RESUMO
BACKGROUND: OZ439 is a new chemical entity which is active against drug-resistant malaria and shows potential as a single-dose cure. However, development of an oral formulation with desired exposure has proved problematic, as OZ439 is poorly soluble (BCS Class II drug). In order to be feasible for low and middle income countries (LMICs), any process to create or formulate such a therapeutic must be inexpensive at scale, and the resulting formulation must survive without refrigeration even in hot, humid climates. We here demonstrate the scalability and stability of a nanoparticle (NP) formulation of OZ439. Previously, we applied a combination of hydrophobic ion pairing and Flash NanoPrecipitation (FNP) to formulate OZ439 NPs 150 nm in diameter using the inexpensive stabilizer hydroxypropyl methylcellulose acetate succinate (HPMCAS). Lyophilization was used to process the NPs into a dry form, and the powder's in vitro solubilization was over tenfold higher than unprocessed OZ439. METHODS: In this study, we optimize our previous formulation using a large-scale multi-inlet vortex mixer (MIVM). Spray drying is a more scalable and less expensive operation than lyophilization and is, therefore, optimized to produce dry powders. The spray dried powders are then subjected to a series of accelerated aging stability trials at high temperature and humidity conditions. RESULTS: The spray dried OZ439 powder's dissolution kinetics are superior to those of lyophilized NPs. The powder's OZ439 solubilization profile remains constant after 1 month in uncapped vials in an oven at 50 °C and 75% RH, and for 6 months in capped vials at 40 °C and 75% RH. In fasted-state intestinal fluid, spray dried NPs achieved 80-85% OZ439 dissolution, to a concentration of 430 µg/mL, within 3 h. In fed-state intestinal fluid, 95-100% OZ439 dissolution is achieved within 1 h, to a concentration of 535 µg/mL. X-ray powder diffraction and differential scanning calorimetry profiles similarly remain constant over these periods. CONCLUSIONS: The combined nanofabrication and drying process described herein, which utilizes two continuous unit operations that can be operated at scale, is an important step toward an industrially-relevant method of formulating the antimalarial OZ439 into a single-dose oral form with good stability against humidity and temperature.
Assuntos
Adamantano/análogos & derivados , Malária/tratamento farmacológico , Sprays Orais , Peróxidos/administração & dosagem , Pós , Adamantano/administração & dosagem , Adamantano/farmacocinética , Administração Oral , Química Farmacêutica , Dessecação , Estabilidade de Medicamentos , Liofilização , Humanos , Nanopartículas/química , Nebulizadores e Vaporizadores , Peróxidos/farmacocinética , Solubilidade , Água/químicaRESUMO
Clofazimine, a drug previously used to treat leprosy, has recently been identified as a potential new drug for the treatment for cryptosporidiosis: a diarrheal disease that contributes to 500 000 infant deaths a year in developing countries. Rapid dissolution and local availability of the drug in the small intestine is considered key to the treatment of the infection. However, the commercially available clofazimine formulation (Lamprene) is not well-suited to pediatric use, and therefore reformulation of clofazimine is desirable. Development of clofazimine nanoparticles through the process of flash nanoprecipitation (FNP) has been previously shown to provide fast and improved drug dissolution rates compared to clofazimine crystals and Lamprene. In this study, we investigate the effects of milk-based formulations (as possible pediatric-friendly vehicles) on the in vitro solubilization of clofazimine formulated as either lecithin- or zein/casein-stabilized nanoparticles. Milk and infant formula were used as the lipid vehicles, and time-resolved synchrotron X-ray scattering was used to monitor the presence of crystalline clofazimine in suspension during in vitro lipolysis under intestinal conditions. The study confirmed faster dissolution of clofazimine from all the FNP formulations after the digestion of infant formula was initiated, and a reduced quantity of fat was required to achieve similar levels of drug solubilization compared to the reference drug material and the commercial formulation. These attributes highlight not only the potential benefits of the FNP approach to prepare drug particles but also the fact that enhanced dissolution rates can be complemented by considering the amount of co-administered fat in lipid-based formulations to drive the solubilization of poorly soluble drugs.
