RESUMEN

Over the last few decades, chemically modified sugars have been incorporated into nucleic acid-based therapeutics to improve their pharmacological potential. Chemical modification can influence the sugar conformation, Watson-Crick hydrogen (W-C) bonding, and nucleobase stacking interactions, which play major roles in the structural integrity and dynamic properties of nucleic acid duplexes. In this study, we categorized 33 uridine (U*) and cytidine (C*) sugar modifications and calculated their sugar conformational parameters. We also calculated the Watson-Crick hydrogen bond energies of the modified RNA-type base pairs (U*:A and C*:G) using DFT and sSAPT0 methods. The W-C base pairing energy calculations suggested that the South-type modified sugar strengthens the C*:G base pair and weakens the U*:A base pair compared to the unmodified one. In contrast, the North-type sugar modifications form weaker C*:G base pair and marginally stronger U*:A base pair compared to the South-type modified sugars. Moreover, intrastrand base stacking energies were calculated for 15 modifications incorporated at the fourth position in 7-mer non-self-complementary RNA duplexes [(GCAU*GAC)2 and (GCAC*GAC)2], utilizing molecular dynamics simulation and quantum mechanical (DFT and sSAPT0) methods. The sugar modifications were found to have minimal effect on the intrastrand base-stacking interactions. However, the glycol nucleic acid modification disturbs the intrastrand base-stacking significantly, corroborating the experimental data.

Asunto(s)

Emparejamiento Base , Enlace de Hidrógeno , Ribosa , Ribosa/química , Teoría Funcional de la Densidad , Conformación de Carbohidratos , Termodinámica , ARN/química , Citidina/química , Uridina/química , Conformación de Ácido Nucleico , Simulación de Dinámica Molecular

RESUMEN

In this study, we designed the 4'-C-acetamidomethyl-2'-O-methoxyethyl (4'-C-ACM-2'-O-MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'-C-ACM-2'-O-MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4'-C-ACM-2'-O-MOE thymidine modification at the penultimate position of the 3'-end of dT20 oligonucleotides in the presence of 3'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2'-O-Me, 2'-O-MOE, and 2'-F. In gene silencing studies, we found that the 4'-C-ACM-2'-O-MOE uridine or thymidine modifications at the 3'-overhang in the passenger strand in combination with two 2'-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3'-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4'-C-ACM-2'-O-MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4'-C-ACM-2'-O-MOE modification at the 3'-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4'-C-ACM-2'-O-MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.

Asunto(s)

Ácidos Nucleicos , ARN Interferente Pequeño/química , ADN , Timidina , Uridina/química

RESUMEN

Herein, we report the synthesis of 2'-O-alkyl/2'-fluoro-N3-methyluridine (2'-O-alkyl/2'-F-m3U) phosphoramidites and their incorporation in DNA and RNA oligonucleotides. The duplex binding affinity and base discrimination studies showed that all 2'-O-alkyl/2'-F-m3U modifications significantly decreased the thermal stability and base-pairing discrimination ability. Serum stability study of dT20 with 2'-O-alkyl-m3U modification exhibited excellent nuclease resistance when incubated with 3'-exonucleases (SVPD) or 5'-exonucleases (PDE-II) as compared to m3U, 2'-F, 2'-OMe modified oligonucleotides. MD simulation studies with RNA tetradecamer duplexes illustrated that the m3U and 2'-O-methyl-m3U modifications reduce the duplex stabilities by disrupting the Watson-Crick hydrogen bonding and base-stacking interactions. Further molecular modelling investigations demonstrated that the 2'-O-propyl-m3U modification exhibits steric interactions with amino acid residues in the active site of 3'- and 5'-exonuclease, leading to enhanced stability. These combined data indicate that the 2'-modified-m3U nucleotides can be used as a promising tool to enhance the stability, silencing efficiency, and drug-like properties of antisense/siRNA-based therapeutics.

