dna lesions can be 
 mutagenic and have been implicated in a variety 
 diseases , most notably cancer , as well as aging . quantifying nucleic acid damage is a valuable exercise as modified 
 nucleosides , nucleobases , and sugar fragments are potential biomarkers .
8-oxo-7,8-dihydro-2-deoxyguanosine ( 8-oxodguo ) is a well - studied , 
 mutagenic lesion that is used as a biomarker .
this 
 lesion is produced from the most readily oxidized of native nucleotides , 
 dg , by a variety of damaging agents .
8-oxodguo 
 is even more readily oxidized than dg and serves as the precursor 
 to a number of highly mutagenic lesions that are of increasing interest 
 to chemical toxicologists and other scientists .
herein , we describe 
 a method for detecting 8-oxodguo that relies upon its tagging by a 
 reagent and subsequent signal detection using quantitative pcr ( qpcr ) .
mass spectrometry is a sensitive and selective method for 
 detecting 
 a large number of dna lesions .
the variety of lesions detectable is expanded when the method is 
 coupled with chemical derivatization techniques .
quantification is greatly facilitated by spiking samples with known 
 quantities of isotopically labeled lesions that require costly independent 
 synthesis .
in addition , mass spectrometers are increasingly powerful , 
 and dna lesion detection methods employing them are proportionally 
 more sophisticated , but the instruments are also expensive . the comet assay is a less costly method that 
 is particularly useful for detecting lesions in cellular dna , but 
 its specificity is limited by the selectivity of chemical reagents 
 and enzymes that cleave dna .
sophisticated methods that take advantage of selective 
 dna lesion tagging and enable the detection of the location of individual 
 lesions in single strands of dna are on the horizon .
reagents that 
 also take advantage of distinctive lesion reactivity 
 but utilize more conventional reporting methods are more common .
aldehyde reactive probe(s ) equipped with fluorophores or biotin 
 have proven useful for quantifying abasic sites and are still being 
 developed .
turn on sensors that exploit the formation of a fluorophore 
 upon reaction with a functional group within a lesion have also been 
 developed .
recently , 
 molecules that selectively recognize 8-oxodguo and incorporate fluorescence 
 reporting have been developed .
there are also efforts 
 reported that utilize aptamers to detect this damaged nucleoside or 
 its respective free base ( 8-oxogua ) .
it is well known that 
 mild oxidation of 8-oxodguo produces the 
 guanidinohydantoin ( gh ) and spiroiminohydantoin ( sp ) lesions that 
 are interesting in their own right .
however , oxidation 
 of 8-oxodguo or fapydg in the presence of an amine nucleophile , 
 such as a spermine ( rnh2 ) , provides a dna adduct ( scheme 1 ) in competition with gh and sp . we previously 
 reported a method for selectively detecting 8-oxodguo and the mechanistically 
 related fapydg lesion by utilizing this chemistry , first reported 
 by burrows , and 1 as a nucleophile to trap the reactive 
 oxidized species . fapydg and
8-oxodguo were distinguished from one another by 
 using an oxidant , k3fe(cn)6 , which oxidizes the latter but not the former lesion .
following 
 the adsorption of tagged dna to a surface , the biotinylated material 
 is used to capture a streptavidin  horseradish peroxidase complex , 
 which yields the signal by oxidizing a profluorescent molecule to 
 a fluorescent one .
the fluorescence intensity is proportional to the 
 amount of horseradish peroxidase bound to the surface , which is dependent 
 upon the amount of biotinylated spermine ( 1 ) that is 
 covalently bound to the dna .
this method was time - consuming due to 
 a lengthy procedure for preparing the plates on which the lesions 
 were quantified .
herein , we have improved the limit of detection to 14 amol and decreased 
 the time required to complete the analysis to 3 h by combining qpcr 
 with a new biotinylated reagent .
taq dna polymerase , onetaq hotstart 
 dna polymerase , and exonuclease i ( exo i ) were from new england biolabs . 