Assuntos
Clofazimina/química , Composição de Medicamentos , Liberação Controlada de Fármacos , Excipientes/química , SolubilidadeRESUMO
More than 40% of newly developed drug molecules are highly hydrophobic and, thus, suffer from low bioavailability. Kinetically trapping the drug as a nanoparticle in an amorphous state enhances solubility. However, enhanced solubility can be compromised by subsequent recrystallization from the amorphous state during drying processes. We combine Flash NanoPrecipitation (FNP) to generate nanoparticles with spray-drying to produce stable solid powders. We demonstrate that the continuous nanofabrication platform for nanoparticle synthesis and recovery does not compromise the dissolution kinetics of the drug. Lumefantrine, an anti-malaria drug, is highly hydrophobic with low bioavailability. Increasing the bioavailability of lumefantrine has the potential to reduce the dose and number of required administrations per treatment, thus reducing cost and increasing patient compliance. The low melting temperature of lumefantrine (Tm = 130 °C) makes the drying of amorphous nanoparticles at elevated temperatures potentially problematic. Via FNP, we produced 200-400 nm nanoparticles using hydroxypropyl methylcellulose acetate succinate (HPMCAS), lecithin phospholipid, and zein protein stabilizers. Zein nanoparticles were spray-dried at 100 °C and 120 °C to study the effect of the drying temperature. For zein powders, at two hours the dissolution kinetics under fasted conditions reached 85% release for the 100 °C sample, but only 60% release for the 120 °C sample. Powder X-ray diffraction, differential scanning calorimetry, and solid state nuclear magnetic resonance indicate that the lumefantrine in the nanoparticle core is amorphous for samples spray-dried at 100 °C. Dissolution under fed state conditions showed similar release kinetics for both temperatures, with 90-95% release at two hours. Zein and HPMCAS nanoparticles spray-dried at 100 °C showed release profiles in fasted and fed state media that are identical to those of lyophilized samples, i.e. those dried at cryogenic conditions where no transformation to the crystalline state can occur. Thus, spray drying 30 °C below the melting transition of lumefantrine is sufficient to maintain the amorphous state. These inexpensive formulations have potential to be developed into future therapies for malaria, and the results also highlight the potential of combining FNP and spray-drying as a versatile platform to assemble and rapidly recover amorphous nanoparticles in a solid dosage form.
RESUMO
We report the use of flash nanoprecipitation (FNP) as an efficient and scalable means of producing Cellax nanoparticles. Cellax polymeric conjugates consisting of carboxymethyl cellulose functionalized with PEG and hydrophobic anticancer drugs, such as cabazitaxel (coined Cellax-CBZ), have been shown to have high potency against several oncology targets, including prostate cancer. FNP, a robust method used to create nanoparticles through rapid mixing, has been used to encapsulate several hydrophobic drugs with block copolymer stabilizers, but has never been used to form nanoparticles from random copolymers, such as Cellax-CBZ. To assess the potential of using FNP to produce Cellax nanoparticles, parameters such as concentration, mixing rate, solvent ratios, and subsequent dilution were tested with a target nanoparticle size range of 60 nm. Under optimized solvent conditions, particles were formed that underwent a subsequent rearrangement to form nanoparticles of 60 nm diameter, independent of Cellax-CBZ polymer concentration. This intraparticle relaxation, without interparticle association, points to a delicate balance of hydrophobic/hydrophilic domains on the polymer backbone. These particles were stable over time, and the random amphiphilicity did not lead to interparticle attractions, which would compromise the stability and corresponding narrow size distribution required for parenteral injection. The amphiphilic nature of these conjugates allows them to be processed into nanoparticles for sustained drug release and improved tumor selectivity. Preferred candidates were evaluated for plasma stability and cytotoxicity against the PC3 prostate cancer cell line in vitro. These parameters are important when assessing nanoparticle safety and for estimating potential efficacy, respectively. The optimal formulations showed plasma stability profiles consistent with long circulating nanoparticles, and cytotoxicity comparable to that of free CBZ. This study demonstrates that FNP is a promising technology for development of Cellax nanoparticles.
