticals/chemical synthesis , Animals , Carcinoma, Squamous Cell/diagnostic imaging , Carcinoma, Squamous Cell/drug therapy , Cell Line, Tumor , Cetuximab/chemistry , Cetuximab/pharmacology , ErbB Receptors/immunology , Humans , Liver/metabolism , Mice , Radioisotopes/chemistry , Radiopharmaceuticals/pharmacology , Radiopharmaceuticals/therapeutic use
4.
Chem Commun (Camb) ; 52(55): 8604-7, 2016 Jun 30.
Article in English | MEDLINE | ID: mdl-27327242

ABSTRACT

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. We report here our results, which have led to the first example involving a short RNA-based catalyst.


Subject(s)
Biocatalysis , Nucleic Acid Conformation , RNA, Catalytic/metabolism , Alkylation , RNA, Catalytic/chemistry , Solvents/chemistry
5.
Chem Commun (Camb) ; 51(28): 6076-9, 2015 Apr 11.
Article in English | MEDLINE | ID: mdl-25613918

ABSTRACT

The challenge in DNA-based asymmetric catalysis is to perform a reaction in the vicinity of the helix by incorporating a small-molecule catalyst anchored to the DNA in a covalent, dative, or non-covalent yet stable fashion in order to ensure high levels of enantio-discrimination. Here, we report the first generation of a DNA-based catalyst bound to a cellulose matrix. The chiral biomaterial is commercially available, trivial to use, fully recyclable and produces high levels of enantioselectivity in various Cu(II)-catalyzed asymmetric reactions including Friedel-Crafts alkylations and Michael additions. A single-pass, continuous-flow process is also reported affording fast conversions and high enantioselectivities at low catalyst loadings thus offering a new benchmark in the field of DNA-based asymmetric catalysis.


Subject(s)
Cellulose/chemistry , Copper/chemistry , DNA/chemistry , Organometallic Compounds/chemistry , Alkylation , Animals , Catalysis , Cattle , Molecular Structure , Stereoisomerism
6.
Cell Rep ; 9(1): 118-128, 2014 Oct 09.
Article in English | MEDLINE | ID: mdl-25263552

ABSTRACT

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). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells.


Subject(s)
Bone Marrow/pathology , Chemokine CXCL12/antagonists & inhibitors , Multiple Myeloma/therapy , Oligonucleotides/pharmacology , Animals , Bone Marrow/metabolism , Bone Marrow Neoplasms/metabolism , Bone Marrow Neoplasms/secondary , Boronic Acids/pharmacology , Bortezomib , Chemokine CXCL12/biosynthesis , Chemokine CXCL12/genetics , Female , Gene Knockdown Techniques , Humans , Male , Mice , Mice, SCID , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Neoplasm Metastasis , Oligonucleotides/chemistry , Oligonucleotides/genetics , Polyethylene Glycols/chemistry , Pyrazines/pharmacology
7.
Angew Chem Int Ed Engl ; 52(44): 11546-9, 2013 Oct 25.
Article in English | MEDLINE | ID: mdl-24030985

ABSTRACT

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.


Subject(s)
DNA, Catalytic/chemistry , DNA/chemistry , Catalysis , Molecular Structure , Stereoisomerism
8.
Curr Protoc Nucleic Acid Chem ; Chapter 4: Unit 4.46.1-30, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21901672

ABSTRACT

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. Critical process parameters are described, as well as time considerations and examples for analytical methods used as in-process and quality controls.


Subject(s)
Aptamers, Nucleotide/chemical synthesis , Aptamers, Nucleotide/isolation & purification , Chromatography, High Pressure Liquid , Freeze Drying , Oligonucleotides/chemistry , Oligonucleotides/isolation & purification , Phosphorothioate Oligonucleotides/chemistry , Polyethylene Glycols/chemistry , Solid-Phase Synthesis Techniques , Ultrafiltration
9.
Nucleic Acids Res ; 39(21): e147, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21948795

ABSTRACT

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. Spiegelmer sequencing is effected by affixing a label or hapten to the 5'-end of the oligonucleotide and chemically degrading the molecule in a controlled fashion to generate fragments that are then resolved and identified using liquid chromatography-mass spectrometry. The Spiegelmer sequence is then derived from these fragments. Examples are shown for two different Spiegelmers (NOX-E36 and NOX-A12), and the specificity of the method is shown using a NOX-E36 mismatch control.


