RESUMO
We analyzed the effect of modified nucleotides within gapmer antisense oligonucleotides on RNase H mediated gene silencing. Additionally, short hairpins were introduced into antisense oligonucleotides as structural motifs, and their influence on biological and physicochemical properties of pre-structured gapmers was investigated for the first time. The results indicate that two LNA residues in specified positions of the gap flanking regions are sufficient and favorable for efficient knock-down of the ß-actin gene. Furthermore, the introduction of other modified nucleotides, i. e. glycyl-amino-LNA-T, 2'-O-propagyluridine, polyamine functionalized uridine, and UNA, in specified positions, also increases the inhibition of ß-actin expression. Importantly, the presence of hairpins within the gapmers improves their silencing properties.
Assuntos
Actinas , Oligonucleotídeos Antissenso , Expressão Gênica , Nucleotídeos , Oligonucleotídeos Antissenso/química , Ribonuclease H/genética , Ribonuclease H/metabolismoRESUMO
The low binding affinity of unmodified triplex-forming oligonucleotides (TFO) is the main drawback to their promising utilization in gene therapy. In the present study, we have synthesized DNA intercalator 5-(pyren-1-ylethynyl)indole Y, known as twisted intercalating nucleic acid (TINA), by a Cu-mediated Sonogashira palladium-catalyzed coupling reaction of 1-ethynylpyrene with 5-iodoindole at a high temperature under anaerobic conditions. Coupling with indole C-5 was far more preferable in obtaining stable TINA-indole than enamine site C-3, as neither hydration of the triple bond to ketones nor competitive Glaser-type homocoupling of acetylenes was observed. The insertion of the new TINA monomer Y as a bulge in the middle or at the 5'-end of the oligodeoxynucleotide sequence via a flexible butane-1,2-diol linker showed extraordinary binding potential, resulting in excellent thermal stabilization of Hoogsteen-type triplex- and duplex-deoxyribonucleic acid (DNA) structures which was detected by thermal denaturation studies and supported by circular dichroism (CD). Molecular dynamics AMBER* revealed the lowest energy conformation in which a pyrenyl residue of the TINA monomer Y stacks in the dsDNA part, while an indolyl unit intercalates between the nucleobases of the TFO pattern. Overall the torsionally rigid conjugated TINA system with a decent twisting of 15.1° around acetylene is confirmed here as a requirement for the best fit inside the intercalation site of the triplex, resulting in high TFO-dsDNA affinity.
Assuntos
Substâncias Intercalantes , Ácidos Nucleicos , Temperatura , Substâncias Intercalantes/química , Oligonucleotídeos/química , Pirenos/química , DNA/química , Ácidos Nucleicos/química , Indóis , Conformação de Ácido Nucleico , Desnaturação de Ácido NucleicoRESUMO
Whereas locked nucleic acid (LNA) has been extensively used to control gene expression, it has never been exploited to control Candida virulence genes. Thus, the main goal of this work was to compare the efficacy of five different LNA-based antisense oligonucleotides (ASO) with respect to the ability to control EFG1 gene expression, to modulate filamentation and to reduce C. albicans virulence. In vitro, all LNA-ASOs were able to significantly reduce C. albicans filamentation and to control EFG1 gene expression. Using the in vivo Galleria mellonella model, important differences among the five LNA-ASOs were revealed in terms of C. albicans virulence reduction. The inclusion of PS-linkage and palmitoyl-2'-amino-LNA chemical modification in these five LNA gapmers proved to be the most promising combination, increasing the survival of G. mellonella by 40%. Our work confirms that LNA-ASOs are useful tools for research and therapeutic development in the candidiasis field.
Assuntos
Candida albicans , Candidíase , Candida albicans/genética , Oligonucleotídeos/farmacologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologiaRESUMO
Attachment of cationic moieties to oligonucleotides (ONs) promises not only to increase the binding affinity of antisense ONs by reducing charge repulsion between the two negatively charged strands of a duplex, but also to augment their in vivo stability against nucleases. In this study, polyamine functionality was introduced into ONs by means of 2'-amino-LNA scaffolds. The resulting ONs exhibited efficient binding towards ssDNA, ssRNA and dsDNA targets, and the 2'-amino-LNA analogue carrying a triaminated linker showed the most pronounced duplex- and triplex-stabilizing effect. Molecular modelling revealed that favourable conformational and electrostatic effects led to salt-bridge formation between positively charged polyamine moieties and the Watson-Hoogsteen groove of the dsDNA targets, resulting in the observed triplex stabilization. All the investigated monomers showed increased resistance against 3'-nucleolytic digestion relative to the non-functionalized controls.