Assuntos
Carboximetilcelulose Sódica/química , Nanopartículas/química , Polietilenoglicóis/química , Polímeros/química , Taxoides/química , Linhagem Celular Tumoral , Humanos , MasculinoRESUMO
Clofazimine, a lipophilic (log P = 7.66) riminophenazine antibiotic approved by the US Food and Drug Administration (FDA) with a good safety record, was recently identified as a lead hit for cryptosporidiosis through a high-throughput phenotypic screen. Cryptosporidiosis requires fast-acting treatment as it leads to severe symptoms which, if untreated, result in morbidity for infants and small children. Consequently, a fast-releasing oral formulation of clofazimine in a water-dispersible form for pediatric administration is highly desirable. In this work, clofazimine nanoparticles were prepared with three surface stabilizers, hypromellose acetate succinate (HPMCAS), lecithin, and zein, using the flash nanoprecipitation (FNP) process. Drug encapsulation efficiencies of over 92% were achieved. Lyophilization and spray-drying were applied and optimized to produce redispersible nanoparticle powders. The release kinetics of these clofazimine nanoparticle powders in biorelevant media were measured and compared with those of crystalline clofazimine and the currently marketed formulation Lamprene. Remarkably improved dissolution rates and clofazimine supersaturation levels up to 90 times equilibrium solubility were observed with all clofazimine nanoparticles tested. Differential scanning calorimetry indicated a reduction of crystallinity of clofazimine in nanoparticles. These results strongly suggest that the new clofazimine nanoparticles prepared with affordable materials in this low-cost nanoparticle formulation process can be used as viable cryptosporidiosis therapeutics.
Assuntos
Antiparasitários/farmacologia , Clofazimina/farmacologia , Criptosporidiose/tratamento farmacológico , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Desenho de Fármacos , Antiparasitários/economia , Antiparasitários/uso terapêutico , Varredura Diferencial de Calorimetria , Química Farmacêutica , Clofazimina/economia , Clofazimina/uso terapêutico , Cristalização , Dessecação , Portadores de Fármacos/economia , Composição de Medicamentos/economia , Liberação Controlada de Fármacos , Excipientes/química , Liofilização , Nanopartículas/química , Nanopartículas/economia , Tamanho da Partícula , Solubilidade , Fatores de TempoRESUMO
The seemingly inevitable protein corona appears to be an insurmountable obstacle to wider application of functional nanomaterials in biotechnology. The accumulation of serum proteins can block targeting functionalities and alter the in vivo fate of these nanomaterials. Here we demonstrate a method to generate non-stick, robustly passivated functional nanoparticles (NPs) using a tailored silica coating. We apply agarose gel electrophoresis to sensitively evaluate protein binding to NPs with different surface chemistry. Using gel banding and retardation as a read-out for protein adsorption, we optimize the surface chemistry to yield a mixed charge surface which displays remarkable binding resistance to a wide range of serum proteins and nucleic acids. The hard silica shell also protects the functional NP core in harsh environments (down to pH 1) while still showing the ability to be targeted for cellular uptake with little or no non-specific binding.
