Solid-phase synthesis of oligonucleotides containing site-specific N-(2'-deoxyguanosin-8-yl)-2-(acetylamino)fluorene adducts using 9-fluorenylmethoxycarbonyl as the base-protecting group

Zhou, Yuanzhong; Romano, Louis J.

doi:10.1021/bi00213a038

Cited by 31 publications

(44 citation statements)

References 30 publications

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“…This data is summarized in Table 2. Fuchs and Romano independently reported T m data for AAF-modified oligonucleotides in the NarI sequence (40,41). The results from the Fuchs study (entries e-g) as well as our previously reported data on 1a and 4a are also listed in Table 2 for comparison.…”

Section: Thermal Melting (T M ) Studies Of Iq-adducted Oligo-nucleotidesmentioning

confidence: 79%

“…To simulate the two-base slippage product, Fuchs and Romano independently examined the thermal stability of an oligonucleotide duplex in which the GpC nucleotides opposite the C8-AAF adduct were deleted (41,46). The C8-AAF adduct at the G 3 -position was found to stabilize the duplex relative to the unmodified oligonucleotide by 15 °C.…”

Section: Thermal Melting (T M ) Studies Of Iq-adducted Oligo-nucleotidesmentioning

confidence: 99%

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Conformational Differences of the C8-Deoxyguanosine Adduct of 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) within the NarI Recognition Sequence

Elmquist

Wang

Stover

et al. 2007

Chem. Res. Toxicol.

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Abstract2-Amino-3-methylimidazo [4,5-f]quinoline (IQ) is a highly mutagenic heterocyclic amine found in cooked meats. The major DNA adduct of IQ is at the C8-position of dGuo. We have previously reported the incorporation of the C8-IQ adduct into oligonucleotides, namely, the G 1 -position of codon 12 of the N-ras oncogene sequence (G 1 G 2 T) and the G 3 -position of the NarI recognition sequence (G 1 G 2 CG 3 CC) (Elmquist et al. (2004) J. Am. Chem. Soc. 126, 11189-11201). Ultraviolet spectroscopy and circular dichroism studies indicated that the conformation of the adduct in the two oligonucleotides was different, and they were assigned as groove-bound and base-displaced intercalated, respectively. The conformation of the latter was subsequently confirmed through NMR and restrained molecular dynamics studies (Wang et al. (2006) J. Am. Chem. Soc. 128, 10085-10095). We report here the incorporation of the C8-IQ adduct into the G 1 -and G 2 -positions of the NarI sequence. A complete analysis of the UV, CD, and NMR chemical shift data for the IQ protons are consistent with the IQ adduct adopting a minor groove-bound conformation at the G 1 -and G 2 -positions of the NarI sequence. To further correlate the spectroscopic data with the adduct conformation, the C8-aminofluorene (AF) adduct of dGuo was also incorporated into the NarI sequence; previous NMR studies demonstrated that the AF-modified oligonucleotides were in a sequence-dependent conformational exchange between major groove-bound and base-displaced intercalated conformations. The spectroscopic data for the IQ-and AF-modified oligonucleotides are compared. The sequence-dependent conformational preferences are likely to play a key role in the repair and mutagenicity of C8-arylamine adducts.

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Section: Thermal Melting (T M ) Studies Of Iq-adducted Oligo-nucleotidesmentioning

confidence: 79%

Section: Thermal Melting (T M ) Studies Of Iq-adducted Oligo-nucleotidesmentioning

confidence: 99%

Conformational Differences of the C8-Deoxyguanosine Adduct of 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) within the NarI Recognition Sequence

Elmquist

Wang

Stover

et al. 2007

Chem. Res. Toxicol.

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“…Oligonucleotides were obtained from Midland Certified, Inc. Sitespecifically modified 12-mer (GTGATG (C8-AAF) ATAAGT) was synthesized and purified as described previously (21). All dNTPs and dideoxyNTPs were ordered from Amersham Pharmacia Biotech.…”

Section: Methodsmentioning

confidence: 99%

Interaction of Escherichia coli DNA Polymerase I (Klenow Fragment) with Primer-Templates ContainingN-Acetyl-2-aminofluorene or N-2-Aminofluorene Adducts in the Active Site

