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1.
J Am Chem Soc ; 146(23): 15897-15907, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38818863

RESUMO

In the RNA World before the emergence of an RNA polymerase, nonenzymatic template copying would have been essential for the transmission of genetic information. However, the products of chemical copying with the canonical nucleotides (A, U, C, and G) are heavily biased toward the incorporation of G and C, which form a more stable base pair than A and U. We therefore asked whether replacing adenine (A) with diaminopurine (D) might lead to more efficient and less biased nonenzymatic template copying by making a stronger version of the A:U pair. As expected, primer extension substrates containing D bound to U in the template more tightly than substrates containing A. However, primer extension with D exhibited elevated reaction rates on a C template, leading to concerns about fidelity. Our crystallographic studies revealed the nature of the D:C mismatch by showing that D can form a wobble-type base pair with C. We then asked whether competition with G would decrease the mismatched primer extension. We performed nonenzymatic primer extension with all four activated nucleotides on randomized RNA templates containing all four letters and used deep sequencing to analyze the products. We found that the DUCG genetic system exhibited a more even product distribution and a lower mismatch frequency than the canonical AUCG system. Furthermore, primer extension is greatly reduced following all mismatches, including the D:C mismatch. Our study suggests that D deserves further attention for its possible role in the RNA World and as a potentially useful component of artificial nonenzymatic RNA replication systems.


Assuntos
2-Aminopurina , RNA , RNA/química , 2-Aminopurina/química , 2-Aminopurina/análogos & derivados , Pareamento de Bases , Moldes Genéticos , Conformação de Ácido Nucleico , Modelos Moleculares
2.
Biotechnol Bioeng ; 121(4): 1384-1393, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38151965

RESUMO

2,6-diaminopurine (Z), a naturally occurring noncanonical nucleotide base found in bacteriophages, enhances DNA hybridization by forming three hydrogen bonds with thymine (T). These distinct biochemical characteristics make it particularly valuable in applications that rely on the thermodynamics of DNA hybridization. However, the practical use of Z-containing oligos is limited by their high production cost and the challenges associated with their synthesis. Here, we developed an efficient and cost-effective approach to synthesize Z-containing oligos of high quality based on an isothermal strand displacement reaction. These newly synthesized Z-oligos are then employed as toehold-blockers in an isothermal genotyping assay designed to detect rare single nucleotide variations (SNV). When compared with their counterparts containing the standard adenine (A) base, the Z-containing blockers significantly enhance the accuracy of identifying SNV. Overall, our innovative methodology in the synthesis of Z-containing oligos, which can also be used to incorporate other unconventional and unnatural bases into oligonucleotides, is anticipated to be adopted for diverse applications, including genotyping, biosensing, and gene therapy.


Assuntos
2-Aminopurina/análogos & derivados , DNA , Nucleotídeos , Genótipo , Hibridização de Ácido Nucleico , DNA/química
3.
Nucleic Acids Res ; 49(20): 11778-11786, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34718727

RESUMO

Single molecule experiments have demonstrated a progressive transition from a B- to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B- to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.


Assuntos
2-Aminopurina/análogos & derivados , DNA Super-Helicoidal/química , Simulação de Dinâmica Molecular
4.
Nucleic Acids Res ; 49(20): 11974-11985, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34751404

RESUMO

All genetic information in cellular life is stored in DNA copolymers composed of four basic building blocks (ATGC-DNA). In contrast, a group of bacteriophages belonging to families Siphoviridae and Podoviridae has abandoned the usage of one of them, adenine (A), replacing it with 2-aminoadenine (Z). The resulting ZTGC-DNA is more stable than its ATGC-DNA counterpart, owing to the additional hydrogen bond present in the 2-aminoadenine:thymine (Z:T) base pair, while the additional amino group also confers resistance to the host endonucleases. Recently, two classes of replicative proteins found in ZTGC-DNA-containing phages were characterized and one of them, DpoZ from DNA polymerase A (PolA) family, was shown to possess significant Z-vs-A specificity. Here, we present the crystallographic structure of the apo form of DpoZ of vibriophage ϕVC8, composed of the 3'-5' exonuclease and polymerase domains. We captured the enzyme in two conformations that involve the tip of the thumb subdomain and the exonuclease domain. We highlight insertions and mutations characteristic of ϕVC8 DpoZ and its close homologues. Through mutagenesis and functional assays we suggest that the preference of ϕVC8 DpoZ towards Z relies on a polymerase backtracking process, more efficient when the nascent base pair is A:T than when it is Z:T.