Assuntos
DNA/química , Nanopartículas/química , Proteínas/química , Dióxido de Silício/química , Animais , Géis , Concentração de Íons de Hidrogênio , Camundongos , Células NIH 3T3 , Pontos Quânticos/químicaRESUMO
The vast majority of deoxyribozyme-based sensors are designed using modified RNA-cleaving deoxyribozymes and detect analytes that act as allosteric regulators of their catalytic activity. These sensors are susceptible to background signals due to catalytic activity in the absence of target or contaminant molecules that cleave the RNA substrate, mimicking the deoxyribozyme reaction. In this manuscript, we introduce a novel system that avoids these problems by using the analyte as the substrate for a deoxyribozyme catalyzed self-phosphorylation reaction. This reaction creates a modified deoxyribozyme product that can be circularized and subjected to massive signal amplification by rolling circle amplification, leading to a sensor system with high sensitivity and low background, which can be coupled to numerous reporter systems. As an example of the potential of this system, we used the self-phosphorylating deoxyribozyme Dk2 to detect as little as 25 nM GTP even in the presence of 1 mM ATP, a potential contaminant. To demonstrate the adaptive properties of this system, we appended another DNA sequence to Dk2, which, once amplified by RCA, codes for a fluorescence generating deoxyribozyme. This two-deoxyribozyme system was able to report the presence of GTP from 4 µM to 1 mM, with specificity over other NTP molecules. Using this model system, we were able to show that small molecule modifying deoxyribozymes can be converted to analyte sensors by coupling their catalytic activity to signal amplification and reporting.
Assuntos
Técnicas Biossensoriais/métodos , DNA Catalítico/metabolismo , Guanosina Trifosfato/análise , DNA Catalítico/química , Fluorescência , Fosforilação , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: The commensal bacterium Faecalibacterium prausnitzii is a prominent member of the microbiome of animals and humans, and it plays an important role in several physiological processes. Numerous studies have correlated the reduction of F.â¯prausnitzii abundance with many disease states, including irritable bowel syndrome, Crohn's disease, obesity, asthma, major depressive disorder, and metabolic diseases in humans. Studies have also correlated F.â¯prausnitzii with diseases in humans involved in altered glucose metabolism, including diabetes. RESEARCH DESIGN AND METHODS: The aim of this study was to investigate the effects of compositions derived from three strains of F.â¯prausnitzii (coined FPZ) on glucose metabolism in diet-induced obese male C57BL/6J prediabetic and type 2 diabetic mice. The primary endpoints of these studies were measuring changes in fasting blood glucose, glucose tolerance (as measured by a glucose tolerance test), and percent hemoglobin A1c (HbA1c) with longer term treatment. Two placebo-controlled trials were carried out using both live cell FPZ and killed cell FPZ and extracts. Two additional placebo-controlled trials were carried out in non-diabetic mice and mice that previously had type 2 diabetes (T2D). RESULTS: Both trials in prediabetic and diabetic mice revealed that peroral administration of live FPZ or extracts from FPZ lowered fasting blood glucose levels and improved glucose tolerance compared with control mice. A trial administering longer FPZ treatment also resulted in lowered percent HbA1c compared with control mice. Additionally, trials in non-diabetic mice treated with FPZ demonstrated that FPZ treatment does not lead to hypoglycemia. CONCLUSIONS: The trial results have shown that treatment with different formulations of FPZ result in lower blood glucose levels, lower percent HbA1c, and improved glucose response in mice compared with control prediabetic/diabetic mice. FPZ is a promising candidate as an orally administered probiotic or postbiotic to manage and improve pre-diabetes and T2D.
Assuntos
Transtorno Depressivo Maior , Diabetes Mellitus Tipo 2 , Estado Pré-Diabético , Humanos , Masculino , Camundongos , Animais , Estado Pré-Diabético/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Hemoglobinas Glicadas , Glicemia/metabolismo , Faecalibacterium prausnitzii/metabolismo , Transtorno Depressivo Maior/tratamento farmacológico , Camundongos Endogâmicos C57BL , ObesidadeRESUMO
When not constrained to long double-helical arrangements, DNA is capable of forming structural arrangements that enable specific sequences to perform functions such as binding and catalysis under defined conditions. Through a process called in vitro selection, numerous catalytic DNAs, known as deoxyribozymes or DNAzymes, have been isolated. Many of these molecules have the potential to act as therapeutic agents and diagnostic tools. As such, a better understanding of the structural arrangements present in these functional DNAs will aid further efforts in the development and optimization of these useful molecules. Structural characterization of several deoxyribozymes through mutagenesis, in vitro re-selection, chemical probing and circular dichroism has revealed many distinct and elaborate structural classes. Deoxyribozymes have been found to contain diverse structural elements including helical junctions, pseudoknots, triplexes, and guanine quadruplexes. Some of these studies have further shown the repeated isolation of similar structural motifs in independent selection experiments for the same type of chemical reaction, suggesting that some structural motifs are well suited for catalyzing a specific chemical reaction. To investigate the extent of structural diversity possible in deoxyribozymes, a group of kinase deoxyribozymes have been extensively characterized. Such studies have discovered some interesting structural features of these DNAzymes while revealing some novel DNA structures.