Asunto(s)

Ácidos Nucleicos , Uridina , Exonucleasas/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/química , ARN/química , ARN Interferente Pequeño/química , Uridina/análogos & derivados , Uridina/química , Uridina/farmacología

RESUMEN

Ribofuranose sugar conformation plays an important role in the structure and dynamics of functional nucleic acids such as siRNAs, AONs, aptamers, miRNAs, etc. To improve their therapeutic potential, several chemical modifications have been introduced into the sugar moiety over the years. The stability of the oligonucleotide duplexes as well as the formation of stable and functional protein-oligonucleotide complexes are dictated by the conformation and dynamics of the sugar moiety. In this review, we systematically categorise various ribofuranose sugar modifications employed in DNAs and RNAs so far. We discuss different stereoelectronic effects imparted by different substituents on the sugar ring and how these effects control sugar puckering. Using this data, it would be possible to predict the precise use of chemical modifications and design novel sugar-modified nucleosides for therapeutic oligonucleotides that can improve their physicochemical properties.

Asunto(s)

Nucleósidos , Oligonucleótidos , Oligonucleótidos/química , Conformación de Ácido Nucleico , Azúcares , ARN/química

RESUMEN

Herein we describe results on the pairing properties of synthetic DNA and RNA oligonucleotides that contain nucleotide analogues with a 7-membered sugar ring (oxepane nucleotides). Specifically, we describe the stereoselective synthesis of a set of three oxepane thymine nucleosides (OxT), their conversion to phosphoramidite derivatives, and their use in solid-phase synthesis to yield chimeric OxT-DNA and OxT-RNA strands. The different regioisomeric OxT phosphoramidites allowed for positional variations of the phosphate bridge and assessment of duplex stability when the oxepane nucleotides were incorporated in dsDNA, dsRNA, and DNA-RNA hybrids. Little to no destabilization was observed when two of the three regioisomeric OxT units were incorporated in the DNA strand of DNA-RNA hybrids, a remarkable result considering the dramatically different structure of oxepanes in comparison to 2'-deoxynucleosides. Extensive molecular modeling and dynamics studies further revealed the various structural features responsible for the tolerance of both OxT modifications in DNA-RNA duplexes, such as base-base stacking and sugar-phosphate H-bond interactions. These studies suggest that oxepane nucleotide analogues may find applications in synthetic biology, where synthetic oligonucleotides can be used to create new tools for biotechnology and medicine.

Asunto(s)

Nucleósidos , ARN , Carbohidratos , ADN/química , Conformación de Ácido Nucleico , Nucleósidos/química , Oligonucleótidos/química , Fosfatos , ARN/química , Azúcares

RESUMEN

Activating mutations in HER2 (ERBB2) drive the growth of a subset of breast and other cancers and tend to co-occur with HER3 (ERBB3) missense mutations. The HER2 tyrosine kinase inhibitor neratinib has shown clinical activity against HER2-mutant tumors. To characterize the role of HER3 mutations in HER2-mutant tumors, we integrate computational structural modeling with biochemical and cell biological analyses. Computational modeling predicts that the frequent HER3E928G kinase domain mutation enhances the affinity of HER2/HER3 and reduces binding of HER2 to its inhibitor neratinib. Co-expression of mutant HER2/HER3 enhances HER2/HER3 co-immunoprecipitation and ligand-independent activation of HER2/HER3 and PI3K/AKT, resulting in enhanced growth, invasiveness, and resistance to HER2-targeted therapies, which can be reversed by combined treatment with PI3Kα inhibitors. Our results provide a mechanistic rationale for the evolutionary selection of co-occurring HER2/HER3 mutations and the recent clinical observations that HER3 mutations are associated with a poor response to neratinib in HER2-mutant cancers.

Asunto(s)

Neoplasias de la Mama/genética , Mutación con Ganancia de Función , Quinolinas/farmacología , Receptor ErbB-2/genética , Receptor ErbB-3/genética , Aminopiridinas/administración & dosificación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Ratones Desnudos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Morfolinas/administración & dosificación , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/administración & dosificación , Multimerización de Proteína , Quinolinas/administración & dosificación , Quinolinas/química , Quinolinas/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/metabolismo , Trastuzumab/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

We report on the synthesis of siRNAs containing both 2'-5'- and 3'-5'-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2'-5' linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5'-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5'p-rN1-(2'-5')-N2 unit in the antisense strand does not alter the 'clover leaf' bend and sugar puckers that are critical for anchoring the 5'-phosphate to Ago 2 MID domain. Importantly, 2'-5'-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2'-5'/3'-5'-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.

