Nuclease P1 Digestion Research Articles

Nuclease P1 Digestion for Bottom-Up RNA Sequencing of Modified siRNA Therapeutics.

siRNA therapeutics provide a selective and powerful approach to reduce the expression of disease-causing genes. For regulatory approval, these modalities require sequence confirmation which is typically achieved by intact tandem mass spectrometry sequencing. However, this process produces highly complex spectra which are difficult to interpret and typically results in less than full sequence coverage. We sought to develop a bottom-up siRNA sequencing platform to ease sequencing data analysis and provide full sequence coverage. Analogous to bottom-up proteomics, this process requires chemical or enzymatic digestion to reduce the oligonucleotide length down to analyzable lengths, but siRNAs commonly contain modifications that inhibit the degradation process. We tested six digestion schemes for their feasibility to digest the 2' modified siRNAs and identified that nuclease P1 provides an effective digestion workflow. Using a partial digestion, nuclease P1 provides high 5' and 3' end sequence coverage with multiple overlapping digestion products. Additionally, this enzyme provides high-quality and highly reproducible RNA sequencing no matter the RNA phosphorothioate content, 2'-fluorination status, sequence, or length. Overall, we developed a robust enzymatic digestion scheme for bottom-up siRNA sequencing using nuclease P1, which can be implemented into existing sequence confirmation workflows.

Establishing stereochemical comparability in phosphorothioate oligonucleotides with nuclease P1 digestion coupled with LCMS analysis.

Stereochemical comparability is critical for ensuring manufacturing consistency in therapeutic phosphorothioate oligonucleotides. Currently, analytical methods for this assessment are limited. We hereby report on a novel protocol capable of detecting a stereochemistry change in a single phosphorothioate linkage by employing nuclease P1 digestion of the oligonucleotide with subsequent LCMS analysis of the resulting fragments. The method proves valuable for establishing stereochemical comparability and for ensuring manufacturing consistency of oligonucleotide therapeutics.

Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine

Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies. Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis. DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time. The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions. Deoxyadenosine monophosphate (dAMP) was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis. The linear range in DNA quantitation by this method is 1.2–5000 ng/mL. In the validation, the inter-day and intra-day accuracies were within 90%–110%, and the inter-day and intra-day precision were acceptable (RSD < 10%). The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model. Compared to the quantitation methods using UV absorbance, the reported method provides an enhanced sensitivity, and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.

RNA Cap Methyltransferase Activity Assay.

Methyltransferases that methylate the guanine-N7 position of the mRNA 5' cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments.

Click chemistry generated model DNA-peptide heteroconjugates as tools for mass spectrometry.

UV cross-linking of nucleic acids to proteins in combination with mass spectrometry is a powerful technique to identify proteins, peptides, and the amino acids involved in intermolecular interactions within nucleic acid-protein complexes. However, the mass spectrometric identification of cross-linked nucleic acid-protein heteroconjugates in complex mixtures and MS/MS characterization of the specific sites of cross-linking is extremely challenging. As a tool for the optimization of sample preparation, ionization, fragmentation, and detection by mass spectrometry, novel synthetic DNA-peptide heteroconjugates were generated to act as mimics of UV cross-linked heteroconjugates. Click chemistry was employed to cross-link peptides to DNA oligonucleotides. These heteroconjugates were fully characterized by high resolution FTICR mass spectrometry and by collision-induced dissociation (CID) following nuclease P1 digestion of the DNA moiety to a single nucleotide monophosphate. This allowed the exact site of the cross-linking within the peptide to be unambiguously assigned. These synthetic DNA-peptide heteroconjugates have the potential to be of use for a variety of applications that involve DNA-peptide heteroconjugates.