Assuntos
DNA Catalítico/química , DNA Catalítico/metabolismo , Estrutura Molecular , Mutagênese , Conformação de Ácido Nucleico , Fosfotransferases , Relação Estrutura-AtividadeRESUMO
We have investigated the effect of the folding of DNA aptamers on the colloidal stability of gold nanoparticles (AuNPs) to which an aptamer is tethered. On the basis of the studies of two different aptamers (adenosine aptamer and K+ aptamer), we discovered a unique colloidal stabilization effect associated with aptamer folding: AuNPs to which folded aptamer structures are attached are more stable toward salt-induced aggregation than those tethered to unfolded aptamers. This colloidal stabilization effect is more significant when a DNA spacer was incorporated between AuNP and the aptamer or when lower aptamer surface graft densities were used. The conformation that aptamers adopt on the surface appears to be a key factor that determines the relative stability of different AuNPs. Dynamic light scattering experiments revealed that the sizes of AuNPs modified with folded aptamers were larger than those of AuNPs modified with unfolded (but largely collapsed) aptamers in salt solution. From both the electrostatic and steric stabilization points of view, the folded aptamers that are more extended from the surface have a higher stabilization effect on AuNP than the unfolded aptamers. On the basis of this unique phenomenon, colorimetric biosensors have been developed for the detection of adenosine, K+, adenosine deaminase, and its inhibitors. Moreover, distinct AuNP aggregation and redispersion stages can be readily operated by controlling aptamer folding and unfolding states with the addition of adenosine and adenosine deaminase.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Coloides/química , Ouro/química , Nanopartículas Metálicas/química , Conformação de Ácido Nucleico , Sequência de Bases , Modelos Moleculares , Estrutura Molecular , Propriedades de Superfície , Fatores de TempoRESUMO
While the formulation of nanoparticle (NP) suspensions has been widely applied in materials and life science, the recovery of NPs from such a suspension into a solid state is practically important to confer long-term storage stability. However, solidification, while preserving the original nanoscale properties, remains a formidable challenge in the pharmaceutical and biomedical applications of NPs. Herein we combined flash nanoprecipitation (FNP) and spray-drying as a nanofabrication platform for NP formulation and recovery without compromising the dissolution kinetics of the active ingredient. Clofazimine was chosen to be the representative drug, which has been recently repurposed as a potential treatment for cryptosporidiosis. Clofazimine was encapsulated in NPs with low-cost surface coatings, hypromellose acetate succinate (HPMCAS) and lecithin, which were required by the ultimate application to global health. Spray-drying and lyophilization were utilized to produce dried powders with good long-term storage stability for application in hot and humid climatic zones. The particle morphology, yield efficiency, drug loading, and clofazimine crystallinity in the spray-dried powders were characterized. The in vitro release kinetics of spray-dried NP powders were compared to analogous dissolution profiles from standard lyophilized NP samples, crystalline clofazimine powder, and the commercially available formulation Lamprene. The spray-dried powders showed a supersaturation level of up to 60 times the equilibrium solubility and remarkably improved dissolution rates. In addition, the spray-dried powders with both surface coatings showed excellent stability during aging studies with elevated temperature and humidity, in view of the dissolution and release in vitro. Considering oral delivery for pediatric administration, the spray-dried powders show less staining effects with simulated skin than crystalline clofazimine and may be made into minitablets without additional excipients. These results highlight the potential of combining FNP and spray-drying as a feasible and versatile platform to design and rapidly recover amorphous NPs in a solid dosage form, with the advantages of satisfactory long-term storage stability, low cost, and easy scalability.
