Hybrid polar compounds, of which hexamethylenebisacetamide (HMBA) is the prototype, are potent inducers of differentiation of murine erythroleukemia (MEL)

Watson-Crick base pairing organizes DNA duplex formation necessary for genetic information storage in biological systems. DNA and RNA templates also direct the specific binding of nucleotide substrates during diverse enzyme-catalyzed reactions in replication, transcription, and DNA repair pathways. Recently, nucleic acid recognition properties have been extended to nonbiological systems, where DNA base pairing has been used to drive the template-directed chemical ligation of oligonucleotides, 1 and the assembly of nanostructures and novel materials. 2 We have been interested in expanding the versatility of nucleic acid base pairing for the addressable synthesis of bioconjugates in aqueous solution. Metallosalen-DNA (4, Scheme 1) represents an ideal system to demonstrate the concept of nucleic acid template-directed molecular synthesis. Salens, which are constructed from two salicylaldehydes and a diamine, serve as ligands for a broad range of metal ions. Many metallosalens are compatible with aqueous conditions 3 and have demonstrated utility as DNA cleavage reagents 4 and versatile catalysts for enantioselective transformations. 3a,5 Template-directed synthesis of metallosalen-DNA conjugates offers a unique approach to a new class of metal-DNA hybrids. Metal-DNA conjugates previously have been employed as probes of DNA structure and electron transfer, 6 "chemical nucleases" for targeted nucleic acid cleavage, 7 and scaffolds for metal-mediated base pairing motifs. 8 Thus, metallosalen-DNA may offer a new bioconjugate platform for DNAorganized materials, nucleic acid cleavage and detection strategies, and in vitro evolution of novel ribozymes and deoxyribozymes. 9 Our approach to template-directed synthesis of metallosalen-DNA is illustrated in Scheme 1. The DNA-metallosalen building blocks consist of two DNA oligonucleotides modified, at either the 3′ or 5′ end, with salicylaldehyde moieties (1 and 2). The modified strands are aligned on a complementary nucleic acid template (3), bringing the salicylaldehyde groups into proximity in a duplex. The metallosalen conjugate then is assembled by addition of an appropriate metal and diamine. Herein we report the efficient DNA and RNA template-directed synthesis and characterization of purified metallosalen-DNA conjugates.A salicylaldehyde phosphoramidite (8, Scheme 2) was synthesized as a precursor to salicylaldehyde-DNA conjugates 1 and 2, necessary for metallosalen-DNA assembly. The protecting groups for 8, including a benzoate ester for the phenol and a 1,3-dioxane for the aldehyde, were chosen for their compatibility with DNA synthesis and postsynthetic deprotection. Starting from known salicylaldehyde derivative 5, 10 dioxane 6 was prepared by alumina-catalyzed acetalization. Direct benzoylation of 6 with benzoyl chloride afforded 7, which was converted to phosphoramidite 8 by standard methods. 11 Oligonucleotide 2 was synthesized by DNA phosphoramidite chemistry (3′-to-5′), using 8 in the final coupling step. Oligonucleotide 1, bearing a 3′-terminal salicylaldehy...

Reactive oxygen species lead to oxidative damage of the nucleobase and sugar components of nucleotides in double-stranded DNA. The 2-deoxyribonolactone (or oxidized abasic site) lesion results from oxidation of the C-1' position of DNA nucleotides and has been implicated in DNA strand scission, mutagenesis, and covalent cross-linking to DNA binding proteins. We previously described a strategy for the synthesis of DNA-containing deoxyribonolactone lesions. We now report an improved method for the site specific photochemical generation of deoxyribonolactone sites within DNA oligonucleotides and utilize these synthetic oligonucleotides to characterize the products and rates of DNA strand scission at the lactone lesion under simulated physiological conditions. A C-1' nitroveratryl cyanohydrin phosphoramidite analogue was synthesized and used for the preparation of DNA containing a photochemically "caged" lactone precursor. Irradiation at 350 nm quantitatively converted the caged analogue into the deoxyribonolactone lesion. The methodology was validated by RP-HPLC and MALDI-TOF mass spectrometry. Incubation of deoxyribonolactone-containing DNA under simulated physiological conditions gave rise to DNA fragmentation by two consecutive elimination reactions. The DNA-containing products resulting from DNA cleavage at the deoxyribonolactone site were isolated by PAGE and unambiguously characterized by MALDI-TOF MS and chemical fingerprinting assays. The rate of DNA strand scission at the deoxyribonolactone site was measured in single- and double-stranded DNA under simulated physiological conditions: DNA cleavage occurred with a half-life of approximately 20 h in single-stranded DNA and 32-54 h in duplex DNA, dependent on the identity of the deoxynucleotide paired opposite the lesion site. The initial alpha,beta-elimination reaction was shown to be the rate-determining step for the formation of methylene furanone and phosphorylated DNA products. These investigations demonstrated that the deoxyribonolactone site represents a labile lesion under simulated physiological conditions and forms the basis for further studies of the biological effects of this oxidative DNA damage lesion.