oligonucleotides were synthesized via standard automated dna synthesis 
 on an applied biosystems model 394 instrument .
dna synthesis 
 reagents , including the phosphoramidite for incorporating 8-oxodguo 
 were obtained from glen research .
radiolabeled oligonucleotides were 
 hybridized with 1.5 equiv of complementary oligonucleotides in 10 
 mm potassium phosphate ( ph 7.2 ) and 100 mm nacl at 90 c for 
 5 min and cooled to room temperature .
solid - phase 
 peptide synthesis was carried out on 10 obtained from 
 cs bio ( 0.45 mmol / g ) .
automated solid - phase peptide synthesis ( spps ) 
 was performed on a symphony peptide synthesizer .
the plasmid containing the p53 gene was obtained as previously 
 described . a solution ( 500 l ) 
 of primers ( 0.2 m each ) , template dna ( 20 fm ) , dntps ( 0.4 mm ) , 
 and onetaq hotstart dna polymerase ( 2.5  10
u / l ) 
 in 1 onetaq standard buffer was partitioned into thin - walled 
 pcr tubes ( 50 l each ) .
the pcr cycle was performed as follows : 
 ( 1 ) 94 c for 30 s , ( 2 ) 94 c for 30 s , ( 3 ) 60 c for 
 30 s , ( 4 ) 68 c for 1.5 min , ( 5 ) steps 24 repeated 45 
 times , ( 6 ) 68 c for 5 min , and ( 7 ) held for 4 c .
the reactions 
 were pooled , incubated with exo i ( 4  10 u / l , 1 
 h , 37 c ) , and evaporated under vacuum .
the dna was purified 
 from the residue by silica spin column chromatography ( qiagen pcr 
 purification kit ) following the manufacturer s protocol , except 
 that an additional wash with 35% guanidinehcl was performed 
 following adsorption to the spin column .
purity and product length were confirmed 
 on 1% agarose gel/1 tbe precasted with etbr ( 0.5 mg / l ) .
typical 
 yields were 20200 nm in 200 l solution , corresponding 
 to 10- to 10-fold amplification .
the p53 pcr fragment containing 8-oxodguo was prepared using the 
 forward primer 5-d(gca gtc aga tcc tag cxt cga gc ) where x 
 = 8-oxodguo and the reverse primer 5-d(ggg cag tgc tcg ctt 
 agt gc ) .
the p53 pcr fragment without 8-oxodguo was prepared using 
 the same reverse primer and 5-d(gca gtc aga tcc tag cgt cga 
 gc ) as the forward primer .
the puc19 pcr fragment was prepared using 
 the forward primer 5-d(bggt gat gac ggt gaa aac ctc ) , where 
 b = biotin linked to the remainder of the primer via tetraethylene 
 glycol .
a chilled acetonitrile 
 solution ( 81 ml ) of fmoc - nhs ester ( 18.1 g , 53.7 mmol ) was added to 
 a vigorously stirred solution of carboxylated spermine ( 5 g , 12.8 mmol ) in 10% na2co3 ( 81 ml ) in an ice bath .
acetonitrile was removed 
 under vacuum , and the residue was acidified with a solution of brine 
 and 0.1 m hcl ( ph 1 , 200 ml ) . the aqueous solution was extracted with 
 dcm ( 5  200 ml ) , and the organic layer was dried over na2so4 and filtered .
the resulting dry powder was applied over 
 a silica gel plug and washed with copious amounts of 1:1 hex / ea .
removal 
 of the nonpolar species was monitored by tlc . after removing the nonpolar 
 species ,
1% formic acid was added to the eluent to elute product - containing 
 fractions .
the eluent was concentrated under vacuum and applied to 
 a silica gel column to further purify the product using 1% formic 
 acid in 1:1 hex / ea .
fractions containing the product were pooled , 
 concentrated under vacuum , and redissolved in ea for a basic wash 
 ( 5 , saturated nahco3/brine ) , followed by an acidic 
 wash ( 2 , brine 0.1 m hcl , ph 1 ) .
the organic layer was concentrated 
 to a sticky white foam ( 5.4 g , 37% yield ) and redissolved in minimal 
 dcm for precipitation into vigorously stirring hexane .
the supernatant 
 was removed following centrifugation and the residue lyophilized to 
 a white powder .