Assuntos
Oligonucleotídeos , Poliaminas , DNA/química , DNA de Cadeia Simples/química , Oligonucleotídeos/químicaRESUMO
OBJECTIVES: There are many mobile health applications (apps) now available and some that use in some way laboratory medicine data. Among them, patient-oriented are of the lowest content quality. The aim of this study was to compare the opinions of non-laboratory medicine professionals (NLMP) with those of laboratory medicine specialists (LMS) and define the benchmarks for quality assessment of laboratory medicine apps. METHODS: Twenty-five volunteers from six European countries evaluated 16 selected patient-oriented apps. Participants were 20-60 years old, 44% were females, with different educational degrees, and no professional involvement in laboratory medicine. Each participant completed a questionnaire based on the Mobile Application Rating Scale (MARS) and the System Usability Scale, as previously used for rating the app quality by LMS. The responses from the two groups were compared using the Mann-Whitney U test and Spearman correlation. RESULTS: The median total score of NLMP app evaluation was 2.73 out of 5 (IQR 0.95) compared to 3.78 (IQR 1.05) by the LMS. All scores were statistically significantly lower in the NLMP group (p<0.05), except for the item Information quality (p=0.1631). The suggested benchmarks for a useful appear: increasing awareness of the importance and delivering an understanding of persons' own laboratory test results; understandable terminology; easy to use; appropriate graphic design, and trustworthy information. CONCLUSIONS: NLMP' evaluation confirmed the low utility of currently available laboratory medicine apps. A reliable app should contain trustworthy and understandable information. The appearance of an app should be fit for purpose and easy to use.
Assuntos
Aplicativos Móveis , Telemedicina , Adulto , Benchmarking , Feminino , Humanos , Laboratórios , Pessoa de Meia-Idade , Smartphone , Adulto JovemRESUMO
Off-target effects remain a significant challenge in the therapeutic use of gapmer antisense oligonucleotides (AONs). Over the years various modifications have been synthesized and incorporated into AONs, however, precise control of RNaseâ H-induced cleavage and target sequence selectivity has yet to be realized. Herein, the synthesis of the uracil and cytosine derivatives of a novel class of 2'-deoxy-2'-fluoro-3'-C-hydroxymethyl-ß-d-lyxo-configured nucleotides has been accomplished and the target molecules have been incorporated into AONs. Experiments on exonuclease degradation showed improved nucleolytic stability relative to the unmodified control. Upon the introduction of one or two of the novel 2'-fluoro-3'-C-hydroxymethyl nucleotides as modifications in the gap region of a gapmer AON was associated with efficient RNaseâ H-mediated cleavage of the RNA strand of the corresponding AON:RNA duplex. Notably, a tailored single cleavage event could be engineered depending on the positioning of a single modification. The effect of single mismatched base pairs was scanned along the full gap region demonstrating that the modification enables a remarkable specificity of RNaseâ H cleavage. A cell-based model system was used to demonstrate the potential of gapmer AONs containing the novel modification to mediate gene silencing.
Assuntos
Inativação Gênica , Nucleotídeos/química , Oligonucleotídeos Antissenso/química , Ribonuclease H/metabolismo , Sequência de Bases , Estabilidade Enzimática , Células HeLa , Humanos , Concentração Inibidora 50 , Desnaturação de Ácido Nucleico , Nucleotídeos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , RNA/química , RNA/metabolismo , Temperatura , TransfecçãoRESUMO
Synthesis of the novel thiophenyl carbazole phosphoramidite DNA building block 5 was accomplished in four steps using a Suzuki-Miyaura cross-coupling reaction from the core carbazole and it was seamlessly accommodated into a 9-mer DNA-based oligonucleotide by incorporation at the flanking 5'-end in combination with a central insertion of an LNA-T nucleotide. The carbazole-containing oligonucleotide was combined in different duplex hybrids, which were characterized by thermal denaturation, circular dichroism and fluorescence studies. The carbazole monomer modulates the duplex stability in various ways. Thus, monomer Z increased the thermal stability of the 9-mer towards the complementary 9-mer/15-mer DNA duplex by 4.2 °C. Furthermore, indications of its intercalation into the duplex were obtained by modeling studies and robust decreases in fluorescence emission intensities upon duplex formation. In contrast, no clear intercalating tendency was corroborated for monomer Z within the DNA/RNA hybrid duplex as indicated by moderate quenching of the fluorescence and similar duplex thermal stabilities relative to the corresponding control duplex. The recognition efficiencies of the carbazole modified oligonucleotide toward single nucleotide mismatches were studied with two 15-mer model targets (DNA and RNA). For both systems, mismatches positioned at the juxtaposition of the carbazole monomer showed pronounced deceases in thermal denaturation temperature. Steady-state fluorescence emission studies of all mismatched duplexes with incorporation of Z monomer typically displayed efficient fluorescence quenching.