Assuntos
2-Aminopurina/análogos & derivados , DNA Polimerase Dirigida por DNA/química , Podoviridae/enzimologia , Siphoviridae/enzimologia , Proteínas Virais/química , 2-Aminopurina/química , Pareamento de Bases , DNA Viral/química , DNA Polimerase Dirigida por DNA/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas Virais/metabolismo
5.
Molecules ; 28(15)2023 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-37570884

RESUMO

Rheumatoid arthritis (RA) remains one of the most prevalent autoimmune diseases worldwide. Janus kinase 3 (JAK3) is an essential enzyme for treating autoimmune diseases, including RA. Molecular modeling techniques play a crucial role in the search for new drugs by reducing time delays. In this study, the 3D-QSAR approach is employed to predict new JAK3 inhibitors. Two robust models, both field-based with R2 = 0.93, R = 0.96, and Q2 = 87, and atom-based with R2 = 0.94, R = 0.97, and Q2 = 86, yielded good results by identifying groups that may readily direct their interaction. A reliable pharmacophore model, DHRRR1, was provided in this work to enable the clear characterization of chemical features, leading to the design of 13 inhibitors with their pIC50 values. The DHRRR1 model yielded a validation result with a ROC value of 0.87. Five promising inhibitors were selected for further study based on an ADMET analysis of their pharmacokinetic properties and covalent docking (CovDock). Compared to the FDA-approved drug tofacitinib, the pharmaceutical features, binding affinity and stability of the inhibitors were analyzed through CovDock, 300 ns molecular dynamics simulations, free energy binding calculations and ADMET predictions. The results show that the inhibitors have strong binding affinity, stability and favorable pharmaceutical properties. The newly predicted molecules, as JAK3 inhibitors for the treatment of RA, are promising candidates for use as drugs.


Assuntos
2-Aminopurina , Antirreumáticos , Desenho Assistido por Computador , Desenho de Fármacos , Janus Quinase 3 , Inibidores de Janus Quinases , 2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacologia , Inibidores de Janus Quinases/química , Inibidores de Janus Quinases/farmacologia , Janus Quinase 3/antagonistas & inibidores , Relação Quantitativa Estrutura-Atividade , Piperidinas/química , Piperidinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Artrite Reumatoide/tratamento farmacológico , Antirreumáticos/química , Antirreumáticos/farmacologia , Farmacóforo
6.
Phys Chem Chem Phys ; 24(7): 4204-4211, 2022 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-35119441

RESUMO

Ultraviolet radiation (UVR) from the sun is essential for the prebiotic syntheses of nucleotides, but it can also induce photolesions such as the cyclobutane pyrimidine dimers (CPDs) to RNA or DNA oligonucleotide in prebiotic Earth. 2,6-Diaminopurine (26DAP) has been proposed to repair CPDs in high yield under prebiotic conditions and be a key component in enhancing the photostability of higher-order prebiotic DNA structures. However, its electronic relaxation pathways have not been studied, which is necessary to know whether 26DAP could have survived the intense UV fluxes of the prebiotic Earth. We investigate the electronic relaxation mechanism of both 26DAP and its 2'-deoxyribonucleoside (26DAP-d) in aqueous solution using steady-state and femtosecond transient absorption measurements that are complemented with electronic-structure calculations. The results demonstrate that both purine derivatives are significantly photostable to UVR. It is shown that upon excitation at 287 nm, the lowest energy 1ππ* state is initially populated. The population then branches following two relaxation coordinates in the 1ππ* potential energy surface, which are identified as the C2- and C6-relaxation coordinates. The population following the C6-coordinate internally converts to the ground state nonradiatively through a nearly barrierless conical intersection within 0.7 ps in 26DAP or within 1.1 ps in 26DAP-d. The population that follows the C2-relaxation coordinate decays back to the ground state by a combination of nonradiative internal conversion via a conical intersection and fluorescence emission from the 1ππ* minimum in 43 ps and 1.8 ns for the N9 and N7 tautomers of 26DAP, respectively, or in 70 ps for 26DAP-d. Fluorescence quantum yields of 0.037 and 0.008 are determined for 26DAP and 26DAP-d, respectively. Collectively, it is demonstrated that most of the excited state population in 26DAP and 26DAP-d decays back to the ground state via both nonradiative and radiative relaxation pathways. This result lends support to the idea that 26DAP could have accumulated in large enough quantities during the prebiotic era to participate in the formation of prebiotic RNA or DNA oligomers and act as a key component in the protection of the prebiotic genetic alphabet.


