RESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos , Quadruplex G , Aptâmeros de Nucleotídeos/química , Trombina/metabolismo , Anticoagulantes/química , DesoxiguanosinaRESUMO
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.
Assuntos
Aptâmeros de Nucleotídeos , Quadruplex G , Ácidos Nucleicos , Anticoagulantes/química , Aptâmeros de Nucleotídeos/química , Peptídeos , Trombina/metabolismo , TimidinaRESUMO
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.
RESUMO
A simple and stereoselective synthesis of a protected 4-(aminomethyl)-1-(2-deoxy-ß-D-ribofuranosyl)-1,2,3-triazole cyanoethyl phosphoramidite was developed for the modification of synthetic oligonucleotides. The configuration of the 1,2,3-triazolyl moiety with respect to the deoxyribose was unambiguously determined in ROESY experiments. The aminomethyl group of the triazolyl nucleotide was fully functional in labelling reactions. Furthermore, the hybridization behavior of 5' triazole-terminated oligonucleotide was similar to that of 5' aminohexyl-terminated oligomer with the same sequence. Internal modifications of the oligonucleotide strands resulted in significant decrease of duplex stability.
Assuntos
Química Click/métodos , Nucleosídeos/química , Compostos Organofosforados/química , Triazóis/química , Química Click/economia , Ciclização , Nucleosídeos/síntese química , Compostos Organofosforados/síntese química , Estereoisomerismo , Triazóis/síntese químicaRESUMO
Recent advances in G-quadruplex (GQ) studies have provided evidence for their important role in key biological processes (replication, transcription, genome stability, and epigenetics). These findings imply highly specific interactions between GQ structures and cellular proteins. The details of the interaction between GQs and cellular proteins remain unknown. It is now accepted that GQ loop elements play a major role in protein recognition. It remains unclear whether and to what extent the GQ core contributes to maintaining the recognition interface. In the current paper, we used the thrombin binding aptamer as a model to study the effect of modification in the quadruplex core on the ability of aptamer to interact with thrombin. We used alpha-2'-deoxyguanosine and 8-bromo-2'-deoxyguanosine to reconfigure the core or to affect syn-anti preferences of selected dG-residues. Our data suggest that core guanines not only support a particular type of GQ architecture, but also set structural parameters that make GQ protein recognition sensitive to quadruplex topology.
Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Trombina/metabolismo , Dicroísmo Circular , Desoxiguanosina/química , Humanos , Ligação ProteicaRESUMO
Post-SELEX modification of DNA aptamers is an established strategy to improve their affinity or inhibitory characteristics. In this study, we examined the possibility of increasing the recognition interface between the thrombin-binding aptamer HD1 (TBA) and thrombin by adding a chemically modified side chain to selected nucleotide residues. A panel of 22 TBA variants with N3-modified residues T3 and T12 was prepared by a two-step modification procedure. Aptamers were characterized by a combination of biophysical and biochemical methods. We identified mutants with enhanced affinity and improved anticoagulant activity. The crystal structures of thrombin complexes with three selected modified variants revealed that the modified pyrimidine base invariably allocates in proximity to thrombin residues Tyr76 and Ile82 due to the directing role of the unmodified TT loop. The modifications induced an increase in the contact areas between thrombin and the modified TBAs. Comparative analysis of the structural, biochemical, and biophysical data suggests that the non-equivalent binding modes of the mutants with thrombin in the T3- and T12-modified series account for the observed systematic differences in their affinity characteristics. In this study, we show that extending the recognition surface between the protein and modified aptamers is a promising approach that may improve characteristics of aptamer ligands.
RESUMO
Replicative strand slippage is a biological phenomenon, ubiquitous among different organisms. However, slippage events are also relevant to non-natural replication models utilizing synthetic polymerase substrates. Strand slippage may notably affect the outcome of the primer extension reaction with repetitive templates in the presence of non-natural nucleoside triphosphates. In the current paper, we studied the ability of Taq, Vent (exo-), and Deep Vent (exo-) polymerases to produce truncated, full size, or expanded modified strands utilizing non-natural 2'-deoxyuridine nucleotide analogues and different variants of the homopolymer template. Our data suggest that the slippage of the primer strand is dependent on the duplex fluttering, incorporation efficiency for a particular polymerase-dNTP pair, rate of non-templated base addition, and presence of competing nucleotides.