A New Photoproduct of 5-Methylcytosine and Adenine Characterized by High-Performance Liquid Chromatography and Mass Spectrometry

The UV portion of sunlight is mutagenic and can modify DNA by producing various photoproducts. UV photodamage often occurs at dipyrimidine sites, to give cyclobutane, pyrimidine-(6-4)-pyrimidone (6-4), and pyrimidine-(6-4)-Dewar pyrimidone (Dewar) photoproducts, and at TA and AA sites. There is no reported evidence, however, of UV photoproduct formation between C or 5-methylC ((m)C) and A. Irradiation of d(GTAT(m)CATGAGGTGC) with UVB light at physiological pH gives an unexpected photoproduct that undergoes fast thermal deamination but does not revert to its original structure under UVC irradiation. Evidence from nuclease P1 digestion coupled with electrospray ionization (ESI)-MS/MS is in accord with product formation between (m)C and A. HPLC analysis indicates that deamination gives a T<>A photoproduct that coelutes on reverse-phase chromatography with the well-known TA* photoproduct, formed from an initial [2 + 2] reaction between C5-C6 and C6-C5 of the adjacent thymine and adenine [as shown by Zhao , X. , et al. ( 1996 ) Nucleic Acids Res. 24 , 1554 - 1560 and Davies , R. J. , et al. ( 2007 ) Nucleic Acids Res. 35 , 1048 - 1053 ]. Furthermore, the deamination product of the unknown (m)C<>A photoproduct and the TA* photoproduct undergo nearly identical fragmentation in tandem MS. The evidence, taken together, indicates that the deamination product of the unknown (m)CA photoproduct has the same chemical structure as the TA* photoproduct. Therefore, the unknown photoproduct is referred to as the (m)CA* photoproduct, which, upon deamination, gives the TA* photoproduct.

LC−MS/MS for the Detection of DNA Interstrand Cross-Links Formed by 8-Methoxypsoralen and UVA Irradiation in Human Cells

DNA interstrand cross-links (ICLs) are induced by many carcinogens and anitcancer drugs. ICL is a covalent linkage of both strands of DNA, preventing DNA strand separation during transcription and replication; thus, it is extremely cytotoxic in vivo. Psoralen and its derivatives are widely applied for the clinical treatment of several skin diseases and cutaneous T cell lymphoma, and they are also commonly used as model compounds for the study of ICL. Upon UVA photoactivation, 8-methoxypsoralen alkylates both strands of DNA at the 5,6-double bond of thymidines at the 5'-TpA-3' site, generating monoadducts and ICLs. Here we developed a method utilizing HPLC-tandem mass spectrometry, combined with nuclease P1 digestion, to assess the formation of ICL in DNA of human skin melanoma cells exposed to 500 ng/mL 8-methoxypsoralen and UVA irradiation. We were able to quantify ICL, in the form of tetranucleotide, at the level of 1 lesion/10(6) unmodified nucleobases using a low-microgram quantity of DNA. In addition, our results revealed that the formation of ICL increased linearly with the UVA dose. The yield of ICL increased by 15-fold from 4.5 to 76 lesions/10(6) nucleotides when the UV dose was increased from 0.5 to 5 J/cm2. This is the first report of an LC-MS assay for the quantification of DNA interstrand cross-links. The specificity and accuracy of this high-throughput approach are advantageous over other methods for the detection of ICLs formed in vitro and in vivo.

Mutagenicity and DNA adduct formation of PAH, nitro-PAH, and oxy-PAH fractions of atmospheric particulate matter from São Paulo, Brazil

Urban particulate matter (UPM) contributes to lung cancer incidence. Here, we have studied the mutagenic activity and DNA adduct-forming ability of fractionated UPM extractable organic matter (EOM). UPM was collected with a high-volume sampler in June 2004 at two sites, one at street level adjacent to a roadway and the other inside a park within the urban area of the city of São Paulo, Brazil. UPM was extracted using dichloromethane, and the resulting EOM was separated by HPLC to obtain PAH, nitro-PAH, and oxy-PAH fractions which were tested for mutagenicity with the Salmonella strains TA98 and YG1041 with and without S9 metabolic activation. The PAH fraction from both sites showed negligible mutagenic activity in both strains. The highest mutagenic activity was found for the nitro-PAH fraction using YG1041 without metabolic activation; however, results were comparable for both sites. The nitro-PAH and oxy-PAH fractions were incubated with calf thymus DNA under reductive conditions appropriate for the activation of nitro aromatic compounds, then DNA adduct patterns and levels were determined with thin-layer chromatography (TLC) 32P-postlabeling method using two enrichment procedures—nuclease P1 digestion and butanol extraction. Reductively activated fractions from both sites produced diagonal radioactive zones (DRZ) of putative aromatic DNA adducts on thin layer plates with both enrichment procedures. No such DRZ were observed in control experiments using fractions from unexposed filters or from incubations without activating system. Total adduct levels produced by the nitro-PAH fractions were similar for both sites ranging from 30 to 45 adducts per 10 8 normal nucleotides. In contrast, the DNA binding of reductively activated oxy-PAH fractions was three times higher and the adduct pattern consisted of multiple discrete spots along the diagonal line on the thin layer plates. However, DNA adduct levels were not significantly different between the sampling sites. Both samples presented the same levels of mutagenic activity. The response in the Salmonella assay was typical of nitroaromatics. Although, more mutagenic activity was related to the nitro-PAH fraction in the Salmonella assay, the oxy-PAH fractions showed the highest DNA adduct levels. More studies are needed to elucidate the nature of the genotoxicants occurring in São Paulo atmospheric samples.