Subject(s)
Chromatography, Liquid , Oligoribonucleotides/chemical synthesis , Sequence Analysis, RNA/methods , Spectrometry, Mass, Electrospray Ionization , Stereoisomerism
10.
J Biol Chem ; 285(51): 40012-8, 2010 Dec 17.
Article in English | MEDLINE | ID: mdl-20961861

ABSTRACT

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. We demonstrate that the best binding Spiegelmers, NOX-A50 and NOX-f33, compete HMGA1b from binding to its natural binding partner, AT-rich double-stranded DNA. We describe a formulation method based on polyplex formation with branched polyethylenimine for efficient delivery of polyethylene glycol-modified Spiegelmers and show improved tissue distribution and persistence in mice. In a xenograft mouse study using the pancreatic cancer cell line PSN-1, subcutaneous administration of 2 mg/kg per day NOX-A50 formulated in polyplexes showed an enhanced delivery of NOX-A50 to the tumor and a significant reduction of tumor volume. Our results demonstrate that intracellular targets can be successfully addressed with a Spiegelmer using polyethylenimine-based delivery and underline the importance of HMGA1 as a therapeutic target in pancreatic cancer.


Subject(s)
Adenocarcinoma/drug therapy , Aptamers, Nucleotide/pharmacology , Drug Delivery Systems , HMGA Proteins/antagonists & inhibitors , Pancreatic Neoplasms/drug therapy , Xenograft Model Antitumor Assays , Animals , Cell Line, Tumor , DNA, Neoplasm/metabolism , Dose-Response Relationship, Drug , HMGA Proteins/metabolism , Humans , Mice , Mice, Mutant Strains , Protein Binding
11.
Bioconjug Chem ; 20(7): 1340-8, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19552458

ABSTRACT

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. In comparison to the N-aryl maleimide-bearing complex [(90)Y]3, N-alkyl maleimide-bearing complex [(90)Y]4 showed an increased hydrolytic stability at pH > or = 7. A slightly higher reactivity was found for [(90)Y]3 by prelabeling of 0.1 and 1 microg glutathione, respectively, in phosphate buffer (pH 7.2) at room temperature. In terms of very high radiochemical yields, the direct radiolabeling of DOTA-L-RNA conjugate with [(90)Y]YCl(3) proved to be more suitable than the prelabeling of the thiol-modified 12mer L-RNA derivative with [(90)Y]4.


Subject(s)
Heterocyclic Compounds/chemistry , Isotope Labeling/methods , Maleimides/chemistry , Oligonucleotides/chemistry , Organometallic Compounds/chemistry , Peptides/chemistry , Glutathione/analysis , Glutathione/chemistry , Heterocyclic Compounds/chemical synthesis , Maleimides/chemical synthesis , Molecular Structure , Oligonucleotides/analysis , Organometallic Compounds/chemical synthesis , Peptides/analysis , RNA/analysis , RNA/chemistry , Solid Phase Extraction , Sulfhydryl Compounds/chemistry , Yttrium Radioisotopes/chemistry
12.
Nucleic Acids Symp Ser (Oxf) ; (52): 61-2, 2008.
Article in English | MEDLINE | ID: mdl-18776253

ABSTRACT

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. Further, they also display a high physicochemical stability in solution. These properties make them interesting substances for drug development.


Subject(s)
Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/pharmacology , Chemokine CCL2/antagonists & inhibitors , Aptamers, Nucleotide/therapeutic use , Cell Line , Chemotaxis/drug effects , Humans , Inflammation/drug therapy
13.
Bioconjug Chem ; 19(4): 928-39, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18345604

ABSTRACT

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. (86)Y radiolabeling of the DOTA-L-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the (86)Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)](-) was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)](-) isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)](-) and conjugated to the L-RNA. The identity of the [(86)Y-DOTA]-L-RNA species was finally established by comparison of the radiometric ((86)Y) and UV-visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [(86)Y((S)-p-NH2-Bn-DOTA)](-) complex isomers, while no significant differences were observed for the [(86)Y-DOTA]-L-RNA isomers. High renal excretions were found for the [(86)Y((S)-p-NH 2-Bn-DOTA)](-) complex isomers as well as for the L-RNA isomers.