RESUMO
Synthetically modified DNA G-quadruplexes (GQs) have great potential in the development of designer molecules for a wide range of applications. Identification of the role of various structural elements in the folding and final topology of artificial GQs is necessary to predict their secondary structure. We report here the results of spectroscopic and electrophoretic studies of GQ scaffolds formed by G-rich sequences comprising four G3-tracts of different polarity connected by either a single-nucleotide thymine loop or a non-natural tetraethyleneglycol loop. Depending on G-strand polarities, loop arrangement and the presence of extra 5'-base G-rich oligonucleotides form monomeric, dimeric, or multimeric species of different topologies. In most cases, oligonucleotides were able to fold into stable parallel or hybrid GQs. However, certain specific arrangements of loops and G-tracts resulted in a diverse mixture of low stable structures. Comparative analysis of topology, stability, and structural heterogeneity of different G-rich sequences suggests the important role of loop type and arrangement, G3-tract polarities, and the presence of 5'-capping residues in the outcome of the folding process. The results also imply that the formation of anti-parallel G-hairpin intermediates is a key event in major favourable folding pathways.
Assuntos
DNA/química , Quadruplex G , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Dimerização , Modelos MolecularesRESUMO
Thrombin-binding aptamer HD1 is a DNA-based thrombin inhibitor that features an antiparallel G-quadruplex (GQ) structure. We recently reported a single-nucleotide G8 to 5-nitroindole (NI) modification of HD1 (N8) that notably improves the anticoagulant properties and binding affinity of the aptamer. Based on molecular modeling and binding studies, it was originally proposed that N8 may acquire the ability to bind thrombin by a modified central loop. To verify this possibility, in this study, we report new variations of the N8 aptamer with intact or damaged TT loops. Anomeric alpha-thymidine was used as a "damaging" residue to disable the primary recognition site of N8. Biophysical characterization of modified aptamers supports the formation of HD1-like antiparallel GQs with varying stability by all studied variants. Binding experiments showed that N8 variants with impaired TT loops lost the ability to bind thrombin, suggesting the primary role of thymines in TT loops for the thrombin-N8 interaction. Aptamer N8α(7/9) bearing NI at position 8 and damaged thymidines 7 and 9 retained thrombin affinity, which was intermediate between N8 and HD1. Fluorescence polarization studies suggest 1:1 stoichiometry for thrombin complexes with either HD1, N8, or N8α(7/9). Further molecular dynamics (MD) study of complexes formed by these three aptamers with thrombin disproves the idea of direct interaction between central loop residues and the protein. Based on MD results, the origin of the NI tuning effect is associated with its ability to promote the formation of compact and rigid structures through hydrophobic interactions with the GQ core and loop thymines.
Assuntos
Anticoagulantes/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Indóis/química , Nitrocompostos/farmacologia , Anticoagulantes/química , Aptâmeros de Nucleotídeos/química , Quadruplex G , Humanos , Simulação de Dinâmica Molecular , Nitrocompostos/química , Timidina/químicaRESUMO
The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3' ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson-Crick base pair at the end. Mismatched duplexes with a 3' dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5' exonuclease activity of the enzyme.
