ABSTRACT
Modification of DNA aptamers is aimed at increasing their thermodynamic stability, and improving affinity and resistance to biodegradation. G-quadruplex DNA aptamers are a family of affinity ligands that form non-canonical DNA assemblies based on a G-tetrads stack. Modification of the quadruplex core is challenging since it can cause complete loss of affinity of the aptamer. On the other hand, increased thermodynamic stability could be a worthy reward. In the current paper, we developed new three- and four-layer modified analogues of the thrombin binding aptamer with high thermal stability, which retain anticoagulant activity against alpha-thrombin. In the modified aptamers, one or two G-tetrads contained non-natural anti-preferred alpha-deoxyguanosines at specific positions. The use of this nucleotide analogue made it possible to control the topology of the modified structures. Due to the presence of non-natural tetrads, we observed some decrease in the anticoagulant activity of the modified aptamers compared to the natural prototype. This negative effect was completely compensated by conjugation of the aptamers with optimized tripeptide sequences.
Subject(s)
Aptamers, Nucleotide , G-Quadruplexes , Aptamers, Nucleotide/chemistry , Thrombin/metabolism , Anticoagulants/chemistry , DeoxyguanosineABSTRACT
Oligonucleotide-peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3-Ser-Phe-Asn (SFN) or T3-Tyr-Trp-Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.
Subject(s)
Aptamers, Nucleotide , G-Quadruplexes , Nucleic Acids , Anticoagulants/chemistry , Aptamers, Nucleotide/chemistry , Peptides , Thrombin/metabolism , ThymidineABSTRACT
A series of new fluorescent symmetric dimeric bisbenzimidazoles DBP(n) bearing bisbenzimidazole fragments joined by oligomethylene linkers with a central 1,4-piperazine residue were synthesized. The complex formation of DBP(n) in the DNA minor groove was demonstrated. The DBP(n) at micromolar concentrations inhibit in vitro eukaryotic DNA topoisomerase I and prokaryotic DNA methyltransferase (MTase) M.SssI. The DBP(n) were soluble well in aqueous solutions and could penetrate cell and nuclear membranes and stain DNA in live cells. The DBP(n) displayed a moderate effect on the reactivation of gene expression.
Subject(s)
Bisbenzimidazole/analogs & derivatives , DNA/chemistry , DNA/drug effects , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Bisbenzimidazole/chemical synthesis , Bisbenzimidazole/pharmacology , Cell Line , DNA/genetics , DNA-Cytosine Methylases/antagonists & inhibitors , Dimerization , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Fluorescent Dyes/chemistry , Gene Expression/drug effects , Humans , MCF-7 Cells , Mice , Microscopy, Fluorescence , Structure-Activity Relationship , Topoisomerase I Inhibitors/chemical synthesis , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/pharmacologyABSTRACT
Currently, oligonucleotide therapy has emerged as a new paradigm in the treatment of human diseases. In many cases, however, therapeutic oligonucleotides cannot be used directly without modification. Chemical modification or the conjugation of therapeutic oligonucleotides is required to increase their stability or specificity, improve their affinity or inhibitory characteristics, and address delivery issues. Recently, we proposed a conjugation strategy for a 15-nt G-quadruplex thrombin aptamer aimed at extending the recognition interface of the aptamer. In particular, we have prepared a series of designer peptide conjugates of the thrombin aptamer, showing improved anticoagulant activity. Herein, we report a new series of aptamer-peptide conjugates with optimized peptide sequences. The anti-thrombotic activity of aptamer conjugates was notably improved. The lead conjugate, TBA-GLE, was able to inhibit thrombin-induced coagulation approximately six-fold more efficiently than the unmodified aptamer. In terms of its anticoagulant activity, the TBA-GLE conjugate approaches NU172, one of the most potent G-quadruplex thrombin aptamers. Molecular dynamics studies have confirmed that the principles applied to the design of the peptide side chain are efficient instruments for improving aptamer characteristics for the proposed TBA conjugate model.
ABSTRACT
The cell wall of Streptomyces sp. MB-8 contains a major teichoic acid, viz., 1,3-poly(glycerol phosphate) substituted with N-acetyl-alpha-D-glucosamine (the degree of substitution is 60%), a minor teichoic acid, viz., non-substituted poly(glycerol phosphate), and a family of Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid)-containing oligomers of the following general structure: [carbohydrate structure: see text]. The composition of the oligomers was established using MALDI-TOF mass spectroscopy. The present study provides the second example of the identification of Kdn as a component of cell wall polymers of streptomycetes, which are the causative agents of potato scab.
Subject(s)
Cell Wall/chemistry , Streptomyces/chemistry , Teichoic Acids/chemistry , Carbohydrate Sequence , Chemical Fractionation , Hydrolysis , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Plant Diseases/microbiology , Solanum tuberosum/microbiology , Teichoic Acids/isolation & purificationABSTRACT
The cell wall of Brevibacterium sp. VKM Ac-2118 isolated from a frozen (mean annual temperature -12 degrees C) late Pliocene layer, 1.8-3 Myr, Kolyma lowland, Russia, contains mannitol teichoic acid with a previously unknown structure. This is 1,6-poly(mannitol phosphate) with the majority of the mannitol residues bearing side phosphate groups at O-4(3). The structure of the polymer was established by chemical methods, NMR spectroscopy, and MALDI-TOF mass spectrometry.