Assuntos
Dímeros de Pirimidina , Raios Ultravioleta , 2-Aminopurina/análogos & derivados , Teoria Quântica , Análise Espectral
7.
Nucleic Acids Res ; 48(4): 2107-2125, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31832686

RESUMO

Ribosomal protein S1 plays important roles in the translation initiation step of many Escherichia coli mRNAs, particularly those with weak Shine-Dalgarno sequences or structured 5' UTRs, in addition to a variety of cellular processes beyond the ribosome. In all cases, the RNA-binding activity of S1 is a central feature of its function. While sequence determinants of S1 affinity and many elements of the interactions of S1 with simple secondary structures are known, mechanistic details of the protein's interactions with RNAs of more complex secondary and tertiary structure are less understood. Here, we investigate the interaction of S1 with the well-characterized H-type pseudoknot of a class-I translational preQ1 riboswitch as a highly structured RNA model whose conformation and structural dynamics can be tuned by the addition of ligands of varying binding affinity, particularly preQ1, guanine, and 2,6-diaminopurine. Combining biochemical and single molecule fluorescence approaches, we show that S1 preferentially interacts with the less folded form of the pseudoknot and promotes a dynamic, partially unfolded conformation. The ability of S1 to unfold the RNA is inversely correlated with the structural stability of the pseudoknot. These mechanistic insights delineate the scope and limitations of S1-chaperoned unfolding of structured RNAs.


Assuntos
Conformação de Ácido Nucleico/efeitos dos fármacos , RNA/química , Proteínas Ribossômicas/genética , Riboswitch/genética , 2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacologia , Sítios de Ligação/genética , Escherichia coli/química , Escherichia coli/genética , Guanina/farmacologia , Ligantes , Pirimidinonas/farmacologia , Pirróis/farmacologia , RNA/efeitos dos fármacos , RNA/genética , Dobramento de RNA/efeitos dos fármacos , Proteínas Ribossômicas/química , Ribossomos/química , Ribossomos/genética , Riboswitch/efeitos dos fármacos , Imagem Individual de Molécula
8.
J Org Chem ; 86(21): 14461-14475, 2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34661407

RESUMO

7-Functionalized 8-aza-7-deaza-2'-deoxyisoguanine and 8-aza-7-deaza-2-aminoadenine 2'-deoxyribonucleosides decorated with fluorescent pyrene or benzofuran sensor tags or clickable side chains with terminal triple bonds were synthesized. 8-Aza-7-deaza-7-iodo-2-amino-2'-deoxyadenosine was used as the central intermediate and was accessible by an improved two-step glycosylation/amination protocol. Functionalization of position-7 was performed either on 8-aza-7-deaza-7-iodo-2-amino-2'-deoxyadenosine followed by selective deamination of the 2-amino group or on 7-iodinated 8-aza-7-deaza-2'-deoxyisoguanosine. Sonogashira and Suzuki-Miyaura cross-coupling reactions were employed for this purpose. Octadiynyl side chains were selected as linkers for click reactions with azido pyrenes. KTaut values calculated from H2O/dioxane mixtures revealed that side chains have a significant influence on the tautomeric equilibrium. Photophysical properties (fluorescence, solvatochromism, and quantum yields) of the new 8-aza-7-deazapurine nucleosides with fluorescent side chains were determined. Remarkably, a strong excimer fluorescence in H2O was observed for pyrene dye conjugates of 8-aza-7-deazaisoguanine and 2-aminoadenine nucleosides with a long linker. In other solvents including methanol, excimer fluorescence was negligible. The 2-aminoadenine and isoguanine nucleosides with the 8-aza-7-deazapurine skeleton expand the class of nucleosides applicable to fluorescence detection with respect to diagnostic and therapeutic purposes.