Assuntos
OligonucleotídeosRESUMO
Alpha-l-Locked nucleic acid (α-l-LNA) is a stereoisomeric analogue of locked nucleic acid (LNA), which possesses excellent biophysical properties and also exhibits high target binding affinity to complementary oligonucleotide sequences and resistance to nuclease degradations. Therefore, α-l-LNA nucleotides could be utilised to develop stable antisense oligonucleotides (AO), which can be truncated without compromising the integrity and efficacy of the AO. In this study, we explored the potential of α-l-LNA nucleotides-modified antisense oligonucleotides to modulate splicing by inducing Dmd exon-23 skipping in mdx mouse myoblasts in vitro. For this purpose, we have synthesised and systematically evaluated the efficacy of α-l-LNA-modified 2'-O-methyl phosphorothioate (2'-OMePS) AOs of three different sizes including 20mer, 18mer and 16mer AOs in parallel to fully-modified 2'-OMePS control AOs. Our results demonstrated that the 18mer and 16mer truncated AO variants showed slightly better exon-skipping efficacy when compared with the fully-23 modified 2'-OMePS control AOs, in addition to showing low cytotoxicity. As there was no previous report on using α-l-LNA-modified AOs in splice modulation, we firmly believe that this initial study could be beneficial to further explore and expand the scope of α-l-LNA-modified AO therapeutic molecules.
Assuntos
Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Éxons , Camundongos , Camundongos Endogâmicos mdx , Mioblastos/efeitos dos fármacos , Nucleotídeos/metabolismoRESUMO
A number of current trends will affect and probably change laboratory medicine, as we know it. Scientific and technological developments, digital health with big data and artificial intelligence, and centralization will change the interfaces among the specialties of laboratory medicine. They might even challenge the identity of some specialties. Other trends such as demographic changes, increased complexity of health care, digital health with electronic health records, and more demanding and well-informed patients will change the way laboratory medicine specialties deliver their services. This paper discusses the possible changes of laboratory medicine in Denmark - a Scandinavian country where almost all hospitals are public. If Danish laboratories grasp the new possibilities instead of trying to avoid them, laboratory medicine is likely to prosper. Such a positive development will call upon good leadership and a genuine willingness among laboratory specialist to adapt to a future where their own specialty might be very different from today.
Assuntos
Atenção à Saúde , Ciência de Laboratório Médico , Dinamarca , HumanosRESUMO
Targeting and invading double-stranded DNA with synthetic oligonucleotides under physiological conditions remain a challenge. Bis-locked nucleic acids (bisLNAs) are clamp-forming oligonucleotides able to invade into supercoiled DNA via combined Hoogsteen and Watson-Crick binding. To improve the bisLNA design, we investigated its mechanism of binding. Our results suggest that bisLNAs bind via Hoogsteen-arm first, followed by Watson-Crick arm invasion, initiated at the tail. Based on this proposed hybridization mechanism, we designed next-generation bisLNAs with a novel linker able to stack to adjacent nucleobases, a new strategy previously not applied for any type of clamp-constructs. Although the Hoogsteen-arm limits the invasion, upon incorporation of the stacking linker, bisLNA invasion is significantly more efficient than for non-clamp, or nucleotide-linker containing LNA-constructs. Further improvements were obtained by substituting LNA with 2'-glycylamino-LNA, contributing a positive charge. For regular bisLNAs a 14-nt tail significantly enhances invasion. However, when two stacking linkers were incorporated, tail-less bisLNAs were able to efficiently invade. Finally, successful targeting of plasmids inside bacteria clearly demonstrates that strand invasion can take place in a biologically relevant context.