Subject(s)
Heterocyclic Compounds/chemistry , Oligonucleotides/chemistry , Oligonucleotides/pharmacokinetics , Organometallic Compounds/chemistry , Animals , Autoradiography , Benzene/chemistry , Heterocyclic Compounds/metabolism , Heterocyclic Compounds/pharmacokinetics , Isomerism , Magnetic Resonance Spectroscopy , Male , Oligonucleotides/metabolism , Organometallic Compounds/metabolism , Organometallic Compounds/pharmacokinetics , RNA/chemistry , RNA/metabolism , RNA/pharmacokinetics , Rats , Rats, Wistar , Tissue Distribution , Yttrium Radioisotopes
14.
Mol Ther ; 15(2): 270-8, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17235304

ABSTRACT

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. Both D-G3139 and L-G3139 bind nonspecifically to basic fibroblast growth factor with approximately the same K(c), and cause highly effective inhibition of net formation in 518A2 melanoma cells on Matrigel. The uptakes of D-G3139 and L-G3139 in melanoma cells were also similar. However, unlike D-G3139, L-G3139 does not produce poly ADP-ribose polymerase-1 and procaspase-3 cleavage at 9.5 h after the initiation of the transfection, but can activate the intrinsic pathway of apoptosis at approximately 48 h. Furthermore, treatment of A375 melanoma human xenografts in severe combined immunodeficiency (SCID) mice demonstrates that tumor growth is not inhibited by L-G3139, whereas D-G3139 significantly inhibits the rate of tumor growth. Furthermore, the immunostimulatory properties of L-G3139 appear to be nil, which differs dramatically from those of D-G3139. In conclusion, profound differences exist between D-G3139 and L-G3139 in vivo despite their similarities in vitro.


Subject(s)
Melanoma, Experimental/drug therapy , Oligonucleotides, Antisense/pharmacology , Proto-Oncogene Proteins c-bcl-2/genetics , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cytochromes c/metabolism , Dose-Response Relationship, Drug , Fibroblast Growth Factor 2/metabolism , Flow Cytometry , Humans , Interleukin-12/metabolism , Interleukin-16/metabolism , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, SCID , Microscopy, Phase-Contrast , Mitochondria/drug effects , Mitochondria/metabolism , Oligonucleotides, Antisense/metabolism , Oligonucleotides, Antisense/pharmacokinetics , Protein Binding , Spleen/cytology , Spleen/drug effects , Spleen/metabolism , Stereoisomerism , Xenograft Model Antitumor Assays
15.
J Med Chem ; 49(12): 3509-19, 2006 Jun 15.
Article in English | MEDLINE | ID: mdl-16759094

ABSTRACT

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. beta-Methylmelatonin (17a) and beta,beta-dimethylmelatonin (17b), though showing a slight decrease in binding at human receptors, show an increase in potency on Xenopus. N-Butanoyl 5-methoxy-1-methyl-beta,beta-trimethylenetryptamine (12c) is an antagonist at human MT(1) receptors but an agonist at MT(2), while N-butanoyl 5-methoxy-1-methyl-beta,beta-tetramethylenetryptamine (13c) is an antagonist at MT(1) but had no action at MT(2) and is one of the first examples of an MT(1) selective antagonist.


Subject(s)
Receptor, Melatonin, MT1/agonists , Receptor, Melatonin, MT1/antagonists & inhibitors , Receptor, Melatonin, MT2/agonists , Receptor, Melatonin, MT2/antagonists & inhibitors , Tryptamines/chemical synthesis , Animals , Binding Sites , Cell Line , Humans , Ligands , Melanophores/drug effects , Melanophores/metabolism , Melatonin/analogs & derivatives , Melatonin/chemical synthesis , Melatonin/pharmacology , Mice , Molecular Conformation , NIH 3T3 Cells , Pigments, Biological/biosynthesis , Radioligand Assay , Recombinant Proteins/agonists , Recombinant Proteins/antagonists & inhibitors , Stereoisomerism , Structure-Activity Relationship , Tryptamines/pharmacology , Xenopus laevis
16.
J Am Chem Soc ; 128(24): 8063-7, 2006 Jun 21.
Article in English | MEDLINE | ID: mdl-16771522

ABSTRACT

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.