Assuntos
Nucleosídeos , Oligonucleotídeos , 2-Aminopurina/análogos & derivados , DNA , Guanina , Purinas , Esqueleto
9.
Nucleic Acids Res ; 47(13): 6618-6631, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31173143

RESUMO

Riboswitches can regulate gene expression by direct and specific interactions with ligands and have recently attracted interest as potential drug targets for antibacterial. In this work, molecular dynamics (MD) simulations, free energy perturbation (FEP) and molecular mechanics generalized Born surface area (MM-GBSA) methods were integrated to probe the effect of mutations on the binding of ligands to guanine riboswitch (GR). The results not only show that binding free energies predicted by FEP and MM-GBSA obtain an excellent correlation, but also indicate that mutations involved in the current study can strengthen the binding affinity of ligands GR. Residue-based free energy decomposition was applied to compute ligand-nucleotide interactions and the results suggest that mutations highly affect interactions of ligands with key nucleotides U22, U51 and C74. Dynamics analyses based on MD trajectories indicate that mutations not only regulate the structural flexibility but also change the internal motion modes of GR, especially for the structures J12, J23 and J31, which implies that the aptamer domain activity of GR is extremely plastic and thus readily tunable by nucleotide mutations. This study is expected to provide useful molecular basis and dynamics information for the understanding of the function of GR and possibility as potential drug targets for antibacterial.


Assuntos
2-Aminopurina/análogos & derivados , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Hipoxantina/metabolismo , Proteínas de Membrana Transportadoras/genética , Simulação de Dinâmica Molecular , Mutação Puntual , Riboswitch/genética , 2-Aminopurina/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Guanina/metabolismo , Ligantes , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Termodinâmica
10.
Rapid Commun Mass Spectrom ; 34(6): e8620, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31658399

RESUMO

RATIONALE: For quality control of oligonucleotide therapeutics, accurate and efficient structural characterization using mass spectrometry techniques, such as liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), is essential. In MALDI MS analysis, matrix selection is critical and a new matrix could enable more efficient and rapid structural analysis. METHODS: We hypothesized that nucleobase derivatives could act as matrices more efficiently than the currently used matrices for oligonucleotides because of structural similarity, which leads to close contact with the analyte. To evaluate their suitability as matrices, 16 nucleobase derivatives were selected and tested as matrix candidates for oligonucleotide analysis. RESULTS: Six of the 16 nucleobase derivatives acted as matrices for oligonucleotides. Particularly, 6-thioguanine (TG) performed well and induced clear in-source decay fragmentation. When TG or 2-amino-6-chloropurine was used as the matrix, oligonucleotides were ionized, and mainly the w and d fragment ions were observed. CONCLUSIONS: Herein we demonstrate that a 10-mer RNA or DNA sequence can be successfully characterized using TG as matrix and suggest the possibility of using nucleobase derivatives as novel matrices in oligonucleotide sequencing.


Assuntos
Oligonucleotídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , DNA/química , RNA/química , Tioguanina/química
11.
Molecules ; 25(10)2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32466298

RESUMO

Irreversible destruction of disease-associated regulatory RNA sequences offers exciting opportunities for safe and powerful therapeutic interventions against human pathophysiology. In 2017, for the first time we introduced miRNAses-miRNA-targeted conjugates of a catalytic peptide and oligonucleotide capable of cleaving an miRNA target. Herein, we report the development of Dual miRNases against oncogenic miR-21, miR-155, miR-17 and miR-18a, each containing the catalytic peptide placed in-between two short miRNA-targeted oligodeoxyribonucleotide recognition motifs. Substitution of adenines with 2-aminoadenines in the sequence of oligonucleotide "shoulders" of the Dual miRNase significantly enhanced the efficiency of hybridization with the miRNA target. It was shown that sequence-specific cleavage of the target by miRNase proceeded metal-independently at pH optimum 5.5-7.5 with an efficiency varying from 15% to 85%, depending on the miRNA sequence. A distinct advantage of the engineered nucleases is their ability to additionally recruit RNase H and cut miRNA at three different locations. Such cleavage proceeds at the central part by Dual miRNase, and at the 5'- and 3'-regions by RNase H, which significantly increases the efficiency of miRNA degradation. Due to increased activity at lowered pH Dual miRNases could provide an additional advantage in acidic tumor conditions and may be considered as efficient tumor-selective RNA-targeted therapeutic.