RESUMO
Malaria poses a major burden on human health and is becoming increasingly difficult to treat due to the development of antimalarial drug resistance. The resistance issue is further exacerbated by a lack of patient adherence to multi-day dosing regimens. This situation motivates the development of new antimalarial treatments that are less susceptible to the development of resistance. We have applied Flash NanoPrecipitation (FNP), a polymer-directed self-assembly process, to form stable, water-dispersible nanoparticles (NPs) of 50-400 nm in size containing OZ439, a poorly orally bioavailable but promising candidate for single-dose malaria treatment developed by Medicines for Malaria Venture (MMV). During the FNP process, a hydrophobic OZ439 oleate ion paired complex was formed and was encapsulated into NPs. Lyophilization conditions for the NP suspension were optimized to produce a dry powder. The in vitro release rates of OZ439 encapsulated in this powder were determined in biorelevant media and compared with the release rates of the unencapsulated drug. The OZ439 NPs exhibit a sustained release profile and several-fold higher release concentrations compared to that of the unencapsulated drug. In addition, XRD suggests the drug was stabilized into an amorphous form within the NPs, which may explain the improvement in dissolution kinetics. Formulating OZ439 into NPs in this way may be an important step toward developing a single-dose oral malaria therapeutic, and offers the possibility of reducing the amount of drug required per patient, lowering delivery costs, and improving dosing compliance.
Assuntos
Adamantano/análogos & derivados , Antimaláricos/administração & dosagem , Composição de Medicamentos , Nanopartículas/química , Peróxidos/administração & dosagem , Adamantano/administração & dosagem , Adamantano/química , Adamantano/farmacocinética , Antimaláricos/química , Antimaláricos/farmacocinética , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Íons/química , Estrutura Molecular , Peróxidos/química , Peróxidos/farmacocinéticaRESUMO
Nanoparticles (NP) are promising contrast agents for positron emission tomography (PET) radionuclide imaging that can increase signal intensity by localizing clusters of PET radionuclides together. However, methods to load NPs with PET radionuclides suffer from harsh loading conditions or poor loading efficacies or result in NP surface modifications that alter targeting in vivo. We present the formation of water-dispersible, polyethylene glycol coated NPs that encapsulate phthalocyanines into NP cores at greater than 50 wt % loading, using the self-assembly technique Flash NanoPrecipitation. Particles from 70 to 160 nm are produced. Phthalocyanine NPs rapidly and spontaneously chelate metals under mild conditions and can act as sinks for PET radionuclides such as 64-Cu to produce PET-active NPs. NPs chelate copper(II) with characteristic rates of 1845 M-1 h-1 at pH 6 and 37 °C, which produced >90% radionuclide chelation within 1 h. NP physical properties, such as core composition, core fluidity, and size, can be tuned to modulate chelation kinetics. These NPs retain 64Cu even in the presence of the strong chelator ethylene diamine tetraacetic acid. The development of these constructs for rapid and facile radionuclide labeling expands the applications of NP-based PET imaging.
