Molecular recognition is central to the design of therapeutics, chemical catalysis and sensors. Motifs for doing so most commonly involve biological structures such as antibodies and aptamers. The key to such biological recognition consists of a folded and constrained heteropolymer that, via intra-molecular forces, forms a unique three dimensional structure that creates a binding pocket or an interface able to recognize a specific molecule. In this work, we demonstrate that synthetic heteropolymers can be alternatively constrained by adsorption around a nanoparticle, and specifically a single walled carbon nanotube (SWNT), forming a corona phase and resulting in a new form of molecular recognition of specific molecules. The phenomenon is shown to be generic, with new heteropolymer recognition complexes demonstrated for three distinct examples: Riboflavin, l-thyroxine, and estradiol, each predicted using a 2D thermodynamic model of surface interactions. The dissociation constants are continuously tunable by perturbing the chemical structure of the heteropolymer. Moreover, these complexes can be used as new types of spatial-temporal sensors based on modulation of SWNT photoemission in the near-infrared, as we show by tracking riboflavin diffusion in murine macrophages.
Structural modifications of cellular macromolecules by chemical carcinogens may represent early and requisite events in neoplastic transformation (1, 2). Through interactions of this nature, qualitative changes could be induced in informational macromolecules such as DNA and RNA, and these lesions could provide a molecular basis for alteration of gene expression in carcinogenesis. Identification of the products of these reactions (herein referred to as adducts) is essential in order to: (i) gain insights into mechanisms of carcinogen activation; (ii) determine the reactive centers in these macromolecules; (iii) follow the kinetics of appearance and disappearance of adducts in the cell; and (iv) relate specific patterns of macromolecule modification with the ultimate development of tumors in target organs of susceptible species.Aflatoxin B1 (AFB1) is a very potent liver carcinogen in several animal species (3), and epidemiologic evidence indicates that it is also an important factor in the etiology of human liver cancer in certain sections of the world (4). AFB1 binds covalently to cellular macromolecules, including DNA, in mvo (5-7) and in vitro after metabolic activation (8-10). The relationship of this type of interaction to its mechanism of action has been emphasized (11). Strong indirect evidence has indicated the production of AFB1-2,3-oxide as a major activated metabolite responsible for macromolecular binding in vitro and in vivo (5-7, 9, 12), but structures of specific adducts formed with nucleic acids or proteins have not been determined. The purpose of the research reported here was to determine the structure of the major adduct formed with DNA by AFB1 activated metabolically in vitro. The results indicate that approximately 90% of the binding in vitro can be attributed to a single adduct, which was isolated in sufficient quantity for structural analysis and identified as 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (structure I).,H0 Ho (c) H3C (c) Hk I MATERIALS AND METHODS Liver microsomes used for metabolic activation of AFB1 were prepared from phenobarbital-treated male Fischer rats (13) by the procedure of Kinoshita et al. (14). The incubation mixture (400 ml) for the binding of AFB1 to DNA included Tris-HCl (pH 7.5,45 mM), MgCl2 (3 mM), glucose-6-phosphate (5 mM), NADP (0.8 mM, Sigma Chemical Co.), glucose-6-phosphate dehydrogenase (0.4 unit/ml, Sigma Chemical Co.), approximately 1 mg of microsomal protein per ml, calf thymus DNA (20 A260 units/ml or a total of 340 mg; type I, Sigma Chemical Co.), AFB1 [224 ,uM added Abbreviations: AFB1, aflatoxin B1; I, 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin Bj; II, 2,3-dihydro-3-hydroxy-2-(4-nitrobenzoxy)-aflatoxin B1; HPLC, high-pressure liquid chromatography; NMR, nuclear magnetic resonance; FD, field-desorption mass spectrometry; EI, electron-impact mass spectrometry.
