5062-5063 Nucleic Acids Research, 1996, Vol. 24, No. 24 © 1996 Oxford University Press

Cytosine-specific chemical probing of DNA using bromide and monoperoxysulfate Steven A. Ross and Cynthia J. Burrows*

Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA

Received July 21, 1996; Revised and Accepted November 5, 1996

ABSTRACT

8romination of cytosine and formation of a piperidine­labile site are observed when two simpie salts, KBr and KHSOs, are allowed to react with single-stranded oligodeoxynucleotides. Selectivity for C compared with T, G or A is typically a factor of 4 or more; selectivity for Cs in a single-stranded region such as a C-bulge is nearly a factor of 10 compared with duplex Cs. low reactivity and little base selectivity are observed using duplex DNA, although increased concentrations of reagents lead to complete degradation of the DNA. The results suggest that these conditions for in situ generation of 81"2 constitute a useful tool for examination of the exposure of a non-duplex cytosine base in folded DNA structures.

Since the initial report of the chemical sequencing of DNA by Maxam and Gilbert (l,2), a series of modifications and improve­ments have been reported (3-6). These new reactions have been developed in order to avoid ambiguities that can occur with the original techniques. In particular, pyrimidine-specific hydrazine reactions have been reported to be less reliable than purine specific reactions (7).

In the course of our studies of metal-mediated DNA oxidation, we discovered a simple cytosine-specific reaction utilizing bromide ion and monoperoxysulfate (KHS05, Oxone), which leads to scission of single-stranded DNA after piperidine treatment. Mechanistic studies (on deoxycytidine) have shown that the reaction proceeds via generation of Br2 in situ, which reacts selectively with the 5,6 double bond to add Br and OH respectively. Under the conditions employed, H20 is then eliminated to give 5-bromodeoxycytidine, which is susceptible to depyrimidination under basic conditions. Although bromination of cytidine and uridine derivatives has been known for some time (8-10), there have been no reports of utilizing this reaction in chemical sequencing or structure probing experiments. In parallel studies, the use of the simple salts KEr + KHS05 to generate Br2 under aqueous conditions has recently been reported for the bromination of alkenes and enones (11). The advantage of this technique lies in avoiding the use of liquid BI2 itself.

Reactions of KErIKHS05 versus aqueous bromine with the 5'-end labelled single-stranded oligodeoxynucleotide 5'_[32p]_ d(ACGTCAGCGTCCGACT) appear to give nearly identical results (Fig. 1). The DNA was purified prior to reaction by anion exchange chromatography followed by 20% polyacrylamide gel electrophoresis under denaturing conditions (7 M urea). The purified oligodeoxynucleotide was then carefully sliced from the gel,

Figure 1. Autoradiogram of 7% denatUling gel electrophoresis experiment using 5'-[32Pl-d(ACGTCAGCGTCCGACT). See text for complete reaction conditions; all lanes include pipelidine treatment after chemical modification. Lane I, controillsing 120 !lM KHSOs; lane 2, 120!lM KHSOs + 240 !lM KBr; lane 3, 240 !lM Br2; lane 4, G lane (12).

extracted with water, dialyzed and lyophilized. The concentrations of the oligonucleotides were determined from the absorbance at 260 nm, and the sequence was labelled at the 5' terminus with [32p] using T4 kinase and [y_32p]ATP. The reaction mixtures (50111) contained 3 11M (strand concentration) unlabelled oligodeoxy­nucleotide, 2 nCi labelled oligodeoxynucleotide, 100 mM NaCI and 10 mM potassium phosphate (pH = 7.0). Reagents were then added (see below), and the mixtures were maintained at ambient conditions for 30 min, followed by addition of 2 mM hydroxy­ethylpiperazine ethanesulfonic acid (HEPES) to quench the oxidant. Samples were then individually dialyzed against water (3 x 3h), lyophilized, treated with 60 111 0.2 M piperidine for 30 min at 90°C, lyophilized, treated with 20 111 water, lyophilized once more, and then resuspended in 80% formamide containing 0.1 % xylene cyanole and bromophenol blue. The DNA product fragments were analyzed using 20% polyacrylamide gel electrophoresis under denaturing conditions (7 M urea) and identified by autoradiography using Kodak X-Omat AR5 film. Scanning densitometry was performed on a Beckman DU 650 spectropho­tometer.

Lane 1 of Figure 1 shows the minimal background reaction of a single-stranded oligodeoxynucleotide using 120 11M monoperoxy­sulfate only, including piperidine treatment; lanes 2 and 3 show C-specific reactivity using either 120 11M KHS05 + 240 11M KEf or 120 11M Br2 respectively, and lane 4 is a G-specific sequencing lane using 60 11M KHS05 + 3 11M NiCR, a metal-mediated reaction reported previously by this group (12). Comparison of lanes 2 and 3 indicates that the KEr/Oxone system is only slightly less reactive than an equivalent concentration of aqueous Br2

*To whom correspondence should be addressed. Tel: + 1 801 585 7290; Fax: + 1 801 5857868; Email: bUITows@atlas.chem.utah.edll

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C

5' 1\ *·A -C-G-T -C-A -G G-T -C-C-G-A -C-T·3' 3'·T-G-C-A-G-T-C-C-A-G-G-C-T-G-A-5'

Figure 2. C-bulged DNA duplex created by annealing of 5' ·end labelled 16mer to a 15mer complement.

itself, and that the pattern of reactivity is similar. Densitometry data is presented in Table 1 in which the most reactive base, C8, is normalized to 100%. In general, all Cs are reactive in single­stranded DNA, and although their reactivities vary by a factor of about 2, they are consistently 2-6-fold more reactive than thymines and other bases. More reactivity was seen at Ts and Gs if the reagent concentration was too high.