2.603.30 ( br , m , 9h ) , 4.13 ( br , m , 4h ) , 4.38 ( br , 
 m , 5h ) , 4.56 ( br , s , 4h ) , 7.247.74 ( m , 32h ) .
c nmr ( cdcl3 ) :  11.4 , 14.2 , 18.8 , 20.7 , 22.6 , 25.3 , 
 29.1 , 31.6 , 34.5 , 34.7 , 36.1 , 47.1 , 47.3 , 47.36 , 47.44 , 77.2 , 119.9 , 
 120.0 , 124.5 , 124.6 , 124.7 , 124.9 , 125.1 , 127.0 , 127.1 , 127.7 , 127.8 , 
 141.31 , 141.34 , 141.37 , 141.39 , 143.8 , 143.9 , 156.5 , 156.6 .
ir ( film ) : 
 3292 ( br ) , 2927 , 1703 , 1644 , 1553 cm .
hrms calcd 
 for c71h67n4o10 ( m + h ) 1135.4852 ; found , 1135.4887 . a thf solution of dmtrcl 
 ( 1.4 g , 4 mmol , 0.2 m ) was added dropwise to a vigorously stirred 
 solution of diamine 4 ( 48 mmol , 10.6 ml ) in thf ( 20 ml ) . 

additional dmtrcl / thf solution ( 6.7 g , 20 mmol , 55 ml thf ) was added 
 in 5 ml portions for a total of 30 ml .
the appropriate amount of dmtrcl added was followed by tlc ( 30% ea / hex ) 
 to maximize monotritylation and minimize ditritylation .
volatiles 
 were removed in vacuo , and the residue diluted with 
 dcm ( 200 ml ) , washed with brine ( 3  200 ml ) , dried with na2so4 , filtered , and concentrated for column chromatography 
 ( isocratic elution : 5% tea / dcm ) , affording 7.7 g ( 91% yield ) of 5 as a clear yellow oil .
h nmr ( cdcl3 ) :  1.701.80 ( m , 7h ) , 2.2 ( t , j = 
 8 hz , 2h ) , 2.762.79 ( t , j = 8 hz , 2h ) , 3.493.59 
 ( m , 12h ) , 3.75 s , 6h ) , 6.766.79 ( m , 4h ) , 7.13 ( m , 1h ) , 7.217.26 
 ( m , 2h ) , 7.337.35 ( m , 4h ) , 7.427.45 ( m , 2h ) .
c nmr ( cdcl3 ) :  30.8 , 33.3 , 39.8 , 41.3 , 
 55.4 , 69.7 , 70.1 , 70.3 , 70.37 , 70.44 , 70.8 , 70.9 , 113.2 , 126.2 , 127.9 , 
 128.7 , 129.9 , 139.0 , 147.1 , 157.9 . hrms calcd for c31h42n2o5na ( m + na ) 545.2986 ; 
 found , 545.3007 . a mixture 
 of biotin ( 770 
 mg , 3.2 mmol ) , dcc ( 710 mg , 3.4 mmol ) , and hobt ( 465 mg , 3.4 mmol ) 
 was dried in vacuo for 30 min prior to suspension 
 in 5% tea / dmf ( 10 ml ) under ar at 40 c .
after 30 min , the suspension 
 was added to 5 ( 1.53 g , 2.94 mmol ) ( predried by azeotropically 
 drying from pyridine ) in 5% tea / dmf ( 10 ml ) .
the precipitate was again removed by passage through celite , and the 
 filtrate was concentrated for column chromatography ( 5% tea / dcm to 
 5% tea/5% meoh / dcm ) , affording 2.2 g ( 100% ) of 6 .
h nmr ( cdcl3 ) :  1.201.23 ( m , 2h ) , 
 1.421.76 ( m , 8h ) , 2.162.19 ( m , 4h ) , 2.802.84 
 ( m , 3h ) , 3.103.15 ( m , 1h ) , 3.323.34 ( m , 1h ) , 3.523.61 
 ( m , 12h ) , 3.77 ( s , 6h ) , 4.264.29 ( dd , 1h ) , 4.434.50 
 ( dd , 1h ) , 5.02 ( br , s , 1h ) , 5.72 ( br , s , 1 h ) , 6.49 ( bd s , 1h ) , 6.77 
 ( d , 4h ) , 7.157.44 ( m , 9h ) .
c nmr ( cdcl3 ) :  24.9 , 25.58 , 25.63 , 28.1 , 28.2 , 28.8 , 33.2 , 33.9 , 35.9 , 
 37.6 , 39.6 , 40.5 , 55.6 , 60.1 , 61.8 , 69.5 , 69.9 , 70.0 , 70.1 , 70.5 , 
 76.7 , 77.0 , 77.2 , 77.3 , 163.8 , 173.1 .
