A decade of chemical biology.

With insights from a panel of experts, the Nature Chemical Biology editors examine the evolution and current era of chemical biology.

Scripps Florida.

A new division of The Scripps Research Institute that is dedicated to biomedical research and drug discovery is taking shape on the shores of southern Florida.

Transcriptional inhibition by an oxidized abasic site in DNA.

2-Deoxyribonolactone (dL) is an oxidized abasic site in DNA that can be induced by gamma-radiolysis, ultraviolet irradiation, and numerous antitumor drugs. Although this lesion is incised by AP endonucleases, suggesting a base-excision repair mechanism for dL removal, subsequent excision and repair synthesis by DNA polymerase beta is inhibited due to accumulation of a protein-DNA cross-link. This raises the possibility that additional repair pathways might be required to eliminate dL from the genome. Transcription-coupled repair (TCR) is a pathway of excision repair specific to DNA lesions present in transcribed strands of expressed genes. A current model proposes that transcription arrest at the site of DNA damage is required to initiate TCR. In support of this model, a strong correlation between transcription arrest by a lesion in vitro and TCR of the lesion in vivo has been found in most cases analyzed. To assess whether dL might be subject to TCR, we have studied the behavior of bacteriophage T3 and T7 RNA polymerases (T3RNAP, T7RNAP) and of mammalian RNA polymerase II (RNAPII) when they encounter a dL lesion or its "caged" precursor located either in the transcribed or in the nontranscribed strand of template DNA. DNA plasmids containing a specifically located dL downstream of the T3, T7 promoter or the Adenovirus major late promoter were constructed and used for in vitro transcription with purified proteins. We found that both dL and its caged precursor located in the transcribed strand represented a complete block to transcription by T3- and T7RNAP. Similarly, they caused more than 90% arrest when transcription was carried out with mammalian RNAPII. Furthermore, RNAPII complexes arrested at dL were subject to the transcript cleavage reaction mediated by elongation factor TFIIS, indicating that these complexes were stable. A dL in the nontranscribed strand did not block either polymerase.

Photochemical generation of ribose abasic sites in RNA oligonucleotides.

[reaction: see text] A general method for the photochemical generation of ribose abasic sites within RNA oligonucleotides is reported. Photochemically caged nucleoside phosphoramidite analogues were prepared and incorporated into RNA oligonucleotides by automated RNA synthesis. Irradiation of the modified RNA at 350 nm efficiently produced ribose abasic sites at specific sites within RNA sequences. The current approach offers a chemical route to RNA abasic lesions for RNA biochemical studies.

Template-directed assembly and characterization of metallosalen-DNA conjugates.

Nucleic acid template-directed synthesis represents a powerful method for the encoded synthesis of new bioconjugates. Our laboratory previously reported a strategy for the synthesis of a new metal-DNA hybrid, metallosalen-DNA, by the DNA or RNA template-directed cross-linking of two salicylaldehyde-modified DNA oligonucleotides. The current manuscript describes the optimal assembly requirements and biophysical characterization of metallosalen-DNA conjugates containing nickel and manganese ions. Competitive assembly reactions demonstrated the template-directed nature of metallosalen-DNA formation. A single metallosalen-DNA conjugate was assembled selectively in the presence of two pairs of salicylaldehyde precursor strands and a single DNA template. Assembly reactions were sensitive to base pair mismatches in the pairing arms. Single base mismatches resulted in a loss of metallosalen-DNA conjugate yield. Metallosalen-DNA assembly yields depended on the identity and length of the template spacer, the reaction pH, and the type of metal and diamine utilized in the assembly reaction. Metallosalen-DNA conjugates were stable to a variety of conditions, including extended incubation at 50 degrees C. Nickel metallosalen-DNA remained unchanged after incubation at 80 degrees C for 24 h, while decomposition of manganese metallosalen-DNA was observed under the same conditions. Circular dichroism (CD) spectroscopy indicated that DNA duplexes containing internal metallosalen moieties adopted B-form double helices. UV thermal denaturation analysis demonstrated that 32-nucleotide duplexes containing internal metallosalen modifications displayed melting temperatures approximately 5 degrees C less than unmodified DNA duplexes.

Site-specific oxidative cleavage of DNA by metallosalen-DNA conjugates.

Ni-salen-DNA conjugates, prepared by template-directed synthesis, targeted oxidative adduct formation and strand scission at deoxyguanosine sites in complementary DNA strands of Watson-Crick duplexes.

Chain termination and inhibition of Saccharomyces cerevisiae poly(A) polymerase by C-8-modified ATP analogs.