Assuntos
DNA Bacteriano/metabolismo , DNA Super-Helicoidal/metabolismo , Glicina/análogos & derivados , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos/metabolismo , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/antagonistas & inibidores , DNA Bacteriano/química , DNA Super-Helicoidal/química , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Oligonucleotídeos/síntese química , Oligonucleotídeos Antissenso/síntese química , Plasmídeos/química , Plasmídeos/metabolismo , Técnicas de Síntese em Fase Sólida , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
The thrombin-binding aptamer (TBA), which shows anticoagulant properties, is one of the most studied G-quadruplex-forming aptamers. In this study, we investigated the impact of different chemical modifications such as a three-carbon spacer (spacer-C3 ), unlocked nucleic acid (UNA) and 3'-amino-modified UNA (amino-UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G-quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer-C3 introduction at the T7 loop position (TBA-SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling experiments provided novel insights into the experimental observations, further supporting the efficacy of TBA-SP7. The results of this study could provide valuable information for future designs of TBA analogues with superior thrombin inhibition properties.
Assuntos
Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/síntese química , Quadruplex G , Coagulação Sanguínea , Modelos Químicos , Simulação de Dinâmica Molecular , Estrutura Molecular , Processos EstocásticosRESUMO
BACKGROUND: There is increasing interest in direct patient engagement including receiving their laboratory medicine results. We previously established an appetite for Specialists in Laboratory Medicine to support patients in understanding results. The aim of this study was to establish whether patients agreed with such an approach, determined through surveying views in eight European countries. METHODS: A standardized five-question survey was administered across eight European countries to a total of 1084 individuals attending medical outpatient clinics, with 100 patients each in Poland, Serbia, Netherlands, Turkey and Czech Republic, 101 in Estonia, 116 in Denmark and 367 in Norway. The responses across countries were compared using the chi-square test (p<0.05). RESULTS: Patients wanting their results ranged from 50% to 94% (mean 65%) of those responding positively, a mean of 72% wanted additional information with their results; direct receipt was preferred over referral to a website. Specialists in Laboratory Medicine providing such information were acceptable to a mean of 62% of those respondents wishing their results; in countries where payment was possible, there was little interest in making additional payment for such a service. CONCLUSIONS: A clear proportion of patients are interested in receiving their laboratory medicine results, the majority with explanatory notes; a role for Specialists in Laboratory Medicine is acceptable and raises the potential for direct engagement by such specialists with patients offering a new paradigm for the provision of laboratory medicine activities.
Assuntos
Laboratórios Hospitalares , Pacientes/psicologia , Europa (Continente) , Humanos , Internet , Especialização , Inquéritos e QuestionáriosRESUMO
Galactose-modified thymidine, LNA-T, and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2'-functionalized 2'-amino-LNA derivatives in particular, showed improved duplex thermal stability against DNA and RNA complements and increased ability to discriminate mismatches. In addition, the 2'-amino-LNA-T derivatives induced remarkable 3'-exonuclease resistance. These results were further investigated using molecular modeling studies.
Assuntos
DNA/química , Galactose/química , Oligonucleotídeos/química , Fenômenos Biofísicos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Modelos Moleculares , Desnaturação de Ácido Nucleico , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray , TemperaturaRESUMO
BACKGROUND: Medicine is a highly professionalized endeavour, by tradition centred on the authority of physicians. Better education and the advent of the information age cater for increased demands on society in general and on health care in particular to enable people to make informed decisions regarding themselves. Participation in medical decisions requires informed knowledge which is hard to obtain without substantial and time consuming professional help. METHODS: We performed a survey amongst the member organizations of European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) in order to investigate the recognition and preparedness of providing help to patients in interpreting their laboratory results. RESULTS: Out of 40 EFLM Member Societies, 27 sent their responses to the survey. In most cases the first line delivery of laboratory results to physicians is by computer link (63%). Patients receive their laboratory results on demand from their physician in 60% of cases. However, 34% of laboratory specialists showed a negative attitude for delivering laboratory results to patients. Yet, in 48% of countries 1-5 patients per day ask a laboratory specialist about the significance of laboratory results outside the reference range. When patients are informed about the purpose of laboratory testing, they seek information primarily from their physician, followed by the internet and the Specialist in Laboratory Medicine. CONCLUSIONS: Changing practices increasingly enabling patient access to their records are on the increase facilitated by recent innovations in information technologies. Successful transfer of some of the responsibilities of physicians, demands a mutual triangular dialogue between the patient, their physician and laboratory medicine.