Asunto(s)

Interferencia de ARN , ARN Interferente Pequeño/química , Proteínas Argonautas/metabolismo , Conformación de Carbohidratos , Células HeLa , Humanos , Luciferasas de Luciérnaga/genética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/inmunología , Proteína p53 Supresora de Tumor/genética

RESUMEN

Stabilization of a G-quadruplex (G4) DNA structure in the proto-oncogene c-MYC using small molecule ligands has emerged as an attractive strategy for the development of anticancer therapeutics. To understand the subtle structural changes in the G4 structure upon ligand binding, molecular dynamics (MD) simulations of c-MYC G4 DNA were carried out in a complex with six different potent ligands: 3AQN, 6AQN, 3APN, 360A, Nap-Et, and Nap-Pr. The results show that the ligands 3AQN, 6AQN, 3APN, and 360A stabilize the G4 structure by making stacking interactions with the top quartet. On the other hand, Nap-Et and Nap-Pr bind at the groove of the G4 structure. These groove binding ligands make crucial H-bond contacts with the guanines and electrostatic interactions with the phosphate backbone. Two-dimensional dynamic correlation maps unraveled the ligand-induced correlated motions between the guanines in the quartet and a di-nucleotide present in the propeller loop-2 of the G4 structure. Cluster analysis and ONIOM calculations revealed the structural dynamics in the loop of the quadruplex upon ligand binding. Overall, the results from the present study suggest that engineering specific contacts with the propeller loop can be an efficient way to design c-MYC G4-specific ligands.

Asunto(s)

Antineoplásicos/química , Diseño de Fármacos , G-Cuádruplex , Proteínas de Neoplasias/química , Proteínas Proto-Oncogénicas c-myc/química , Bibliotecas de Moléculas Pequeñas/química , Sitios de Unión , Guanina , Humanos , Enlace de Hidrógeno , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Proteínas de Neoplasias/antagonistas & inhibidores , Unión Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Electricidad Estática , Termodinámica

RESUMEN

The use of chemical modifications in small interfering RNAs (siRNAs) is warranted to impart drug-like properties. However, certain chemical modifications especially those on the sugar have deleterious effects on the RNA interference (RNAi) when they are placed at key positions in the seed region of an siRNA guide strand. In order to probe the effect of chemically modified siRNAs [(2'-O-methyl, 4'-C-aminomethyl-2'-O-methyl, 2'-O-(2-methoxyethyl), and 2'-O-benzyl] on human Argonaute 2 (hAGO2), the catalytic engine of RNAi, we have developed a model of its open conformation. Results from microsecond MD simulations of 15 different siRNA-hAGO2 complexes provide insights about how the key noncovalent interactions and conformational changes at the seed region are modulated, depending upon the nature and position of chemical modifications. Such modification induced structural changes can affect siRNA loading into hAGO2, which may influence RNAi activity. Our studies show that microsecond MD simulations can provide useful information for the design of therapeutically relevant siRNAs.

Asunto(s)

Proteínas Argonautas/metabolismo , Simulación de Dinámica Molecular , ARN Interferente Pequeño/metabolismo , Proteínas Argonautas/química , Proteínas Argonautas/deficiencia , Proteínas Argonautas/genética , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Fosfatos/metabolismo , Unión Proteica , Conformación Proteica , Interferencia de ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Termodinámica

RESUMEN

In the last decade, an increasing number of cardiac conditions have been shown to have a genetic basis. Cardiovascular genetic counseling has emerged as a subspecialty aiming to identify unaffected at-risk individuals. An important sector of this at-risk population also includes expectant mothers, in whom unique clinical challenges may arise. Genetic counselors, especially those in cardiovascular and prenatal settings, have an opportunity to identify and assist women who may benefit from cardiovascular care during pregnancy. This paper provides basic management and genetic evaluation principles for affected women, as well as guidance on identifying those who are at risk. We provide considerations for cardiac surveillance in pregnancy and the post-partum period. Finally, key psychosocial issues that appraise how to best provide support to at risk women as they make informed decisions are discussed. We propose that a team approach including cardiology, maternal fetal medicine, and genetic counseling best serves this patient population. Ongoing questions addressing an evidence based approach to cardiovascular genetic conditions in pregnancy still remain. Thus, well-designed research protocols are essential to mark progress in this area.

