RESUMEN
The formation of two different minor groove complexes between netropsin and A2T2 DNA has been attributed to specific binding and hydration effects. In this study, we have examined the effect of added osmolyte (e.g., TEG or betaine) on the binding of netropsin to a hairpin DNA, d(CGCGAATTCGCGTC-TCCGCGAATTCGCG)-3, having a single A2T2 binding site. Netropsin binding to this DNA construct is described by a two fractional site model with a saturation stoichiometry of 1:1. Free energy changes, ΔGi, for formation of both complex I and complex II decrease continuously as osmolyte is added (e.g., ΔG1 decreases by 1.3 kcal/mol and ΔG2 decreases by 0.8 kcal/mol in 4 m osmolyte vs buffer). The negative ΔCp values for formation of both complexes, I and II, are largely unaffected by the addition of osmolyte. Formation of complex I is accompanied by the acquisition of 31 water molecules vs 19 waters for complex II. The most significant difference between the two osmolytes is that betaine diminishes the fractional formation of the complex II species, virtually eliminating complex II at 2 m. Addition of osmolyte or a decrease in the temperature have approximately the same effect on DNA hydration and on the thermodynamics of netropsin binding.
Asunto(s)
ADN/metabolismo , Netropsina/metabolismo , Agua/química , Secuencia de Bases , ADN/química , Netropsina/química , Conformación de Ácido Nucleico , Presión Osmótica , TermodinámicaRESUMEN
Synthetic N-methyl imidazole and N-pyrrole containing polyamides (PAs) that can form "stacked" dimers can be programmed to target and bind to specific DNA sequences and control gene expression. To accomplish this goal, the development of PAs with lower molecular mass which allows for the molecules to rapidly penetrate cells and localize in the nucleus, along with increased water solubility, while maintaining DNA binding sequence specificity and high binding affinity is key. To meet these challenges, six novel f-ImPy*Im PA derivatives that contain different orthogonally positioned moieties were designed to target 5'-ACGCGT-3'. The synthesis and biophysical characterization of six f-ImPy*Im were determined by CD, ΔTM, DNase I footprinting, SPR, and ITC studies, and were compared with those of their parent compound, f-ImPyIm. The results gave evidence for the minor groove binding and selectivity of PAs 1 and 6 for the cognate sequence 5'-ACGCGT-3', and with strong affinity, Keq = 2.8 × 10(8) M(-1) and Keq = 6.2 × 10(7) M(-1), respectively. The six novel PAs presented in this study demonstrated increased water solubility, while maintaining low molecular mass, sequence specificity, and binding affinity, addressing key issues in therapeutic development.
Asunto(s)
Secuencia de Bases , Nylons , Dicroismo Circular , ADN/química , Resonancia por Plasmón de SuperficieRESUMEN
Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal ß-alanines as polyamide building blocks in place of N-methylpyrrole; ß-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327-339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with ß-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with ß-alanine to generate a ß/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that ß-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition.
Asunto(s)
Ciclooxigenasa 2/química , ADN/química , Papillomavirus Humano 16/química , Nylons/química , Proteína Proto-Oncogénica c-ets-1/química , Pirroles/química , beta-Alanina/química , Secuencia de Bases , Ciclooxigenasa 2/genética , Huella de ADN , Papillomavirus Humano 16/genética , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Secuencias Invertidas Repetidas/genética , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Nylons/síntesis química , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-ets-1/genética , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
N-Methyl imidazole (Im) and N-methyl pyrrole (Py)-containing polyamides that can form stacked dimers can be programmed to target specific DNA sequences in the minor groove of DNA and control gene expression. Polyamides are being investigated as potential medicinal agents for treating diseases including cancer. The naturally occurring polyamide distamycin binds as a dimer in the minor groove of DNA and recognizes sequences rich in A/T and T/A base pairs indiscriminately. Synthetic analogs of distamycin that incorporate N-methylimidazole into the heterocyclic core have been shown to bind to G/C rich sequences with a high degree of specificity. The purpose of this study is to investigate the behavior of polyamides containing the 2,5-linked N-methylpyrrole-2-carboxamide or pyrrole(H) [Py(H)] moiety upon binding to DNA. The synthesis and biophysical characteristics of two polyamides PyPyPyPy(H) 2 and ImPyPyPy(H) 3 designed to test the binding preference of a Py/Pyrrole(H) pairing [Py/Py(H)] and a [Im/Py(H)] is described. Studies utilizing circular dichroism, thermal denaturation (ΔT(M)), biosensor-surface plasmon resonance and DNase I footprinting show that an [Im/Py(H), 3] pairing prefers a G/C or C/G pairing whilst a [Py/Py(H), 2] pairing tolerates A/T or T/A base pairs and avoids a G/C base pair.
Asunto(s)
Composición de Base , ADN/química , Nylons/química , Pirroles/química , Secuencia Rica en At , Sitios de Unión , Dicroismo Circular/métodos , Secuencia Rica en GC , Imidazoles/química , Modelos Moleculares , Estructura Molecular , Resonancia por Plasmón de Superficie/métodosRESUMEN
This case report describes a patient who presented to the Emergency Department (ED) after a high-speed motor vehicle crash (MVC), whose initial ultrasound examination was interpreted as being positive for fluid in Morison's pouch. Subsequent ultrasound examinations and computed tomography scans further delineated this finding to be fluid-filled bowel juxtaposed between the liver and right kidney. With greater implementation of ED ultrasound, it is important to identify entities that cause false-positive and false-negative examinations.