Sequence Verification of Oligonucleotides Containing Multiple Arylamine Modifications by Enzymatic Digestion and Liquid Chromatography Mass Spectrometry (LC/MS)
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Sequence Verification of Oligonucleotides Containing Multiple Arylamine Modifications by Enzymatic Digestion and Liquid Chromatography Mass Spectrometry (LC/MS) Lan Gao,a Li Zhang,b Bongsup P. Cho,b and M. Paul Chiarellia a Department of Chemistry, Loyola University, Chicago, Illinois, USA b Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island, USA An analytical method for the structure differentiation of arylamine modified oligonucleotides (ODNs) using on-line LC/MS analysis of raw exonuclease digests is described. Six different dodeca ODNs derived from the reactionN-acetoxy- of N-(trifluoroacetyl)-2-aminofluorene with the dodeca oligonucleotide=-CTCGGCGCCATC-3 5 = are isolated and sequenced with this LC/MS method using=- and3 = -exonucleases.5 When the three products modified by a single aminofluorene (AF) are subjected= -exonucleaseto 3 digestion, the exonuclease will cleave a modified nucleotide but when di-AF modified ODNs are analyzed=-exonuclease the ceases3 to cleave nucleotides when the first modification is exposed=-terminus. at theSmall 3 abundances of ODN fragments formed by the cleavage of an AF-modified nucleotide were observed when two of the three di-AF modified ODNs were= -exonucleasesubjected to 5 digestion. The results of =-exonucleasethe 5 studies of the three di-AF modified ODNs suggest that as the number of unmodified bases between two modifications in an ODN sequence increases, the easier it becomes to sequence beyond the modification=-terminus. closest to the 5 The results of this study indicate that the LC/MS method described here would be useful in sequencing ODNs modified by multiple arylamines to be used as templates for site-specific mutagenesis studies. (J Am Soc Mass Spectrom 2008, 19, 1147–1155) © 2008 American Society for Mass Spectrometry nterest in the analysis of carcinogen-modifieddepend nu- on the number of guanine bases present in the cleic acids is motivated by the belief that thesequence. forma- Ition of these compounds may lead to the develop-Sequence verification using tandem MS methods ment of tumors. The ability of a particular modificationhave been used with varying degrees of success in to induce cancer is believed to be influenced obtainingin part bystructural information from modified oligo- the nucleotide sequence surrounding the modified nucleotidesbase. [6–8]. Applications of tandem MS methods In particular, the identities of the bases surroundingfor the study of carcinogen-modified ODNs have been the lesion site are strongly suggested to influencelimited the due to the occurrence of nonspecific fragmen- rate of mutation and DNA [1,repair 2]. The most tation that may make sequence assignments very diffi- rigorous way to carry out site-specificmutagenesis cult. To date, most efforts to sequence damaged, short studies would be to insert a short DNA strandoligomers contain- (25nucleotides or less) have involved exonu- ing well-characterized modifications at specific cleasesites digestion followed by a sample purification step into a particular vector and then determine thecombined repair with matrix-assisted laser desorption ionization and mutation outcomes[1, 3, . 4]The success of these(MALDI) combined with time-of-flight (TOF) mass spec- studies is predicated on the preparation and character-trometry [9 –13]. In these studies=-o r53=-exonuclease ization of carcinogen-modified oligodeoxyribonucleoti-digests are sampled at different time intervals and des (ODNs) with known sequences. Arylamines, spottedsuch on a laser probe for mass analysis. The as 2-aminofluorene, will bind to the C8 positionMALDI of massa spectra show a series of ODNs whose guanine base through a nitrenium[5] toion form AF mass differences correspond to the nucleotides adduct. The number of sequential isomers obtainedcleaved in from the terminus. This strategy is often the reaction of an activated arylamine with an referredODN will to as ladder sequencing. Previous ESI-MS studies of ODN digestion products [14–18] have been based on off-line preparation and Address reprint requests to Dr. M. P. Chiarelli, Department of Chemistry, Loyola University, 1068 W Sheridan Rd., Chicago, IL 60626, USA. E-mail: desalting followed by mass analysis via direct infusion. [email protected] Direct on-line HPLC-ESI-MS for structure identification Published online May 10, 2008 © 2008 American Society for Mass Spectrometry. Published by Elsevier Inc. Received October 29, 2007 1044-0305/08/$32.00 Revised April 25, 2008 doi:10.1016/j.jasms.2008.04.034 Accepted April 25, 2008 1148 GAO ET AL. J Am Soc Mass Spectrom 2008, 19, 1147–1155 of DNA adducts using exonuclease digestion has not employed a