Unconventional Mechanism of mRNA Capping by the RNA-Dependent RNA Polymerase of Vesicular Stomatitis Virus

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

Contrasting Genome-Wide Distribution of 8-Hydroxyguanine and Acrolein-Modified Adenine during Oxidative Stress-Induced Renal Carcinogenesis

Oxidative stress is a persistent threat to the genome and is associated with major causes of human mortality, including cancer, atherosclerosis, and aging. Here we established a method to generate libraries of genomic DNA fragments containing oxidatively modified bases by using specific monoclonal antibodies to immunoprecipitate enzyme-digested genome DNA. We applied this technique to two different base modifications, 8-hydroxyguanine and 1,N6-propanoadenine (acrotein-Ade), in a ferric nitrilotriacetate-induced murine renal carcinogenesis model. Renal cortical genomic DNA derived from 10- to 12-week-old male C57BL/6 mice, of untreated control or 6 hours after intraperitoneal injection of 3 mg iron/kg ferric nitrilotriacetate, was enzyme digested, immunoprecipitated, cloned, and mapped to each chromosome. The results revealed that distribution of the two modified bases was not random but differed in terms of chromosomes, gene size, and expression, which could be partially explained by chromosomal territory. In the wild-type mice, low GC content areas were more likely to harbor the two modified bases. Knockout of OGG1, a repair enzyme for genomic 8-hydroxyguanine, increased the amounts of acrolein-Ade as determined by quantitative polymerase chain reaction analyses. This versatile technique would introduce a novel research area as a high-throughput screening method for critical genomic loci under oxidative stress.

Detection and quantitation of RNA base modifications.

Using a new combination of previously published techniques, we developed a method for quantitating modified nucleotides in RNAs. First, an RNA is cleaved with RNase H at the 5' side of a nucleotide of interest. Next, 32P is substituted for the phosphate at the 5' end of this nucleotide. Finally, after nuclease P1 digestion, the released radiolabeled nucleotide is analyzed by thin layer chromatography and quantitated by PhosphorImager. Using this method, we showed that the analysis of a pseudouridine at a specific site within an in vitro synthesized U2 RNA is indeed quantitative. We also applied this technique to cellular U2 RNA isolated from mouse liver, and showed that position U34 is approximately 90% pseudouridylated. This method, combined with previously described reverse transcription-based methods, constitutes a powerful tool for detecting and quantifying modified nucleotides in RNAs. With minor modifications, this method can serve as an effective assay to study RNA modifying enzymes.

Synthesis, characterization, and 32P-postlabeling of N-(deoxyguanosin)-4-aminobiphenyl 3'-phosphate adducts.