Asunto(s)

Enfermedades Cardiovasculares/congénito , Enfermedades Cardiovasculares/diagnóstico , Consejeros/normas , Asesoramiento Genético/normas , Complicaciones Cardiovasculares del Embarazo/diagnóstico , Diagnóstico Prenatal/normas , Adulto , Femenino , Humanos , Embarazo , Factores de Riesgo

RESUMEN

Stabilization of G-quadruplex DNA structures by small molecules has emerged as a promising strategy for the development of anticancer drugs. Since G-quadruplex structures can adopt various topologies, attaining specific stabilization of a G-quadruplex topology to halt a particular biological process is daunting. To achieve this, we have designed and synthesized simple structural scaffolds based on an indolylmethyleneindanone pharmacophore, which can specifically stabilize the parallel topology of promoter quadruplex DNAs (c-MYC, c-KIT1, and c-KIT2), when compared to various topologies of telomeric and duplex DNAs. The lead ligands (InEt2 and InPr2) are water-soluble and meet a number of desirable criteria for a small molecule drug. Highly specific induction and stabilization of the c-MYC and c-KIT quadruplex DNAs (ΔT1/2 up to 24 °C) over telomeric and duplex DNAs (ΔT1/2 ∼ 3.2 °C) by these ligands were further validated by isothermal titration calorimetry and electrospray ionization mass spectrometry experiments (Ka ∼ 10(5) to 10(6) M(-1)). Low IC50 (∼2 µM) values were emerged for these ligands from a Taq DNA polymerase stop assay with the c-MYC quadruplex forming template, whereas the telomeric DNA template showed IC50 values >120 µM. Molecular modeling and dynamics studies demonstrated the 5'- and 3'-end stacking modes for these ligands. Overall, these results demonstrate that among the >1000 quadruplex stabilizing ligands reported so far, the indolylmethyleneindanone scaffolds stand out in terms of target specificity and structural simplicity and therefore offer a new paradigm in topology specific G-quadruplex targeting for potential therapeutic and diagnostic applications.

Asunto(s)

ADN/química , G-Cuádruplex , Indanos/química , Indoles/química , Proteínas Proto-Oncogénicas c-kit/química , Proteínas Proto-Oncogénicas c-myc/química , Humanos , Simulación de Dinámica Molecular , Regiones Promotoras Genéticas

RESUMEN

N(2)-Furfuryl-deoxyguanosine (fdG) is carcinogenic DNA adduct that originates from furfuryl alcohol. It is also a stable structural mimic of the damage induced by the nitrofurazone family of antibiotics. For the structural and functional studies of this model N(2)-dG adduct, reliable and rapid access to fdG-modified DNAs are warranted. Toward this end, here we report the synthesis of fdG-modified DNAs using phosphoramidite chemistry involving only three steps. The functional integrity of the modified DNA has been verified by primer extension studies with DNA polymerases I and IV from E. coli. Introduction of fdG into a DNA duplex decreases the Tm by ∼1.6 °C/modification. Molecular dynamics simulations of a DNA duplex bearing the fdG adduct revealed that though the overall B-DNA structure is maintained, this lesion can disrupt W-C H-bonding, stacking interactions, and minor groove hydrations to some extent at the modified site, and these effects lead to slight variations in the local base pair parameters. Overall, our studies show that fdG is tolerated at the minor groove of the DNA to a better extent compared with other bulky DNA damages, and this property will make it difficult for the DNA repair pathways to detect this adduct.

Asunto(s)

Aductos de ADN/química , ADN Forma B/química , ADN/química , Desoxiguanosina/análogos & derivados , Escherichia coli/química , Emparejamiento Base , ADN/metabolismo , Aductos de ADN/metabolismo , ADN Forma B/metabolismo , Desoxiguanosina/química , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular

RESUMEN

Lucidin is a genotoxic and mutagenic hydroxyanthraquinone metabolite, which originates from the roots of Rubia tinctorum L. (madder root). It reacts with exocyclic amino groups of DNA nucleobases and forms adducts/lesions leading to carcinogenesis. To study the effect of lucidin-induced DNA damage, herein, we report the first synthesis of a structural analogue of lucidin [N(2)-methyl-(1,3-dimethoxyanthraquinone)-deoxyguanosine, LdG] embedded DNAs utilizing phosphoramidite strategy. LdG modification in a DNA duplex imparts destabilization (ΔTm ∼5 °C/modification), which is attributed to the unfavorable contribution from the enthalpy. Primer extension studies using the Klenow fragment (exo(-)) of Escherichia coli DNA polymerase I demonstrate that bypass of LdG modification is error prone as well as slow compared to that across the unmodified sites. Molecular dynamics simulations of the binary complex of Bacillus fragment polymerase (homologue of the Klenow fragment) and LdG-DNA duplex elucidate the structural fluctuations imparted by the LdG lesion, as well as the molecular mechanism of bypass at the lesion site. Overall, the results presented here show that the lucidin adduct destabilizes DNA structure and reduces fidelity and processivity of DNA synthesis.