Assuntos
Carbocianinas/química , Nucleotídeos de Desoxiuracil/química , Nucleotídeos de Desoxiuracil/metabolismo , Corantes Fluorescentes/química , Taq Polimerase/metabolismo , Pareamento Incorreto de Bases , Carbocianinas/metabolismo , Clonagem Molecular , Nucleotídeos de Desoxiuracil/genética , Corantes Fluorescentes/metabolismoRESUMO
BACKGROUND: Improving the efficacy of anticancer therapy remains an urgent and very important task. Screening of the individual genetic metabolism of cancer patients allows for prescribing adequate medication in the correct dose as well as for decreasing side effects associated with drug toxicity. OBJECTIVE: Estimation of a microarray-based method for genotyping of the UGT1A1, DPYD, GSTP1, and ABCB1 metabolic regulation genes to evaluate for an increased risk of toxicity of anticancer drugs. METHODS: The microarray was used to conduct genotyping of specimens taken from 115 cancer patients and 31 healthy donors. RESULTS: A microarray-based method for identification of the rs8175347, rs3918290, rs1695, and rs1045642 polymorphisms in the corresponding UGT1A1, DPYD, GSTP1, and ABCB1 genes has been developed for genotyping. The results obtained were in full concordance with those obtained using control sequencing. The frequencies of the rs8175347, rs3918290, rs1695, and rs1045642 genetic variations were 0.38, 0, 0.35, and 0.56, respectively. CONCLUSION: The implementation of this biochip-based method in diagnostic practice should increase the overall survival and quality of life of cancer patients, decrease the length of their hospital stay, and reduce treatment costs.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Glucuronosiltransferase/genética , Glutationa S-Transferase pi/genética , Hidrogel de Polietilenoglicol-Dimetacrilato , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Genótipo , Humanos , Neoplasias/genéticaRESUMO
Non-natural nucleotide substrates are widely used in the enzymatic synthesis of modified DNA. The terminal activity of polymerases in the presence of modified nucleotides is an important, but poorly characterized, aspect of enzymatic DNA synthesis. Here, we studied different types of polymerase activity at sequence ends using extendable and non-extendable synthetic models in the presence of the Cy5-dUTP analog Y. In primer extension reactions with selected exonuclease-deficient polymerases, nucleotide Y appeared to be a preferential substrate for non-templated 3'-tailing, as determined by MALDI mass-spectrometry and gel-electrophoresis. This result was further confirmed by the 3'-tailing of a non-extendable hairpin oligonucleotide model. Additionally, DNA polymerases induce an exchange of the 3' terminal thymidine for a non-natural nucleotide via pyrophosphorolysis in the presence of inorganic pyrophosphate. In primer extension reactions, the proofreading polymerases Vent, Pfu, and Phusion did not support the synthesis of Y-modified primer strand. Nevertheless, Pfu and Phusion polymerases were shown to initiate terminal nucleotide exchange at the template. Unlike non-proofreading polymerases, these two enzymes recruit 3'-5' exonuclease functions to cleave the 3' terminal thymidine in the absence of pyrophosphate.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Conformação de Ácido Nucleico , Archaea/enzimologia , Proteínas Arqueais/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Carbocianinas/metabolismo , DNA/análise , DNA/química , Nucleotídeos de Desoxiuracil/metabolismo , Espectrometria de Massas , Taq Polimerase/metabolismoRESUMO
DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10 degrees C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in T(m) values as compared with unmodified complexes.
Assuntos
Pareamento de Bases/fisiologia , DNA/química , Oligonucleotídeos/química , DNA/metabolismo , Ligação de Hidrogênio , Oligonucleotídeos/metabolismoRESUMO
Aptamers are nucleic acid-based scaffolds that can bind with high affinity to a variety of biological targets. Aptamers are identified from large DNA or RNA libraries through a process of directed molecular evolution (SELEX). Chemical modification of nucleic acids considerably increases the functional and structural diversity of aptamer libraries and substantially increases the affinity of the aptamers. Additionally, modified aptamers exhibit much greater resistance to biodegradation. The evolutionary selection of modified aptamers is conditioned by the possibility of the enzymatic synthesis and replication of non-natural nucleic acids. Wild-type or mutant polymerases and their non-natural nucleotide substrates that can support SELEX are highlighted in the present review. A focus is made on the efforts to find the most suitable type of nucleotide modifications and the engineering of new polymerases. Post-SELEX modification as a complementary method will be briefly considered as well.
Assuntos
Aptâmeros de Nucleotídeos , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos/químicaRESUMO
Noncanonically structured DNA aptamers to thrombin were examined. Two different approaches were used to improve stability, binding affinity and biological activity of a known thrombin-binding aptamer. These approaches are chemical modification and the addition of a duplex module to the aptamer core structure. Several chemically modified aptamers and the duplex-bearing ones were all studied under the same conditions by a set of widely known and some relatively new methods. A number of the thrombin-binding aptamer analogs have demonstrated improved characteristics. Most importantly, the study allowed us to compare directly the two approaches to aptamer optimization and to analyze their relative advantages and disadvantages as well as their potential in drug design and fundamental studies.
Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Quadruplex G , Trombina/metabolismo , Triazóis/química , Ensaio de Desvio de Mobilidade Eletroforética , Microscopia de Força Atômica , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade , Trombina/antagonistas & inibidoresRESUMO
Thrombin-binding aptamer (TBA) is a 15-nt DNA oligomer that efficiently inhibits thrombin. It has been shown that TBA folds into an anti-parallel unimolecular G-quadruplex. Its three-dimensional chair-like structure consists of two G-tetrads connected by TT and TGT loops. TBA undergoes fast degradation by nucleases in vivo. To improve the nuclease resistance of TBA, a number of modified analogs have been proposed. Here, we describe anomeric modifications of TBA. Non-natural α anomers were used to replace selected nucleotides in the loops and core. Significant stabilization of the quadruplex was observed for the anomeric modification of TT loops at T4 and T13. Replacement of the core guanines either prevents quadruplex assembly or induces rearrangement in G-tetrads. It was found that the anticoagulant properties of chimeric aptamers could be retained only with intact TT loops. On the contrary, modification of the TGT loop was shown to substantially increase nuclease resistance of the chimeric aptamer without a notable disturbance of its anticoagulant activity.
Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Anticoagulantes/química , Anticoagulantes/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Sequência de Bases , Dicroísmo Circular , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Termodinâmica , Tempo de TrombinaRESUMO
A series of DNA aptamers bearing triazole internucleotide linkages that bind to thrombin was synthesized. The novel aptamers are structurally analogous to the well-known thrombin-inhibiting G-quadruplexes TBA15 and TBA31. The secondary structure stability, binding affinity for thrombin and anticoagulant effects of the triazole-modified aptamers were measured. A modification in the central loop of the aptamer quadruplex resulted in increased nuclease resistance and an inhibition efficiency similar to that of TBA15. The likely aptamer-thrombin binding mode was determined by molecular dynamics simulations. Due to their relatively high activity and the increased resistance to nuclease digestion imparted by the triazole internucleotide linkages, the novel aptamers are a promising alternative to known DNA-based anticoagulant agents.
Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Nucleotídeos/química , Inibidores de Serina Proteinase/farmacologia , Trombina/antagonistas & inibidores , Trombina/metabolismo , Triazóis/química , Aptâmeros de Nucleotídeos/síntese química , Aptâmeros de Nucleotídeos/metabolismo , Sítios de Ligação , Relação Dose-Resposta a Droga , Quadruplex G , Conformação Molecular , Simulação de Dinâmica Molecular , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Relação Estrutura-Atividade , Trombina/química , Triazóis/farmacologiaRESUMO
Oligonucleotide analogs containing one or a few glycine, L-, and D-alanine or L-and D-phenylalanine residues instead of phosphodiesterinternucleotide linkages were synthesized. The stability of the duplexes formed by modified oligonucleotides and their wildtype complements was studied. Oligonucleotides with D-alanine residues in internucleotide linkages form duplexes more stable than native ones (ΔT(m) +0.2 °C per modification), whereas other modifications destabilize the duplexes.
Assuntos
Oligonucleotídeos/química , Oligonucleotídeos/síntese química , Ácidos Nucleicos Peptídicos/síntese química , Alanina/química , Glicina/química , Hibridização de Ácido Nucleico , Fenilalanina/químicaRESUMO
This unit describes a simple and efficient synthesis of the phosphoramidite derivative of N(1)-methyl-2'-deoxyadenosine from 2'-deoxyadenosine. The synthesis starts with the monomethoxytritylation of 2'-deoxyadenosine followed by methylation of 5'-O-protected nucleoside at N-1. Subsequent N-chloroacetylation leads to N(6)-chloroacetyl-N(1)-methyl-5'-O-(p-anisyldiphenylmethyl)-2'-deoxyadenosine, which is finally converted to its 3' phosphoramidite derivative. This phosphoramidite is used to incorporate N(1)-methyl-2'-deoxyadenosine into synthetic oligonucleotides. N-Chloroacetyl protection and controlled anhydrous deprotection conditions are used to avoid the Dimroth rearrangement.