The chemical stability of aflatoxin B1 bound to calf thymus DNA was studied over a 48-hour exposure to phosphate buffers at pH 6.8-8.0 (37C). During this time, aliquots of the aflatoxin B1-modified DNA were acid-hydrolyzed and analyzed for the presence of 2,3-dihydro-2-N7-guanyl)-3-hydroxyflatoxin B1, 2,3-dihydro-2,3-dihydroxy-aflatoxin B1, and the tentatively identified, 2,3-dihydro-2-N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl-3-hyvdroxyflatoxin B1 and 2,3-dihydro-2-(8,9-dihydro-8-hydroxy-N-guanyl)-3-hydroxyaflatoxin B1. Initial experiments determined the stability of 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 in DNA at levels of modification of one residue per 60 and 1500 nucleotides. The acid-hydrolysis products obtained from these modified nucleic acids were qualitatively similar, but their proportional concentrations were different. These quantitative differences were dependent upon both pH and the initial level of modification of the DNA. During the first 6 hr of incubation, under all conditions examined, the formation of 2,3-dihydro-2,3-dihydroxyaflatoxin B was responsible for the initial decrease of the 2,3-dihydro-2-(N -guanyl)-3-hydroxyaflatoxin B1 adduct in DNA. After 48 hr of incubation at pH 7.0, the major reaction of the modified DNA was depurination of the 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 adduct. However, at pH 8.0, the predominant reaction product formed in 48 hr was the putative 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxy-aflatoxin B1. The putative DNA-bound products resulting from the elimination of the positive charge in the imidazole ring of the aflatoxin-N7-guanine adduct [2,3-dihydro-2-(N5-formyl-2',5',6'-triamino4'-oxo-N5-pyimidyl)3-hydroxyaflatoxin B1 and 2,3-dihydro-248,9-dihydro-8-hydroy-N7-guanyl) 3-hydroxyaflatoxin B1] were found to be stable in DNA for at least 24 hr at both pH 6.8 and 7.4. Taken together with observed patterns ofstability ofaflatoxin B1 adducts in vivo, these observations strongly suggest the involvement of enzymatic repair processes in removal of the N7-guanyl adduct and also emphasize the possible biological significance of the stable imidazole ring-opened adduct. The aflatoxins are produced as secondary fungal metabolites by specific strains of Aspergillusflavus and A. parasiticus and are structurally a group of substituted coumarins containing a fused dihydrofurofuran moiety. Aflatoxin B1 (AFB1) is the most biologically potent of these compounds and is toxic, hepatocarcinogenic, and mutagenic in a wide range oforganisms (1, 2). This mycotoxin is a consistent contaminant ofthe human food supply in many areas of the world (3) and is epidemiologically linked to increased incidences ofhuman liver cancer in Asia and Africa (1, 2).Experimental evidence indicates that many of the biological effects ofAFB1 are mediated through formation ofcovalent derivatives with cellular macromolecules. Covalent products that are formed in DNA have received particular attention. Modification of DNA by AFB1 requires metabol...
Aflatoxin B 1 (AFB 1 ) and/or hepatitis B and C viruses are risk factors for human hepatocellular carcinoma (HCC). Available evidence supports the interpretation that formation of AFB 1 -DNA adducts in hepatocytes seeds a population of mutations, mainly G:C→T:A, and viral processes synergize to accelerate tumorigenesis, perhaps via inflammation. Responding to a need for early-onset evidence predicting disease development, highly accurate duplex sequencing was used to monitor acquisition of high-resolution mutational spectra (HRMS) during the process of hepatocarcinogenesis. Four-day-old male mice were treated with AFB 1 using a regimen that induced HCC within 72 wk. For analysis, livers were separated into tumor and adjacent cellular fractions. HRMS of cells surrounding the tumors revealed predominantly G:C→T:A mutations characteristic of AFB 1 exposure. Importantly, 25% of all mutations were G→T in one trinucleotide context (CGC; the underlined G is the position of the mutation), which is also a hotspot mutation in human liver tumors whose incidence correlates with AFB 1 exposure. The technology proved sufficiently sensitive that the same distinctive spectrum was detected as early as 10 wk after dosing, well before evidence of neoplasia. Additionally, analysis of tumor tissue revealed a more complex pattern than observed in surrounding hepatocytes; tumor HRMS were a composite of the 10-wk spectrum and a more heterogeneous set of mutations that emerged during tumor outgrowth. We propose that the 10-wk HRMS reflects a short-term mutational response to AFB 1 , and, as such, is an early detection metric for AFB 1 -induced liver cancer in this mouse model that will be a useful tool to reconstruct the molecular etiology of human hepatocarcinogenesis. duplex sequencing | mycotoxins | cancer | mutagenesis | mouse model
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