Assuntos
Sistemas de Informação em Laboratório Clínico , Acesso dos Pacientes aos Registros , Educação de Pacientes como Assunto , Europa (Continente) , Humanos , Informática Médica , Relações Médico-Paciente , Médicos , Inquéritos e QuestionáriosRESUMO
In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasion into duplex DNA (DSI). We thus report on the development of a clamp type of LNA ON-bisLNA-with capacity to bind and invade into supercoiled double-stranded DNA. The bisLNA links a triplex-forming, Hoogsteen-binding, targeting arm with a strand-invading Watson-Crick binding arm. Optimization was carried out by varying the number and location of LNA nucleotides and the length of the triplex-forming versus strand-invading arms. Single-strand regions in target duplex DNA were mapped using chemical probing. By combining design and increase in LNA content, it was possible to achieve a 100-fold increase in potency with 30% DSI at 450 nM using a bisLNA to plasmid ratio of only 21:1. Although this first conceptual report does not address the utility of bisLNA for the targeting of DNA in a chromosomal context, it shows bisLNA as a promising candidate for interfering also with cellular genes.
Assuntos
DNA Super-Helicoidal/química , Oligonucleotídeos/química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Soluções Tampão , DNA/química , Clivagem do DNA , Enzimas de Restrição do DNA/química , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oligonucleotídeos/síntese química , Plasmídeos/química , Temperatura de TransiçãoRESUMO
BACKGROUND: Information about the cost-effectiveness of early intervention programmes for first-episode psychosis is limited. AIMS: To evaluate the cost-effectiveness of an intensive early-intervention programme (called OPUS) (trial registration NCT00157313) consisting of enriched assertive community treatment, psychoeducational family treatment and social skills training for individuals with first-episode psychosis compared with standard treatment. METHOD: An incremental cost-effectiveness analysis of a randomised controlled trial, adopting a public sector perspective was undertaken. RESULTS: The mean total costs of OPUS over 5 years (123,683, s.e. = 8970) were not significantly different from that of standard treatment (148,751, s.e. = 13073). At 2-year follow-up the mean Global Assessment of Functioning (GAF) score in the OPUS group (55.16, s.d. = 15.15) was significantly higher than in standard treatment group (51.13, s.d. = 15.92). However, the mean GAF did not differ significantly between the groups at 5-year follow-up (55.35 (s.d. = 18.28) and 54.16 (s.d. = 18.41), respectively). Cost-effectiveness planes based on non-parametric bootstrapping showed that OPUS was less costly and more effective in 70% of the replications. For a willingness-to-pay up to 50,000 the probability that OPUS was cost-effective was more than 80%. CONCLUSIONS: The incremental cost-effectiveness analysis showed that there was a high probability of OPUS being cost-effective compared with standard treatment.
Assuntos
Serviços Comunitários de Saúde Mental/economia , Intervenção Médica Precoce/economia , Custos de Cuidados de Saúde/estatística & dados numéricos , Serviços de Saúde/estatística & dados numéricos , Avaliação de Processos e Resultados em Cuidados de Saúde , Transtornos Psicóticos/economia , Adolescente , Adulto , Antipsicóticos/uso terapêutico , Serviços Comunitários de Saúde Mental/organização & administração , Análise Custo-Benefício , Dinamarca , Grupos Diagnósticos Relacionados/economia , Terapia Familiar/economia , Humanos , Análise de Intenção de Tratamento , Pessoa de Meia-Idade , Equipe de Assistência ao Paciente/economia , Educação de Pacientes como Assunto/economia , Transtornos Psicóticos/terapia , Esquizofrenia/economia , Esquizofrenia/terapia , Método Simples-Cego , Socialização , Adulto JovemRESUMO
Huntington's disease is a neurodegenerative, trinucleotide repeat (TNR) disorder affecting both males and females. It is caused by an abnormal increase in the length of CAGâ¢CTG TNR in exon 1 of the Huntingtin gene (HTT). The resultant, mutant HTT mRNA and protein cause neuronal toxicity, suggesting that reduction of their levels would constitute a promising therapeutic approach. We previously reported a novel strategy in which chemically modified oligonucleotides (ONs) directly target chromosomal DNA. These anti-gene ONs were able to downregulate both HTT mRNA and protein. In this study, various locked nucleic acid (LNA)/DNA mixmer anti-gene ONs were tested to investigate the effects of varying ON length, LNA content, and fatty acid modification on HTT expression. Altering the length did not significantly influence the ON potency, while LNA content was critical for activity. Utilization of palmitoyl-modified LNA monomers enhanced the ON activity relatively to the corresponding nonmodified LNA under serum starvation conditions. Furthermore, the number of palmitoylated LNA monomers and their positioning greatly affected ON potency. In addition, we performed RNA sequencing analysis, which showed that the anti-gene ONs affect the "immune system process, mRNA processing, and neurogenesis." Furthermore, we observed that for repeat containing genes, there is a higher tendency for antisense off-targeting. Taken together, our findings provide an optimized design of anti-gene ONs that could potentially be developed as DNA-targeting therapeutics for this class of TNR-related diseases.