hrms calcd for c41h57n4o7s ( m + h ) 749.3942 ; 
 found , 749.3968 . a methanolic 
 solution ( 10 
 ml ) of 6
( 1.3 g , 1.8 mmol ) was detritylated within minutes 
 following the addition of 1 m hcl / meoh ( 20 ml ) .
the reaction was diluted 
 with water ( 100 ml ) , washed with dcm ( 3  100 ml ) , and the ph 
 adjusted to 4 with 4 m naoh .
dcm washes were repeated , the ph adjusted 
 to 12 , and the solvent evaporated to dryness . the product was extracted 
 from the residue triturating with dcm ( 5 ) , followed by filtration 
 over a glass frit .
the filtrate was concentrated to provide 7 as a pale yellow amorphous solid ( 480 mg , 60% yield ) .
h nmr ( cdcl3 ) :  1.421.46 ( m , 3h ) , 
 1.631.78 ( m , 13h ) , 2.172.20 ( t , j = 6 hz , 2h ) , 2.712.80 ( m , 4h ) , 2.882.93 ( dd , j = 6.7 , 2 hz , 1h ) , 3.143.15 ( m , 1h ) , 3.323.36 
 ( q , j = 5.3 hz , 3h ) , 3.533.65 ( m , 14h ) , 4.31 
 ( m , 1h ) , 4.48 ( m , 1h ) , 5.21 ( s , 1h ) , 6.09 ( s , 1h ) , 6.79 ( m , 1h ) . hrms 
 calcd for c20h39n4o5s 
 ( m + h ) 447.2636 ; found , 447.2651 .
a solution of 7 ( 482 mg , 1.1 mmol ) , edci ( 345 
 mg , 2.2 mmol ) , and hobt ( 300 mg , 2.2 
 mmol ) in dmf ( 5 ml ) under ar was added dropwise to 3 ( 1.7 
 g , 1.5 mmol ) in dmf ( 10 ml ) . after 16 h ,
the reaction was quenched 
 with a solution of brine and 0.1 m hcl ( 100 ml ) and extracted with 
 dcm ( 5  100 ml ) .
the organic layer was dried with na2so4 , filtered , and concentrated for column chromatography . 

( the column was packed with 1% hco2h / ea and then eluted 
 with ea to 10% meoh / dcm . ) depending on the purity of the product , 
 an optional second column chromatography was performed ( chcl3 to 5% meoh / chcl3 ) .
fractions of pure product were identified 
 by esi , pooled , and concentrated to a white foam ( 1 g , 55% yield ) 
 that was redissolved in dcm and precipitated by dripping the solution 
 into hexanes .
the solution was centrifuged , the solvent decanted , 
 and the residue lyophilized over 1 week into a white powder .
some 
 fmoc cleavage was detected in the powder and confirmed by esi / ms ( 1363.8 m / z = [ m - fmoc + 
 na ] 1363.6 ) .
hrms calcd for c91h103n8o14nas ( m + na ) 1563.7309 ; found , 
 1563.7302 ; confirmed the desired product .
fmoc - cleavage of the 
 protected form of 2 ( 200 mg , 128 mol ) was carried 
 out in 50% cyclohexylamine / dcm ( 15 ml ) .
after 15 min , the reaction 
 was diluted with dcm ( 20 ml ) and water ( 100 ml ) . after increasing 
 the ph of the aqueous layer to 12 with naoh ( 4 m ) ,
the aqueous layer was filtered through a 0.2 
 m syringe filter , concentrated under vacuum , and azeotropically 
 dried with toluene ( 3  50100 ml ) , to afford 2 as a pale yellow , amorphous solid that was difficult to completely 
 dry ( 264 mg , > 100% recovery ) .
h nmr ( d2o ) : 
  1.33 ( br , m , 6h ) , 1.451.56 ( m , 13h ) , 2.172.19 
 ( t , j = 4 hz , 2h ) , 2.372.54 ( m , 10h ) , 2.682.71 
 ( d , j = 12 hz , 1h ) , 2.892.93 ( dd , j = 4 , 8 hz , 1h ) , 3.033.07 ( t , j = 6 hz , 1h ) , 3.163.19 ( t , j = 6 hz , 4h ) , 
 3.203.26 ( m , 1h ) , 3.49 ( t , j = 6 hz , 4h ) , 
 3.583.61 ( m , 8h ) , 4.324.35 ( dd , j = 6 , 2 hz , 1h ) , 4.514.54 ( dd , j = 4 , 2 
 hz , 1h ) .