Assuntos
Nanopartículas , Cobre , Radioisótopos de Cobre , Tomografia por Emissão de PósitronsRESUMO
The process of in vitro selection has led to the discovery of many aptamers with potential to be developed into inhibitors and biosensors, but problems in isolating aptamers against certain targets with desired affinity and specificity still remain. One possible improvement is to use libraries enhanced for motifs repeatedly isolated in aptamer molecules. One such frequently observed motif is the two-tiered guanine quadruplex. In this study we investigated whether DNA libraries could be designed to contain a large fraction of molecules capable of folding into two-tiered guanine quadruplexes. Using comprehensive circular dichroism analysis, we found that DNA libraries could be designed to contain a large proportion of sequences that adopt guanine quadruplex structures. Analysis of individual sequences from a small library revealed a mixture of quadruplexes of different topologies providing the diversity desired for an in vitro selection. We also found that primer-binding sites are detrimental to quadruplex formation and devised a method for post-selection amplification of primer-less quadruplex libraries. With the development of guanine quadruplex enriched DNA libraries, it should be possible to improve the chances of isolating aptamers that utilize a quadruplex scaffold and enhance the success of in vitro selection experiments.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Quadruplex G , Biblioteca Gênica , Dicroísmo Circular , Conformação de Ácido Nucleico , TermodinâmicaRESUMO
Here we report a deoxyribozyme with a unique structure that contains a two-tiered guanine quadruplex interlinked to a Watson-Crick duplex. Through in vitro selection, sequence mutation, and methylation interference, we show the presence of both the two-tiered guanine-quadruplex and two helical regions contained in the active structure of this self-phosphorylating deoxyribozyme. Interestingly, one GG element of the quadruplex is part of a hairpin loop within one of the identified helical regions. Circular dichroism analysis showed that antiparallel quadruplex formation was dependent on this helix. To our knowledge, this is the first report of a pseudoknot nucleic acid structure that involves a guanine quadruplex. Our findings indicate that guanine quadruplexes can be part of complex structural arrangements, increasing the likelihood of finding more complex guanine quadruplex arrangements in biological systems.
Assuntos
DNA Catalítico/química , Quadruplex G , Guanina/química , Conformação de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , Sítios de Ligação , Soluções Tampão , Catálise , Dicroísmo Circular , DNA Catalítico/genética , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Metilação , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Oligonucleotídeos , Fosforilação , Seleção Genética , Especificidade por SubstratoRESUMO
The catalytic and structural characteristics of two new self-phosphorylating deoxyribozymes (referred to as deoxyribozyme kinases), denoted "Dk3" and "Dk4", are compared to those of Dk2, a previously reported deoxyribozyme kinase. All three deoxyribozymes not only utilize GTP as the source of activated phosphate and Mn(II) as the divalent metal cofactor but also share a common secondary structure with significant sequence variations. Multiple Watson-Crick helices are identified within the secondary structure, and these helical interactions confine three extremely conserved sequence elements of 8, 5, and 14 nucleotides in length, presumably for the formation of the catalytic core for GTP binding and the self-phosphorylating reaction. The locations of the conserved regions suggest that these three deoxyribozymes arose independently from in vitro selection. The existence of three sequence variants of the same deoxyribozyme from the same in vitro selection experiment implies that these catalytic DNAs may represent the simplest structural solution for the DNA self-phosphorylation reaction when GTP is used as the substrate.
Assuntos
DNA Catalítico/química , DNA Catalítico/metabolismo , Sequência de Bases , Cátions Bivalentes/metabolismo , Sequência Conservada , DNA Catalítico/síntese química , DNA de Cadeia Simples/síntese química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Guanosina Trifosfato/metabolismo , Manganês/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , FosforilaçãoRESUMO
Dk1 and Dk2 are two catalytically proficient, manganese-dependent, guanine-rich deoxyribozymes previously isolated for DNA phosphorylation. In this study, we carried out a series of experiments that aimed to understand the structural properties of Dk1 and Dk2 and compare the structural similarities or differences of these two distinct deoxyribozymes that carry out similar catalytic functions. First, we performed reselections from two partially randomized DNA libraries on the basis of the original Dk1 and Dk2 sequences to isolate catalytically active sequence variants and identify nucleotides that are invariable, well-conserved, or highly mutagenized. Sequence analysis of these variants assisted by secondary-structure predictions led to the identification of possible Watson-Crick base-pairing regions within each deoxyribozyme. Sequence truncation and base-pair partner exchange experiments were conducted to confirm, or rule out, the existence of the predicted secondary-structure elements. Finally, methylation interference experiments were applied to identify nucleotides that are potentially important for the tertiary structure folding of the deoxyribozymes. Our data suggest that Dk1 and Dk2, despite the differences in their primary sequences and NTP requirements, use an analogous stem-loop element to anchor a structural domain of substantial tertiary interactions to execute their catalytic functions.