The (32)P-postlabeling assay is an extremely sensitive technique for detecting carcinogen-DNA adducts. However, for the assignment of DNA adduct structures and the accurate determination of DNA adduct levels by (32)P-postlabeling, authentic adduct standards are needed. For most (32)P-postlabeling applications, such verified synthetic standard compounds are required in the form of their deoxynucleoside 3'-phosphates because they represent substrates for the polynucleotide kinase for transfer of [(32)P]phosphate from [gamma-(32)P]ATP. Three N-(deoxyguanosin)-4-aminobiphenyl 3'-phosphate adducts were prepared and fully characterized by (1)H NMR and mass spectroscopy to serve as standards for the (32)P-postlabeling assay. Apart from the C8- and the N(2)-deoxyguanosine 3'-phosphate adducts of the mutagenic human bladder carcinogen 4-aminobiphenyl (dG3'p-C8-4-ABP and dG3'p-N(2)-4-ABP), the C8-deoxyguanosine 3'-phosphate adduct of the nonmutagenic 4'-tert-butyl-4-aminobiphenyl (dG3'p-C8-4'tBu-4-ABP) was included in the study. Both C8-deoxyguanosine 3'-phosphate adducts were prepared by the in situ formation of deoxyguanosine 3'-phosphate and its subsequent reaction with the appropriate electrophilic amination agent (N-acetoxy compound). The N(2)-deoxyguanosine 3'-phosphate adduct was obtained by a modification of a previously described procedure for the synthesis of N(2)-deoxyguanosine adducts of aromatic amines. The three adduct standards were added at different concentrations to calf thymus DNA, and adduct recoveries were determined by the (32)P-postlabeling assay under conditions routinely used in the standard methodology, enhancement by nuclease P1 digestion and enhancement by butanol extraction. The dG3'p-C8-4-ABP adduct was recovered irrespective of the concentration with approximately 30% in both the standard and the butanol extraction version of the assay. Both C8-deoxyguanosine 3'-phosphate adducts were sensitive to nuclease P1 digestion resulting in recoveries of only 1-3%. In contrast, the dG3'p-N(2)-4-ABP adduct was resistant to nuclease P1 digestion; however, recovery in all three versions was poor (1-2%) resulting in a detection limit of one adduct/10(6) nucleotides. These results demonstrate that the (32)P-postlabeling assay underestimates the level of DNA adducts formed by 4-ABP and indicates that there is a need to determine the recovery for each adduct to be analyzed by the (32)P-postlabeling technique.

Analysis of 8-Hydroxyguanine (8-OH-Gua) Released from DNA by the Formamidopyrimidine DNA Glycosylase (Fpg) Protein: A Reliable Method to Estimate Cellular Oxidative Stress

To improve the analyses of a form of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua), we treated isolated DNA with formamidopyrimidine DNA glycosylase (Fpg) and analyzed the released 8-OH-Gua by using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The human lung carcinoma cells (A549) and human keratinocyte (HaCaT) were irradiated with gamma-rays. After the isolated DNA was treated with the Fpg protein, we analyzed the released 8-OH-Gua by using an HPLC-ECD. With this method, the background level of 8-OH-Gua in DNA from human lung carcinoma cells was determined to be 3.4 residues per 10(7) guanine (Gua). A similar background level of 8-OH-Gua (3.1 residues per 10(7) Gua) was also detected in human keratinocyte DNA with this method. These background 8-OH-Gua levels in cellular DNA are comparable to that obtained previously by an analysis of 8-OH-dGMP after nuclease P1 digestion of cellular DNA (4.3 residues per 10(7) dCMP). A dose-dependent increase of 8-OH-Gua (0.17/10(7) Gua/Gy) was observed after cells were irradiated with gamma-rays. Twenty hours after gamma-irradiation with 60 Gy, 75% of the 8-OH-Gua produced in keratinocyte DNA was repaired. With our new analysis method, it is possible to detect the small changes in the 8-OH-Gua levels in cellular DNA induced by various environmental factors.

Structure elucidation of DNA interstrand cross-link by a combination of nuclease P1 digestion with mass spectrometry.

DNA interstrand cross-link reagents are among the most powerful agents for cancer treatment. Here we report a combined nuclease P1 digestion/mass spectrometry method for the structure elucidation of duplex oligodeoxynucleotides (ODNs) containing an interstrand cross-link. Our results demonstrate that nuclease P1 digestion of a double-stranded ODN containing an interstrand cross-link (ICL) of 4,5',8-trimethylpsoralen or mitomycin C gives a tetranucleotide bearing the cross-linked nucleobase moiety. Product ion spectra of the deprotonated ions of the tetranucleotides provide information about the structure of the cross-link. Furthermore, product-ion spectra of tetranucleotides containing two orientation isomers of mitomycin C interstrand cross-link are distinctive. We believe that the method described in this paper can be generally applicable for investigating the structures of other DNA ICLs.