Subject(s)
Benzimidazoles/chemistry , Metals/chemistry , Oligonucleotides, Antisense/chemistry , RNA/chemistry , Amides/chemistry , Amides/metabolism , Base Sequence , Catalysis , DNA/chemistry , Disulfides/chemistry , Disulfides/metabolism , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides, Antisense/metabolism , RNA/metabolism , Substrate Specificity
17.
Proc Natl Acad Sci U S A ; 103(13): 5173-8, 2006 Mar 28.
Article in English | MEDLINE | ID: mdl-16547136

ABSTRACT

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. Elevated plasma levels of AVP are implicated in several pathological conditions, mainly cardiovascular diseases. In congestive heart failure, AVP is part of a neuroendocrine imbalance that is responsible for progressive worsening of the disease. Employing in vitro selection techniques, RNA aptamers that bind to the unnatural d-configuration of AVP were isolated. The best aptamer displayed an affinity to d-AVP of approximately 560 pM at 37 degrees C. The corresponding Spiegelmer, a 38-mer mirror-image oligonucleotide (l-RNA) termed NOX-F37, inhibits vasopressin-dependent activation of V(1a) as well as V(2) receptors with IC(50) values of 6.1 nM and 1 nM, respectively. NOX-F37 administered to healthy rats effectively neutralized AVP and increased diuresis dose-dependently for 24 h. The mode of action was strictly aquaretic, i.e., the increase in urine volume was not accompanied by an increase in electrolytes. These results clearly prove the in vivo efficacy of NOX-F37 and points out its potential as a drug in the treatment of diseases that are associated with body fluid overload.


Subject(s)
Oligoribonucleotides/pharmacology , RNA/pharmacology , Vasopressins/antagonists & inhibitors , Animals , Antidiuretic Hormone Receptor Antagonists , Base Sequence , Binding Sites , Carbohydrate Conformation , Cell Line , Kidney/drug effects , Kidney/metabolism , Male , Molecular Sequence Data , Mutation/genetics , Oligoribonucleotides/chemistry , RNA/chemistry , RNA/genetics , Rats , Rats, Sprague-Dawley , Receptors, Vasopressin/metabolism , Swine , Vasopressins/pharmacology
18.
Eur J Nucl Med Mol Imaging ; 32(4): 470-7, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15614506

ABSTRACT

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. Their metabolic profile was explored in primates and rats, and ex vivo autoradiography of [(125)I]-L-RNA was performed in rat kidneys, the major organ for Spiegelmer uptake. RESULTS: Both [18F]-L-DNA and [18F]-L-RNA Spiegelmers were metabolically stable in plasma during 2 h after injection. No evidence of non-specific binding was found with either type of Spiegelmer in any tissue. CONCLUSION: The biodistribution and metabolic profiles of [18F]-L-DNA and [18F]-L-RNA Spiegelmers highlight their potential as radiotracers for in vivo imaging applications.


Subject(s)
Fluorine Radioisotopes/pharmacokinetics , Iodine Radioisotopes/pharmacokinetics , Oligonucleotides/pharmacokinetics , Animals , Male , Metabolic Clearance Rate , Organ Specificity , Papio , Radiopharmaceuticals/pharmacokinetics , Rats , Rats, Sprague-Dawley , Species Specificity , Tissue Distribution
19.
Proc Natl Acad Sci U S A ; 101(36): 13174-9, 2004 Sep 07.
Article in English | MEDLINE | ID: mdl-15329412

ABSTRACT

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. l-NOX-B11 is highly specific for the bioactive, n-octanoylated form of ghrelin. Like the GHS receptor, it does not recognize the inactive unmodified peptide and requires only the N-terminal five amino acids for the interaction. The i.v. administration of polyethylene glycol modified l-NOX-B11 efficiently suppresses ghrelin-induced growth hormone release in rats. These results demonstrate that the neutralization of circulating bioactive ghrelin leads to inhibition of ghrelin's secretory effects in the CNS.


Subject(s)
Oligonucleotides/pharmacology , Peptide Hormones/antagonists & inhibitors , Amino Acid Sequence , Animals , Ghrelin , Growth Hormone/metabolism , Male , Molecular Sequence Data , Oligonucleotides/metabolism , Peptide Hormones/chemistry , Peptide Hormones/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction
20.
RNA ; 10(3): 516-27, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14970396

ABSTRACT

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. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC(50) values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPgammaS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes from Xenopus laevis, NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K(+) channels.


Subject(s)
Opioid Peptides/antagonists & inhibitors , RNA/metabolism , Animals , Base Sequence , Humans , Ligands , Molecular Sequence Data , Nucleic Acid Conformation , Oocytes , Receptors, Opioid/metabolism , Tritium/metabolism , Xenopus , Nociceptin Receptor , Nociceptin
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