Assuntos
MicroRNAs/metabolismo , Oligonucleotídeos/metabolismo , Peptídeos/metabolismo , Ribonucleases/metabolismo , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , 2-Aminopurina/metabolismo , Sequência de Bases , Biocatálise , Domínio Catalítico , Humanos , Concentração de Íons de Hidrogênio , Oligonucleotídeos/síntese química , Peptídeos/síntese química , Estabilidade de RNA , Ribonucleases/síntese química
12.
Molecules ; 25(3)2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32033464

RESUMO

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9- -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.


Assuntos
2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacologia , Escherichia coli/efeitos dos fármacos , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Tubercidina/análogos & derivados , Tubercidina/farmacologia , 2-Aminopurina/síntese química , Acetaldeído/análogos & derivados , Acetaldeído/química , Antibacterianos/química , Antibacterianos/farmacologia , Escherichia coli/enzimologia , Pirimidinas/química , Tubercidina/síntese química
13.
Biophys J ; 116(5): 760-771, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30795872

RESUMO

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.


Assuntos
2-Aminopurina/análogos & derivados , DNA/química , Fenômenos Mecânicos , 2-Aminopurina/química , Fenômenos Biomecânicos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Temperatura de Transição
14.
Chemistry ; 25(30): 7387-7395, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-30942502

RESUMO

Nucleotides that contain two nucleobases (double-headed nucleotides) have the potential to condense the information of two separate nucleotides into one. This presupposes that both bases must successfully pair with a cognate strand. Here, double-headed nucleotides that feature cytosine, guanine, thymine, adenine, hypoxanthine, and diaminopurine linked to the C2'-position of an arabinose scaffold were developed and examined in full detail. These monomeric units were efficiently prepared by convergent synthesis and incorporated into DNA oligonucleotides by means of the automated phosphoramidite method. Their pairing efficiency was assessed by UV-based melting-temperature analysis in several contexts and extensive molecular dynamics studies. Altogether, the results show that these double-headed nucleotides have a well-defined structure and invariably behave as functional dinucleotide mimics in DNA duplexes.


Assuntos
Pareamento de Bases , Nucleotídeos/química , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , Adenina/química , Pareamento Incorreto de Bases , Citosina/química , DNA/química , Guanina/química , Hipoxantina/química , Modelos Moleculares , Conformação de Ácido Nucleico , Timina/química
15.
Biochemistry ; 57(9): 1517-1522, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29389111

RESUMO

DNAzymes are catalytic DNA molecules that can perform a variety of reactions. Although advances have been made in obtaining DNAzymes via in vitro selection and many of them have been developed into sensors and imaging agents for metal ions, bacteria, and other molecules, the structural features responsible for these enzymatic reactions are still not well understood. Previous studies of the 8-17 DNAzyme have suggested conserved guanines close to the phosphodiester transfer site may play a role in the catalytic reaction. To identify the specific guanine and functional group of the guanine responsible for the reaction, we herein report the effects of replacing G1.1 and G14 (G; p Ka,N1 = 9.4) with analogues with a different p Ka at the N1 position, such as inosine (G14I; p Ka,N1 = 8.7), 2,6-diaminopurine (G14diAP; p Ka,N1 = 5.6), and 2-aminopurine (G14AP; p Ka,N1 = 3.8) on pH-dependent reaction rates. A comparison of the pH dependence of the reaction rates of these DNAzymes demonstrated that G14 in the bulge loop next to the cleavage site, is involved in proton transfer at the catalytic site. In contrast, we did not find any evidence of G1.1 being involved in acid-base catalysis. These results support general acid-base catalysis as a feasible strategy used in DNA catalysis, as in RNA and protein enzymes.