In vitro evolution was used to develop a DNA enzyme that catalyzes the site-specific depurination of DNA with a catalytic rate enhancement of about 10 6 -fold. The reaction involves hydrolysis of the N-glycosidic bond of a particular deoxyguanosine residue, leading to DNA strand scission at the apurinic site. The DNA enzyme contains 93 nucleotides and is structurally complex. It has an absolute requirement for a divalent metal cation and exhibits optimal activity at about pH 5. The mechanism of the reaction was confirmed by analysis of the cleavage products by using HPLC and mass spectrometry. The isolation and characterization of an Nglycosylase DNA enzyme demonstrates that single-stranded DNA, like RNA and proteins, can form a complex tertiary structure and catalyze a difficult biochemical transformation. This DNA enzyme provides a new approach for the site-specific cleavage of DNA molecules.depurination ͉ DNA cleavage ͉ DNA repair ͉ in vitro evolution ͉ nucleic acid catalysis A lthough the sugar-phosphate backbone of DNA is highly resistant to hydrolysis, DNA is not impervious to degradation by other means. Damage can occur to either the sugar or nucleobase components of DNA, compromising its strand continuity and information content. Various environmental factors, such as high temperature, oxidative conditions, ionizing radiation, and reactive chemical agents, all can lead to alteration of DNA (1). One of the simplest and best studied forms of DNA damage is spontaneous depurination. This reaction involves hydrolytic cleavage of the N-glycosidic bond of a purine nucleoside ( Fig. 1), giving rise to an apurinic (AP) site (compound 1). The reaction is catalyzed by acid and facilitated by heating. It proceeds by a S N 1-like loss of the purine to give an oxocarbenium ion, which subsequently is trapped with water.Production of AP sites in DNA can result in the loss of genetic information, both because AP sites themselves are mutagenic (2, 3) and because these sites can lead to DNA strand scission (4). As illustrated in Fig. 1, hydrolysis of the N-glycosidic bond unmasks the latent aldehyde functionality at the C1Ј position, rendering the 3Ј-phosphate group susceptible to ␤-elimination (compound 2). It has been estimated that about 10,000 AP sites are produced in a typical mammalian cell each day (5, 6). Not surprisingly, organisms have evolved elaborate biochemical pathways to repair and͞or minimize the effects of these lesions (7-9).Although the primary role of DNA is information storage, it also has structural and functional properties. In recent years, the laboratory evolution of catalytic DNA molecules has demonstrated that single-stranded DNA is capable of forming tertiary structural motifs that catalyze a variety of chemical transformations with rate enhancements comparable to those of naturally occurring enzymes (10, 11). It is reasonable to suppose that more complex DNA enzymes could be developed, including ones that function analogously to DNA repair enzymes.Starting with a population of 10 14 random-seq...

[reaction: see text] An efficient method for the site-specific generation of 2-deoxyribonolactone oxidative DNA damage lesions from a "photocaged" nucleoside analogue was developed. A nucleoside phosphoramidite bearing a C-1' nitrobenzyl cyanohydrin was prepared and incorporated into DNA oligonucleotides using automated DNA synthesis. The caged analogue, which was stable in aqueous solution, was converted to the 2-deoxyribonolactone lesion by UV irradiation. DNA containing the caged analogue and the deoxyribonolactone site were characterized by electrospray mass spectrometry (ES-MS).