Asunto(s)

Antraquinonas/farmacología , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxiguanosina/metabolismo , Antraquinonas/química , Antraquinonas/metabolismo , Daño del ADN , ADN Polimerasa Dirigida por ADN/química , Desoxiguanosina/química , Simulación de Dinámica Molecular , Estructura Molecular

RESUMEN

Various potential G-quadruplex forming sequences present in the genome offer a platform to modulate their function by means of stabilizing molecules. Though G-quadruplex structures exhibit diverse structural topologies, the presence of G-quartets as a common structural element makes the design of topology specific ligands a daunting task. To address this, the subtle structural variations of loops and grooves present in the quadruplex structures can be exploited. To this end, we report the design and synthesis of quadruplex stabilizing agents based on bisbenzimidazole carboxamide derivatives of pyridine, 1,8-naphthyridine, and 1,10-phenanthroline. The designed ligands specifically bind to and stabilize promoter quadruplexes having parallel topology over any of the human telomeric quadruplex topologies (parallel, hybrid, or antiparallel) and duplex DNAs. CD melting studies indicate that ligands could impart higher stabilization to c-MYC and c-KIT promoter quadruplexes (up to 21 °C increment in Tm) than telomeric and duplex DNAs (ΔTm ≤ 2.5 °C). Consistent with a CD melting study, ligands bind strongly (Kb = ∼10(4) to 10(5) M(-1)) to c-MYC quadruplex DNA. Molecular modeling and dynamics studies provide insight into how the specificity is achieved and underscore the importance of flexible N-alkyl side chains attached to the benzimidazole-scaffold in recognizing propeller loops of promoter quadruplexes. Overall, the results reported here demonstrate that the benzimidazole scaffold represents a potent and powerful side chain, which could judiciously be assembled with a suitable central core to achieve specific binding to a particular quadruplex topology.

Asunto(s)

Amidas/química , Bencimidazoles/química , G-Cuádruplex , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-kit/química , Proteínas Proto-Oncogénicas c-myc/química , Humanos , Cinética , Simulación de Dinámica Molecular , Desnaturalización de Ácido Nucleico , Fenantrolinas/química , Piridinas/química , Termodinámica

RESUMEN

The formation of G-quadruplex structures can regulate telomerase activity and the expression of oncogenes at the transcriptional and translational levels. Therefore, stabilization of G-quadruplex DNA structures by small molecules has been recognized as a promising strategy for anticancer drug therapy. One of the major challenges in this field is to impart stabilizing molecules with selectivity toward quadruplex structures over duplex DNAs, and to maintain specificity toward a particular quadruplex topology. Herein we report the synthesis and binding interactions of indenopyrimidine derivatives, endowed with drug-like properties, with oncogenic promoters of c-myc and c-kit, telomeric and duplex DNAs. The results show specific stabilization of promoter over telomeric quadruplexes and duplex DNAs. Molecular modeling studies support the experimental observations by unraveling the dual binding mode of ligands by exploiting the top and bottom quartets of a G-quadruplex structure. This study underscores the potential of the indenopyrimidine scaffold, which can be used to achieve specific G-quadruplex-mediated anticancer activity.

Asunto(s)

G-Cuádruplex , Proteínas Proto-Oncogénicas c-kit/genética , Pirimidinas/química , Sitios de Unión , Dicroismo Circular , Enlace de Hidrógeno , Ligandos , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Transición de Fase , Regiones Promotoras Genéticas , Pirimidinas/síntesis química , Termodinámica

RESUMEN

Herein, we report the synthesis of 4'-C-aminomethyl-2'-deoxy-2'-fluorouridine, a therapeutically appealing RNA modification. Conformational analysis by DFT calculations and molecular dynamics simulations using trinucleotide model systems revealed that modified sugar adopts C3'-endo conformation. In this conformer, a weak intramolecular C-H···F H-bond between the hydrogen atom of the 4'-C-CH2 group and the F atom at the 2' position is observed. Comparative studies with unmodified, 2'-fluoro-, 2'-O-methyl-, and 4'-C-aminomethyl-2'-O-methyluridine showed the chemical nature of 2'-substituent dictates the sugar puckering of 2',4'-modified nucleotides.