Assuntos
DNA Catalítico/química , DNA Catalítico/metabolismo , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , 2-Aminopurina/metabolismo , Sequência de Bases , Domínio Catalítico , Concentração de Íons de Hidrogênio , Inosina/química , Inosina/metabolismo , Cinética , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , RNA/química , RNA/metabolismo , Relação Estrutura-Atividade
16.
Chemistry ; 24(16): 4157-4164, 2018 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-29451325

RESUMO

A series of purine-based spin labels was prepared for noncovalent spin-labeling of abasic sites of duplex nucleic acids through hydrogen bonding to an orphan base on the opposing strand and π-stacking interactions with the flanking bases. Both 1,1,3,3-tetramethylisoindolin-2-yloxyl and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were conjugated to either the C2- or C6-position of the purines, yielding nitroxide derivatives of guanine, adenine, or 2,6-diaminopurine. The isoindoline-derived spin labels showed extensive or full binding to abasic sites in RNA duplexes, whereas the TEMPO-derived spin labels showed limited binding. An adenine-derived spin label (5) bound fully at low temperature to abasic sites in both DNA and RNA duplexes when paired with thymine and uracil, respectively, complementing the previously described guanine-derived spin label Ç´, which binds efficiently opposite cytosine. Compound Ç´ was also shown to bind to abasic sites in DNA-RNA hybrids, either in the DNA- or the RNA-strand. Ç´ showed only a minor flanking-sequence effect upon binding to abasic sites in RNA. When the abasic site was placed close to the end of the RNA duplex, the affinity of the spin label Ç´ was reduced; full binding was observed at the fourth position from the duplex end. In summary, spin labels 5 and Ç´ showed full binding to abasic sites in both DNA and RNA duplexes and are promising spin labels for structural studies of nucleic acids by pulsed EPR methods.


Assuntos
DNA/química , Ácidos Nucleicos/química , Purinas/química , RNA/química , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , Adenina/química , Óxidos N-Cíclicos/química , Citosina/química , Guanina/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Marcadores de Spin , Timina/química
17.
Chembiochem ; 18(19): 1917-1922, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28748559

RESUMO

MicroRNAs (miRNAs) are endogenous small RNAs that regulate gene expression at the post-transcriptional level by sequence-specific hybridisation. Anti-miRNA oligonucleotides (AMOs) are inhibitors of miRNA activity. Chemical modification of AMOs is required to increase binding affinity and stability in serum and cells. In this study, we synthesised AMOs with our original acyclic nucleic acid, serinol nucleic acid (SNA), backbone and with the artificial nucleobase 2,6-diaminopurine. The AMO composed of only SNA had strong nuclease resistance and blocked endogenous miRNA activity. A significant improvement in anti-miRNA activity of the AMO was achieved by introduction of a 2,6-diaminopurine residues into the SNA backbone. In addition, we found that the enhancement in AMO activity depended on the position of the 2,6-diaminopurine residue in the sequence. The high potency of the SNA-AMOs suggests that these oligomers will be useful as therapeutic reagents for control of miRNA function in patients and as tools for investigating the roles of microRNAs in cells.


Assuntos
2-Aminopurina/análogos & derivados , MicroRNAs/antagonistas & inibidores , Ácidos Nucleicos/química , Ácidos Nucleicos/farmacologia , Propanolaminas/química , Propilenoglicóis/química , 2-Aminopurina/química , Células HeLa , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia
18.
Bioorg Med Chem Lett ; 27(3): 406-412, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28049589

RESUMO

The splicing of pre-mRNA is a critical process in normal cells and is deregulated in cancer. Compounds that modulate this process have recently been shown to target a specific vulnerability in tumors. We have developed a novel cell-based assay that specifically activates luciferase in cells exposed to SF3B1 targeted compounds, such as sudemycin D6. This assay was used to screen a combined collection of approved drugs and bioactive compounds. This screening approach identified several active hits, the most potent of which were CGP-74514A and aminopurvalanol A, both have been reported to be cyclin-dependent kinases (CDKs) inhibitors. We found that these compounds, and their analogs, show significant cdc2-like kinase (CLK) inhibition and clear structure-activity relationships (SAR) at CLKs. We prepared a set of analogs and were able to 'dial out' the CDK activity and simultaneously developed CLK inhibitors with low nanomolar activity. Thus, we have demonstrated the utility of our exon-skipping assay and identified new molecules that exhibit potency and selectivity for CLK, as well as some structurally related dual CLK/CDK inhibitors.


Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , 2-Aminopurina/metabolismo , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Sítios de Ligação , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Éxons , Genes Reporter , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Luciferases/genética , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Splicing de RNA , Relação Estrutura-Atividade
19.
Phys Chem Chem Phys ; 19(30): 20224-20240, 2017 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-28726897

RESUMO

We report deep UV initiated excited state dynamics of the canonical nucleobase adenine (Ade) through Resonance Raman (RR) intensity analysis. RR spectra of Ade at excitation wavelengths throughout the Bb absorption band in the 210-230 nm wavelength range are measured and subsequently converted to scattering cross-sections. The time-dependent wave packet (TDWP) formalism has been employed for self-consistent simulations of the resulting wavelength dependent Raman excitation profiles (REP) and absorption spectrum of Ade. These simulations yield instantaneous nuclear dynamics of Ade within tens of femtoseconds (fs) of photoabsorption as structural distortions, linewidth broadening and solvation parameters. The instantaneous geometrical distortions of the purine ring following photoexcitation into the Bb state are analyzed vis-à-vis the low energy La state (∼260 nm) of Ade. We find that while photoabsorption by the La state causes major distortions of the imidazole ring, pyrimidine ring suffers maximal changes following Bb excitation. Seven in-plane stretching vibrations out of fifteen resonantly enhanced modes of Ade are found to contribute 76% of the total internal reorganization energy (981 cm-1) in the Bb excited state. In addition, the inertial response of the solvation shell to photoexcitation is found to be of 1190 cm-1 in magnitude, and with a relaxation time of 26.5 fs. A parallel comparison is drawn between the UV-C initiated photodynamics of Ade (6-aminopurine) with that of two substituted purines, viz., 6-chloroguanine (6-ClG or 2-amino-6-chloropurine) and guanine (2-amino-6-oxo-purie) which were reported earlier.


Assuntos
Adenina/química , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , Guanina/análogos & derivados , Guanina/química , Teoria Quântica , Análise Espectral Raman , Raios Ultravioleta
20.
Health Econ ; 26(4): 450-468, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-26893065

RESUMO

Spending on prescription drugs (Rx) represents one of the fastest growing components of US healthcare spending and has coincided with an expansion of pharmaceutical promotional spending. Most (83%) of Rx promotion is directed at physicians in the form of visits by pharmaceutical representatives (known as detailing) and drug samples provided to physicians' offices. Such promotion has come under increased public scrutiny, with critics contending that physician-directed promotion may play a role in raising healthcare costs and may unduly affect physicians' prescribing habits towards more expensive, and possibly less cost-effective, drugs. In this study, we bring longitudinal evidence to bear upon the question of how detailing impacts physicians' prescribing behaviors. Specifically, we examine prescriptions and promotion for a particular drug class based on a nationally representative sample of 150,000 physicians spanning 24 months. The use of longitudinal physician-level data allows us to tackle some of the empirical concerns in the extant literature, virtually all of which have relied on aggregate national data. We estimate fixed-effects specifications that bypass stable unobserved physician-specific heterogeneity and address potential targeting bias. In addition, we also assess differential effects at both the extensive and intensive margins of prescribing behaviors and differential effects across physician-level and market-level characteristics, questions that have not been explored in prior work. The estimates suggest that detailing has a significant and positive effect on the number of new scripts written for the detailed drug, with an elasticity magnitude of 0.06. This effect is substantially smaller than those in the literature based on aggregate information, suggesting that most of the observed relationship between physician-directed promotion and drug sales is driven by selection bias. We find that detailing impacts selective brand-specific demand but does not have any substantial effects on class-level demand. The increase in brand-specific demand appears to crowd out demand for the substitute branded drug although not for the generic alternative. Results also indicate that most of the detailing response may operate at the extensive margin; detailing affects the probability of prescribing the drug more than it affects the number of prescriptions conditional on any prescribing. We draw some implications from these estimates with respect to effects on healthcare costs and public health. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Comércio , Indústria Farmacêutica , Estudos Longitudinais , Padrões de Prática Médica/estatística & dados numéricos , Medicamentos sob Prescrição , 2-Aminopurina/análogos & derivados , 2-Aminopurina/uso terapêutico , Antivirais/classificação , Antivirais/uso terapêutico , Conflito de Interesses , Uso de Medicamentos/economia , Famciclovir , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medicamentos sob Prescrição/uso terapêutico
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