Waters (Milford, MA) XTerra C18 column been accomplished to date. In all cases, adduct ions (10 ϫ 50 mm, 5 m) with a 60-min gradient system formed from salts used in the digestion process have involving 3% to 15% acetonitrile in pH 7.0 ammonium limited the amount of structural information that may acetate buffer (0.10 M) with a flow rate of 2.0 mL/min. be obtained in a mass spectrum. An off-line desalting Each product was purified by repeated HPLC injec- step [16, 17] is often used to clean up the samples before tions. The chromatograms associated with the product ESI-MS (or MALDI) analysis. Here we demonstrate the isolation and purification are shown in Figure 1. Six first methodology using on-line HPLC sample clean up different AF-modified products were isolated and and separation coupled with ESI-MS for analysis of the throughout the following discussion they are referred enzyme digestion products of DNA adducts. Methods to as products (P1-P6) based about their elution (Table based on LC/MS are attractive because they would 1). Throughout the discussion that follows, the position permit the direct analyses of ODN digests as a function of the guanines within the chain will be referred to as of time without any sample pretreatment. The LC/MS G1, G2, and G3 going from the 5=- to the 3=-end, analyses of a variety of dodeca ODNs containing the consistent with Figure 2. NarI sequence, 5=-CTCG1G2CG3CCATC-3=, containing different aminofluorene modifications in different posi- tions are presented. The NarI sequence is a well-known Enzymatic Digestion hot spot for deletion mutations in Escherichia coli [19–21]. Snake venom phosphodiesterase (3=-PDE) and bovine The mono-AF adducts characterized here have been spleen phosphodiesterase (5=-PDE) were acquired from used for structure studies [22]. Worthington Biochemical (Lakewood, NJ) and used without purification. The 3=-PDE cleaves terminal nu- Materials and Methods cleotides sequentially from the 3=-end of the ODN, Sample Preparation while the 5=-PDE cleaves terminal nucleotides sequen- tially from the 5=-end of the ODN. Enzymatic digestions An unmodified dodeca ODN, 5=-CTCGGCGCCATC-3=, were carried out in polypropylene 96-well plates at was treated with incremental amounts of N-acetoxy-N- ambient temperature (24 °C). Each well contained the (trifluoroacetyl)-2-aminofluorene in a pH 6.0 sodium following reagents: 0.75 L of 1 mg/mL aqueous solu- citrate buffer at 37 °C for 18 h [4]. Formation of a tion of each ODN, 3.75 L of 1 unit/mL snake venom covalent adduct at the C8-position of guanine was exonuclease aqueous solution, 3.75 L of 20 mM MgCl2 followed by hydrolysis of the trifluoroacetyl group to solution, and 3.75 L of 0.5% ammonium hydroxide. yield an AF-modified ODN. The reaction mixtures were The final pH of the reaction mixture was 8.8. After purified by reverse phase HPLC, which consisted of a mixing for 30 s, the 96-well plate was loaded onto an Hitachi (Pleasanton, CA) EZChrom Elite system with a autosampler, and the untreated digest samples were L2450 diode array as a detector. The purification system injected at 10 min intervals up to 200 min. The bovine Figure 1. (a) HPLC chromatogram of a mixture from reaction between a 12-mer sequence and N-trifuloroacetyl-N-acetoxy-2-aminofluorene. The mono- (G3, G1, G2) and di- (G1G3, G2, G3, G1G2) AF adducts eluted in the 30–-40 and 50–60 min ranges, respectively. (b) On-line UV spectra of the control (unmodified dodecamer), mono- and di-AF adducts. (c) Purity check of all the fractions. J Am Soc Mass Spectrom 2008, 19, 1147–1155 LC/MS ANALYSES OF MODIFIED OLIGONUCLEOTIDES 1149 Table 1. Calculated and measured molecular weights for the AF-modified dodecaODNs used in this study Sample number Retention time (minutes) Sequence (5’–3’) Calculated mass Measured mass Error (ppm) P1 34.0 CTCGGCG(AF)CCATC 3767.69 3767.67 5.3 P2 34.9 CTCG(AF)GCGCCATC 3767.69 3767.67 5.3 P3 38.0 CTCGG(AF)CGCCATC 3751.69 3751.59 27 P4 49.1 CTCGG(AF)CG(AF)CCATC 3727.68 3727.63 13 P5 57.2 CTCG(AF)G(AF)CGCCATC 3742.68 3742.59 24 P6 58.9 CTCG(AF)GCG(AF)CCATC 3743.68 3743.73 13 spleen exonuclease digests were prepared like the delivered acetonitrile at 50 L/min to increase the snake venom digests described above. The following ionization efficiency by post-column mixing with LC components were added to each well: 0.75 Lofthe eluent. A 2.0 m pore size stainless steel frit was placed ODN aqueous solution at a concentration of 1 mg/mL, in front of the column to prevent clogging. A Waters 1.6 L of 1 unit/mL bovine spleen exonuclease solution (Milford, MA) LCT time-of-flight (TOF) mass spectrom- and 10 L of 10 mM NH4OAc (pH 6.7) buffer. The eter was utilized to detect oligodeoxynucleotides intro- mixture was allowed to react for various time periods duced into the system from HPLC through an electro- up to 200 min at ambient temperature before injection.