Assuntos
Doença de Huntington , Oligonucleotídeos , Masculino , Humanos , Oligonucleotídeos/genética , Oligonucleotídeos/farmacologia , Oligonucleotídeos/química , Oligonucleotídeos Antissenso/farmacologia , DNA/uso terapêutico , Expressão Gênica , RNA Mensageiro/metabolismo , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/terapiaRESUMO
A new intercalating nucleic acid monomer M comprising a 4-(1-indole)-butane-1,2-diol moiety was synthesized via a classical alkylation reaction of indole-3-carboxaldehyde followed by a condensation reaction with phenanthrene-9,10-dione in the presence of ammonium acetate to form a phenanthroimidazole moiety linked to the indole ring. Insertion of the new intercalator as a bulge into a Triplex Forming Oligonucleotide resulted in good thermal stability of the corresponding Hoogsteen-type triplexes. Molecular modeling supports the possible intercalating ability of M. Hybridisation properties of DNA/DNA and RNA/DNA three-way junctions (TWJ) with M in the branching point were also evaluated by their thermal stability at pH 7. DNA/DNA TWJ showed increase in thermal stability compared to wild type oligonucleotides whereas this was not the case for RNA/DNA TWJ.
Assuntos
Imidazóis/química , Indóis/química , Substâncias Intercalantes/química , Oligonucleotídeos/química , DNA/química , Substâncias Intercalantes/síntese química , Modelos Moleculares , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Raios UltravioletaRESUMO
A new locked pyranosyl nucleoside was synthesized by phenylsulfinyl-assisted chemistry. The novel building block was inserted into oligonucleotides and provides new insight on conformational restricted pyranosyl nucleosides on duplex formation.
Assuntos
Oligonucleotídeos/síntese química , Piranos/química , Sequência de Bases , Modelos Moleculares , Oligonucleotídeos/química , Ozônio , Transição de Fase , Temperatura de TransiçãoRESUMO
Oligonucleotides with the sequences 5'-GTG AUPA TGC, 5'-GCA TAUP CAC and 5'-GUPG ATA UPGC, where UP is 2'-O-propargyl uridine, were subjected to post-synthetic Cu(I)-catalyzed azide-alkyne cycloaddition to attach 1,4,7,10-tetraazacyclododecane (cyclen) and two well-known DNA intercalating dyes: thioxanthone and 1,8-naphthalimide. We propose a convenient cyclen protection-deprotection strategy that allows efficient separation of the resulting polyamine-oligonucleotide conjugates from the starting materials by RP-HPLC to obtain high-purity products. In this paper, we present hitherto unknown macrocyclic polyamine-oligonucleotide conjugates and their hybridization properties reflected in the thermal stability of thirty-two DNA duplexes containing combinations of labeled strands, their unmodified complementary strands, and strands with single base pair mismatches. Circular dichroism measurements showed that the B-conformation is retained for all dsDNAs consisting of unmodified and modified oligonucleotides. An additive and destabilizing effect of cyclen moieties attached to dsDNAs was observed. Tm measurements indicate that placing the hydrophobic dye opposite to the cyclen moiety can reduce its destabilizing effect and increase the thermal stability of the duplex. Interestingly, the cyclen-modified U showed significant selectivity for TT mismatch, which resulted in stabilization of the duplex. We conclude the paper with a brief review and discussion in which we compare our results with several examples of oligonucleotides labeled with polyamines at internal strand positions known in the literature.