Asunto(s)

Nucleósidos/síntesis química , Nucleótidos/síntesis química , Uridina/análogos & derivados , Uridina/síntesis química , Secuencia de Bases , Enlace de Hidrógeno , Modelos Moleculares , Conformación Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Conformación de Ácido Nucleico , Nucleósidos/química , Nucleótidos/química , Uridina/química

RESUMEN

The quest for a G-quadruplex specific fluorescent sensor among other DNA forms under physiological salt conditions has been addressed in this article. We demonstrate for the first time the application of a water-soluble fluorogenic dye, Thioflavin T (ThT), in a dual role of exclusively inducing quadruplex folding in the 22AG human telomeric DNA, both in the presence and absence of Tris buffer/salt, and sensing the same through its fluorescence light-up having emission enhancement of the order of 2100-fold in the visible region. Appropriate conditions allow an apparent switch over of the parallel quadruplex structure in 22AG-ThT (50 mM Tris, pH 7.2) solution to the antiparallel form just by the addition of K(+) ions in the range 10-50 mM. Moreover, addition of ThT cooperatively stabilizes the K(+) induced antiparallel quadruplexes by a ΔT(m) ∼11 °C. The distinction of ThT as a quadruplex inducer has been contrasted with the erstwhile used structurally related dye, Thiazole Orange (TO), which did not induce any quadruplex folding in the 22AG strand in the absence of salt. The striking fluorescence light-up in ThT on binding to the human telomeric G-quadruplex is shown to be highly specific compared to the less than 250-fold enhancement observed with other single/double strand DNA forms. This work has implication in designing new generation dyes based on the ThT scaffold, which are highly selective for telomeric DNA, for potential diagnostic, therapeutic, and ion-sensing applications.

Asunto(s)

Colorantes Fluorescentes/química , G-Cuádruplex , Telómero , Tiazoles/química , Secuencia de Bases , Benzotiazoles , Humanos , Modelos Moleculares , Estructura Molecular

RESUMEN

The linear syntheses of 4'-C-aminomethyl-2'-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T(m) (∼1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3' end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.

Asunto(s)

Citidina/análogos & derivados , Nucleótidos/síntesis química , Nucleótidos/genética , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Uridina/análogos & derivados , Secuencia de Bases , Citidina/síntesis química , Citidina/química , Silenciador del Gen , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Uridina/síntesis química , Uridina/química

RESUMEN

Various biologically relevant G-quadruplex DNA structures offer a platform for therapeutic intervention for altering the gene expression or by halting the function of proteins associated with telomeres. One of the prominent strategies to explore the therapeutic potential of quadruplex DNA structures is by stabilizing them with small molecule ligands. Here we report the synthesis of bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine and their interaction with human telomeric DNA and promoter G-quadruplex forming DNAs. The interactions of ligands with quadruplex forming DNAs were studied by various biophysical, biochemical, and computational methods. Results indicated that bisquinolinium ligands bind tightly and selectively to quadruplex DNAs at low ligand concentration (∼0.2-0.4 µM). Furthermore, thermal melting studies revealed that ligands imparted higher stabilization for quadruplex DNA (an increase in the T(m) of up to 21 °C for human telomeric G-quadruplex DNA and >25 °C for promoter G-quadruplex DNAs) than duplex DNA (ΔT(m) ≤ 1.6 °C). Molecular dynamics simulations revealed that the end-stacking binding mode was favored for ligands with low binding free energy. Taken together, the results indicate that the naphthyridine-based ligands with quinolinium and pyridinium side chains form a promising class of quadruplex DNA stabilizing agents having high selectivity for quadruplex DNA structures over duplex DNA structures.

Asunto(s)

Excipientes/química , Compuestos Heterocíclicos/química , Naftiridinas/química , Compuestos de Piridinio/química , Compuestos de Quinolinio/química , Secuencia de Bases , G-Cuádruplex , Ligandos , Modelos Moleculares