Dzantiev

Romano

1999

Journal of Biological Chemistry

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DNA adducts formed by aromatic amines such as N-acetyl-2-aminofluorene (AAF) and N-2-aminofluorene (AF) are known to cause mutations by interfering with the process of DNA replication. To understand this phenomenon better, a gel retardation assay was used to measure the equilibrium dissociation constants for the binding of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) to DNA primertemplates modified with an AAF or AF adduct. The results indicate that the nature of the adduct as well as the presence and nature of an added dNTP have a significant influence on the strength of the binding of the polymerase to the DNA. More specifically, it was found that the binding is 5-10-fold stronger when an AAF adduct, but not an AF adduct, is positioned in the enzyme active site. In addition, the polymerase was found to bind the unmodified primer-template less strongly in the presence of a noncomplementary dNTP than in the presence of the correct nucleotide. The same trend holds true for the primer-template having an AF adduct, although the magnitude of this difference was lower. In the case of the AAF adduct, the interaction of the polymerase with the primer-template was stronger and almost independent of the nucleotide present.It is well established that the presence of DNA adducts in the template strand can impede or block DNA synthesis at a replication fork. Although most bulky adducts inhibit DNA synthesis strongly, some can be bypassed readily in vitro. The well studied carcinogen N-acetyl-2-aminofluorene (AAF) 1 can form both types of adducts in DNA; N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adducts (dG-C8-AAF) are known to be strong blocks to DNA synthesis, whereas N-(deoxyguanosin-8-yl)-2-aminofluorene adducts (dG-C8-AF) can be bypassed by all polymerases tested (1). The mutagenic consequences of each adduct are also quite distinct. The dG-C8-AAF adduct results in mostly frameshift mutations in bacteria, whereas the dG-C8-AF adduct produces predominantly base substitution mutations (2-4). These different properties are likely to be the result of differences in the structures that these adducts assume in DNA in the active site of the DNA polymerase. Structural and enzymatic studies on duplex DNA molecules have demonstrated that the AF adduct produces less distortion in the DNA helix than the AAF adduct (2, 5). Multidimensional NMR experiments show that the guanine bearing the C8-AAF adduct rotates from anti to syn conformation in the doublestranded DNA helix with the fluorene ring inserted into the helix (base displacement model (6)). This contrasts with the AF adduct, which can adopt interchangeable conformations: in one the fluorene remains outside the helix (outside binding model), whereas the other has the fluorene ring stacked within the helix (5). The ratio of these conformations seems to be dependent on the sequence within which the adduct lies (7). Although it has been assumed that the structural differences between the AAF and AF adducts are responsible for the observed in the...

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“…Early studies along those lines quickly revealed that the ammonia deprotection step required after solid-phase synthesis compromised the integrity of the N 8 acetyl group of the dG-AAF adduct (32). In 1993, Zhou and Romano reported the successful incorporation of an Fmoc-protected dG-AAF phosphoramidite in a 12mer oligonucleotide (33,34). The use of Fmoc as a protecting group for the exocyclic amines of all the bases, made it possible to perform a mildly basic deprotection step, devised to preserve the N 8 acetyl group of dG-AAF (33).…”

Section: Introductionmentioning

confidence: 99%

Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis

Gillet

Alzeer

Schärer

2005

Nucleic Acids Research

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Aromatic amino and nitro compounds are potent carcinogens found in the environment that exert their toxic effects by reacting with DNA following metabolic activation. One important adduct is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF), which has been extensively used in studies of the mechanisms of DNA repair and mutagenesis. Despite the importance of dG-AAF adducts in DNA, an efficient method for its incorporation into DNA using solid-phase synthesis is still missing. We report the development of a modified ‘ultra-mild’ DNA synthesis protocol that allows the incorporation of dG-AAF into oligonucleotides of any length accessible by solid-phase DNA synthesis with high efficiency and independent of sequence context. Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of β-mercaptoethanol) designed to remove protecting groups of commercially available ‘ultra-mild’ phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF. We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction. Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.

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Solid-phase synthesis of oligonucleotides containing site-specific N-(2'-deoxyguanosin-8-yl)-2-(acetylamino)fluorene adducts using 9-fluorenylmethoxycarbonyl as the base-protecting group

Cited by 31 publications

References 30 publications

Conformational Differences of the C8-Deoxyguanosine Adduct of 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) within the NarI Recognition Sequence

Conformational Differences of the C8-Deoxyguanosine Adduct of 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) within the NarI Recognition Sequence

Interaction of Escherichia coli DNA Polymerase I (Klenow Fragment) with Primer-Templates ContainingN-Acetyl-2-aminofluorene or N-2-Aminofluorene Adducts in the Active Site

Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis

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