Table 1. Relative reactivities of nucleobases toward brominating reagents'

Base Single-stranded DNA C-Bulge duplexb

KBr/KHSOSc Br2d KBrlKHSOs

CI2 51 45 7 CII 98 97 12

TIO 29 32

C8 100 100 100

G7 15 43 16

C5 55 58 9 T4 16 24

aReactions were carried out as described in the text using 5' .[32Pl-d(ACGT­CAGCGTCCGACT). Reactivities are calculated relative to C8, the most reactive site, set as 100. Bands are observed but values are not reported for C2 and C 15 due to the large error in reading the extremities of the gel. bFigure 2; CFigure I, lane 2; dFigure I, lane 3.

We have also employed this technique as a probe for detecting C-bulges in double-stranded DNA. The 16-residue oligodeoxy­nucleotide used in the single-stranded DNA reactions described above was annealed with a 15-residue oligodeoxynucleotide to generate the C-bulged, double-stranded fragment illustrated in Figure 2. The method and conditions for double-strand reactions were as described above. Prior to reaction, the labelled strand was annealed with the non-labelled complementary strand, d(AGTC­GGACCTGACGT) (3 ~), by heating to 90°C for 3 min, then allowing to cool to 25°C over a 3-4 h period. Treatment with KHS05 (240 /-1M) and KBr (240 /-1M) gave 5-15% reactivity predominantly at the bulged C8 residue. A densitometry scan of lanes representing bulged duplex versus single-stranded C reactivity is presented in Table 1. Reactivity at the bulged position was confirmed by running sequencing lanes with the same single-stranded oligodeoxynucleotide in adjacent lanes. For the C-bulge duplex, the extrahelical cytosine base (C8) is -lO-fold more reactive than other Cs in the duplex, and other nucleobases show negligible reactivity with the exception of a small amount of reaction at G7 adjacent to the bulge site.

In order to examine the reactivity of these reagents with doubled-stranded DNA, we have carried out an exhaustive range of experiments with a 5'-end labelled 167 bp restriction fragment from the plasmid pBR322 (obtained from BRL). Restriction fragments were prepared by digestion with EcoRI restriction endonuclease followed by treatment with alkaline calf intestinal phosphatase. 5'-End-labelling was then carried. out with T4 polynucl~oti~e kin~se and [y_32~]~TP, which was followed by a second dlgestIOn Wlth RsaI restnctIOn endonuclease to yield a 167

Nucleic Acids Research, 1996, Vol. 24, No. 24 5063

and a 514 bp fragment. The 167 bp fragment was purified by 8% preparative non-denaturing gel electrophoresis and isolated by the crush and soak method. All enzymes were obtained from New England Bio1abs. Reactions with bromide and monoperoxysulfate were generally carried out as 50 /-11 mixtures containing 20 ~ calf thymus DNA (base pair concentration), 15 nCi 5'-end labelled restriction fragment, 100 mM NaCI, and 10 mM sodium phosphate (pH 7.0). Little C-specific reaction was observed using these conditions although high concentrations of bromide (6oo~) and monoperoxysulfate (300 ~) led to complete and non-specific degradation of the restriction fragment. Reactions were also carried out at pH values ranging from 4.0 to 7.0 without change. Attempts were then made to denature the duplex restriction fragments to single-stranded DNA. This was done by heating the samples to 90°C for several minutes, followed by immediate cooling to O°C and then carrying out the reaction at either 0, 20 or 3rc. The duration of heating (1-60 min) and cooling (0.5-15 min) times was varied, along with reagent concentrations, and some reactions were also carried out in the presence of denaturants (urea, formarnide and dimethylformamide) and at low ionic strength (0-10 mM NaCl). Although C-specific reactions were occasionally observed under these denaturing conditions, we found these experiments to be difficult to reproduce. However, the general conclusion that can be drawn from the data is that double-stranded DNA is much less reactive than single-stranded, as we found in the reactions of C-bulged duplex oligodeoxy­nucleotides described above. Therefore, the reaction is most applicable to studies of cytosine in single-stranded regions such as bulges, hairpin loops or dangling ends. It can be used as a sequencing agent with short oligonucleotides that are easily denatured, but denaturation of long restriction fragments presents a problem, and reagents are too unreactive with duplex DNA.

While electrophilic bromination of cytosine is an old reaction, its new application as a sequencing agent for cytosine in single­stranded DNA and as a structural probe for the accessibility of cytosine bases in duplex DNA should find widespread application due to the ease of use of the reagents, KBr and KHS05, both stable, solid inorganic salts. Studies directed to the application of this chemistry to RNA structural studies and to site-specific synthesis of 5-bromocytidine for bioconjugate chemistry is in progress.

ACKNOWLEDGEMENTS

We thank the NSF for continued support (CHE-952l2l6), the Royal SocietylNATO for a postdoctoral fellowship to SAR, and J. G. Muller (Utah) for technical assistance.

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