The nucleotide substrate specificity of yeast poly(A) polymerase (yPAP) toward various C-2- and C-8-modified ATP analogs was examined. 32P-Radiolabeled RNA oligonucleotide primers were incubated with yPAP in the absence of ATP to assay polyadenylation using unnatural ATP substrates. The C-2-modified ATP analogs 2-amino-ATP and 2-chloro (Cl)-ATP were excellent substrates for yPAP. 8-Amino-ATP, 8-azido-ATP, and 8-aza-ATP all produced chain termination of polyadenylation, and no primer extension was observed with the C-8-halogenated derivatives 8-Br-ATP and 8-Cl-ATP. The effects of modified ATP analogs on ATP-dependent poly(A) tail synthesis by yPAP were also examined. Whereas C-2 substitution (2-amino-ATP and 2-Cl-ATP) had little effect on poly(A) tail length, C-8 substitution produced moderate (8-amino-ATP, 8-azido-ATP, and 8-aza-ATP) to substantial (8-Br-ATP and 8-Cl-ATP) reduction in poly(A) tail length. To model the biochemical consequences of 8-Cl-Ado incorporation into RNA primers, a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP residue was prepared. Polyadenylation of this modified RNA primer by yPAP in the presence of ATP was blocked completely. To probe potential mechanisms of inhibition, two-dimensional NMR spectroscopy experiments were used to examine the conformation of two C-8-modified AMP nucleotides, 8-Cl-AMP and 8-amino-AMP. C-8 substitution in adenosine analogs shifted the ribose sugar pucker equilibrium to favor the DNA-like C-2'-endo form over the C-3'-endo (RNA-like) conformation, which suggests a potential mechanism for polyadenylation inhibition and chain termination. Base-modified ATP analogs may exert their biological effects through polyadenylation inhibition and thus may provide useful tools for investigating polyadenylation biochemistry within cells.

Half-life and DNA strand scission products of 2-deoxyribonolactone oxidative DNA damage lesions.

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.

Chemoenzymatic synthesis and antibody detection of DNA glycoconjugates.

A chemoenzymatic approach for the efficient synthesis of DNA-carbohydrate conjugates was developed and applied to an antibody-based strategy for the detection of DNA glycoconjugates. A phosphoramidite derivative of N-acetylglucosamine (GlcNAc) was synthesized and utilized to attach GlcNAc sugars to the 5'-terminus of DNA oligonucleotides by solid-phase DNA synthesis. The resulting GlcNAc-DNA conjugates were used as substrates for glycosyl transferase enzymes to synthesize DNA glycoconjugates. Treatment of GlcNAc-DNA with beta-1,4-galactosyl transferase (GalT) and UDP-Gal produced N-acetyllactosamine-modified DNA (LacNAc-DNA), which could be converted quantitatively to the trisaccharide Lewis X (LeX)-DNA conjugate by alpha-1,3-fucosyltransferase VI (FucT) and GDP-Fuc. The facile enzymatic synthesis of LeX-DNA from GlcNAc-DNA also was accomplished in a one-pot reaction by the combined action of GalT and FucT. The resulting glycoconjugates were characterized by gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and glycosidase digestion experiments. Covalent modification of the 5'-terminus of DNA with carbohydrates did not interfere with the ability of DNA glycoconjugates to hybridize with complementary DNA, as indicated by UV thermal denaturation analysis. The trisaccharide DNA glycoconjugate, LeX-DNA, was detected by a dual DNA hybridization/monoclonal antibody (mAb) detection protocol ("Southwestern"): membrane-immobilized LeX-DNA was visualized by Southern detection with a radiolabeled complementary DNA probe and by Western chemiluminescence detection with a mAb specific for the LeX antigen. The efficient chemoenzymatic synthesis of DNA glycoconjugates and the Southwestern detection protocol may facilitate the application of glycosylated DNA to cellular targeting and DNA glycoconjugate detection strategies.

Effects of 8-chlorodeoxyadenosine on DNA synthesis by the Klenow fragment of DNA polymerase I.

8-chloro-2'-deoxyadenosine (8-Cl-dAdo) was incorporated into synthetic DNA oligonucleotides to determine its effects on DNA synthesis by the 3'-5' exonuclease-free Klenow fragment of Escherichia coli DNA Polymerase I (KF-). Single nucleotide insertion experiments were used to determine the coding potential of 8-Cl-dAdo in a DNA template. KF- inserted TTP opposite 8-Cl-dAdo in the template, but with decreased efficiency relative to natural deoxyadenosine. Running-start primer extensions with KF- resulted in polymerase pausing at 8-Cl-dAdo template sites during DNA synthesis. The 2'-deoxyribonucleoside triphosphate analogue, 8-Cl-dATP, was incorporated opposite thymidine (T) approximately two-fold less efficiently than dATP.

Synthesis and hybridization properties of RNA containing 8-chloroadenosine.

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3'- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by approximately 5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.