Identification of N-(deoxyguanosin-8-yl)-4-azobiphenyl by (32)P-postlabeling analyses of DNA in human uroepithelial cells exposed to proximate metabolites of the environmental carcinogen 4-aminobiphenyl.

DNA adducts formed in human uroepithelial cells (HUC) following exposure to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP), were analyzed by the (32)P-postlabeling method. Two adducts detected by (32)P-postlabeling were previously identified as the 3',5'-bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (dA-C8-ABP) (Frederickson S et al. [1992] Carcinogenesis 13: 955-961; Hatcher and Swaminathan [1995b] Carcinogenesis 16: 295-301). In contrast to the dG-C8-ABP adduct, which was 3'-dephosphorylated by nuclease P1, dA-C8-ABP was resistant to nuclease P1, thus providing an enrichment step before postlabeling. Autoradiography of the two-dimensional thin-layer chromatogram of the postlabeled products obtained following nuclease P1 digestion revealed several minor adducts, one of which has been identified in the present study. Postlabeling analyses following nuclease P1 digestion of the products obtained from the reaction of N-acetoxy-4-aminobiphenyl with deoxyguanosine-3'-monophosphate (dGp) demonstrated the presence of this minor adduct. The 3'-monophosphate derivative of the adduct was subsequently chromatographically purified and subjected to spectroscopic analyses. Based on proton NMR and mass spectroscopic analyses of the synthetic product, the chemical structure of the adduct has been identified as N-(deoxyguanosin-N(2)-yl)-4-azobiphenyl (dG-N==N-ABP). (32)P-Postlabeling analysis of the nuclease P1-enriched DNA hydrolysate of HUCs treated with N-OH-ABP or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) showed the presence of the dG-N==N-ABP adduct. It was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl-CoA, or incubated with HUC microsomes and N-OH-AABP. These results demonstrate that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP and N-OH-AABP are bioactivated by acyltransferases to reactive arylnitrenium ions that covalently interact at the N2 position of deoxyguanosine in DNA.

Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses

Estimates of 8-oxo-2′-deoxyguanosine (8-oxo-dG) in DNA vary at least one order of magnitude using different quantitative methods or even the same method. Our hypothesis is that an incomplete DNA hydrolysis to nucleosides by the conventional nuclease P1 (NP1) and alkaline phosphatase (AP) digestion system plays an important role in contributing to the variability of measurements using HPLC coupled with UV and electrochemical (EC) detection. We show here that factors, such as the amount of DNA, choice of enzymes, their activities, and incubation time, can affect DNA digestion and, thus, cause variability in 8-oxo-dG levels. The addition of DNase I and phosphodiesterases I and II to the NP1 + AP system improves the DNA digestion by completely releasing normal nucleosides and 8-oxo-dG, thereby reducing the interday variations of 8-oxo-dG levels. Diethylenetriamine pentaacetic acid (DTPA), an iron chelator, prevented background increases of 8-oxo-dG during DNA digestion, as well as during the waiting period in the autosampler when a batch of DNA samples is analyzed by HPLC. After optimization of the DNA digestion conditions, the interday variability of 8-oxo-dG measurements using commercially available salmon testes DNA (ST DNA) were 26% over a period of 2 years. Under these optimal conditions, our laboratory variability may contribute as little as 13% to the overall variability as shown by assessment of oxidative DNA damage in a population of smokers. Based on our results, we believe that the modified DNA digestion conditions will provide much more accurate 8-oxo-dG determinations and, thus, more reliable estimates of cancer risk.

DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by32P-postlabeling

Diesel exhaust is known to induce tumors in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust and in airborne particulate matter, 3-nitrobenzanthrone (3-NBA), is a particularly powerful mutagen. We investigated the capacity of 3-NBA to form DNA adducts in vivo that could be used as agent-specific biomarkers of exposure. Female Sprague-Dawley rats were treated orally with 2 mg/kg body weight of 3-NBA, and DNA from various organs was analyzed by (32)P-postlabeling. High levels of 3-NBA-specific adducts were detectable in all organs. Both enrichment versions nuclease P1 digestion and n-butanol extraction resulted in patterns consisting of either 3 or 4 adducts remarkably similar in all tissues examined. The highest level of DNA adducts was found in the small intestine (38 adducts per 10(8) nucleotides) followed by forestomach, glandular stomach, kidney, liver, lung and bladder. To provide information on the nature of the adducts formed in vivo in rats, DNA adducts were cochromatographed in 2 independent systems with standardized deoxyguanosine adducts and deoxyadenosine adducts produced by reaction of 3-NBA in the presence of xanthine oxidase with deoxyribonucleoside 3'-monophosphates in vitro. In both systems, each of the rat adducts comigrated either with a deoxyguanosine or a deoxyadenosine-derived 3-NBA adduct. Our results demonstrate that 3-NBA binds covalently to DNA after metabolic activation, forming multiple DNA adducts in vivo, all of which are products derived from reductive metabolites bound to the purine bases (deoxyguanosine 60% and deoxyadenosine 40%).

Double-Base Lesions Are Produced in DNA by Free Radicals

Evidence has been accumulating at the oligomer level that free radical-initiated DNA damage includes lesions in which two adjacent bases are both modified. Prominent examples are lesions in which a pyrimidine base is degraded to a formamido remnant and an adjacent guanine base is oxidized. An assay has been devised to detect double-base lesions based on the fact that the phosphoester bond 3′ to a nuclesoside bearing the formamido lesion is resistant to hydrolysis by nuclease P1. The residual modified dinucleoside monophosphates obtained from a nuclease P1 (plus acid phosphatase) digest of DNA can be 32P-postlabeled using T4 polynucleotide kinase. Using this assay the formamido single lesion and the formamido–8-oxoguanine double lesion were detected in calf thymus DNA after X-irradiation in oxygenated aqueous solution. The lesions were measured in the forms d(PFpG) and d(PFpGH), where PF stands for a pyrimidine nucleoside having the base degraded to a formamido remnant and GH stands for 8-oxo-deoxyguanosine. The yields in calf thymus DNA irradiated 60 Gy were 8.6 and 3.2 pmol/μg DNA, respectively.

Identification of unexpected modifications of fluorescein-labeled oligodeoxynucleotides by nuclease P1 digestion and mass spectrometric techniques.

Fluorescein-labeled oligodeoxynucleotides (ODNs) from automated synthesis commonly produce multiple peaks in high performance liquid chromatography (HPLC) chromatograms. We found that these peaks are due to chemical modifications of the ODNs instead of the common perception of isomers. To identify the modifications, a model ODN, fluorescein-T(25), was synthesized and five compounds were isolated. Nuclease P1 (NP1) digestion was employed to cleave these compounds into nucleotides and fluorescein-nucleotides in order that the modifications be determined by mass spectrometry (MS). Analyses of NP1 digestion products containing fluorescein by MS revealed the expected product F1-T (M) and four unexpected compounds with MWs at M-1, M-17, M-16 and M + 16, respectively. Collision-induced dissociation (CID) spectra of these digestion products indicate that all modifications occur on the thiourea linkage [-NH-C( = S)-NH-] to the fluorescein moiety and the adjacent phosphate group, and the modifications were determined. The modifications were also confirmed by accurate mass measurement with Fourier transform mass spectrometry (FT-MS), by the synthesis of a reference compound, and by a mechanistic study using model compounds. These results demonstrate the power of the mass spectrometric techniques by determining the structures of two pairs of ODNs with MW difference of 1 Da. The results also suggest that fluorescein phosphoramidite with a thiourea linkage is not appropriate for the automated synthesis of fluorescein-labeled ODNs of high purity.

Nuclease P1 digestion combined with tandem mass spectrometry for the structure determination of DNA photoproducts.

UV irradiation of oligodeoxynucleotides at 254 nm generates several different types of DNA photoproducts, such as cis-syn cyclobutane pyrimidine dimers, pyrimidine[6-4]pyrimidone photoproducts and their Dewar valence isomers, and thymine-adenine photoproducts (TA). Nuclease P1 degrades the oligodeoxynucleotide photoproducts to small photoproduct-containing trinucleotides which are more amenable to tandem mass spectrometry (MS/MS) and HPLC. Product-ion mass spectra of these digestion products give characteristic fragmentations, allowing us to identify quickly the types of photomodifications. The results also show that mass spectrometry will be a tool for studying enzyme reaction mechanisms because it can determine rapidly and with high sensitivity the structures of the products that are generated.