c nmr ( d2o ) :  22.4 , 22.6 , 
 25.2 , 25.4 , 25.7 , 25.8 , 27.8 , 29.1 , 29.2 , 33.0 , 33.6 , 36.1 , 36.1 , 
 37.2 , 42.0 , 43.5 , 45.68 , 52.9 , 57.67 , 59.2 , 59.5 , 65.8 , 65.9 , 66.8 , 
 66.9 , 67.01 , 67.02 , 162.7 , 165.9 , 173.8 , 173.9 .
hrms calcd for c31h62n8o6nas ( m + na ) 697.4405 ; found , 697.4429 . for 
 the preparation of 1113 , the arginine(s ) 
 and lysine residues were added via automated 
 spps from 10 ( 150 mg , 0.45 mmol / g , 70 
 mol - scale ) .
all residues were coupled ( 2  30 min ) with amino acid ( 0.2 
 m , 5 equiv ) , hbtu ( 0.2 m , 5 equiv ) , and dipea ( 0.4 
 m , 10 equiv ) in nmp .
the 
 remaining portions of the syntheses of 1113 and all of 9 were carried out manually as 
 follows .
alloc group cleavage was performed with me2nhbh3 ( 6 eq 24 mg , 0.4 mmol ) and ( ph3p)4pd(0 ) 
 ( 0.05 equiv , 4 mg , 3.4 mol , 15 min , 3 ) , where the former 
 was added first to a dmf suspension of resin with ar bubbling . following 
 washing as described below , fmoc - protected aminocaproic acid ( 3 equiv ) 
 was then preactivated with pybop ( 3 equiv ) and tea ( 6 equiv ) in dmf 
 ( 0.1 m ) for 1 h with ar bubbling .
the fmoc group was removed by treating 
 the preswollen resin with 20% piperidine / dmf ( 5 min , 3 ) .
unreacted amine was acetylated 
 by treatment with 50% ac2o / dcm 3 times for 3 , 3 , and 7 
 min , with dcm washing in between each treatment . after each coupling
, 
 the resin was washed sequentially with dmf , dcm , meoh , dry dcm , and 
 dry dmf ( 2 ) . the lysine residue in 9
was coupled 
 as described above for aminocaproic acid prior to the removal of the 
 alloc group .
the amino group was quantified indirectly by the released 
 fulvene chromophore ( 300 7.8  10 mcm ) .
peptide 
 cleavage / deprotection was performed with a cleavage cocktail ( 88% 
 tfa , 2% tips , 5% h2o , and 5% phenol ) .
hplc purification 
 was performed on a c18 semipreparatory column ( waters 300  7.8 
 mm i.d . ) using h2o ( solvent a ) and acetonitrile ( solvent 
 b ) with 0.1% tfa in an elution gradient optimized for each probe at 
 3 ml / min .
probe - containing fractions were lyophilized , redissolved 
 in water and titrated to ph 9 with 4 m naoh , and analyzed by ms in 
 the positive mode . the following gradients ( time , % b )
esi - ms [ m + h ] : calcd , 501.2 ; obsd , 501.2 .
11 : 0 , 0 ; 5 , 0 ; 
 10 , 20 ; 20 , 20 .
esi - ms [ m + h ] : calcd , 
 770.1 ; obsd , 770.5 .
esi - ms [ m + h ] : calcd , 926.3 ; obsd , 926.6 .
esi - ms [ m + h ] : calcd , 1082.5 ; obsd , 1082.8 .
manual spps from 10 ( 0.3 g , 135 mol ) 
 was identical to that described 
 above .
detritylation of the cysteine thiol was carried out with 2% 
 tfa / dcm ( 12 ml , 12 min ) and repeated until 
 the cleavage solution ran clear ( 10 ) .
the cysteine thiol 
 was converted into the disulfide by treating with cystamine2 
 hcl ( 5 equiv , 0.51.0 h , 3 ) and dipea ( 15 equiv ) in dmso 
 ( 5 ml ) .
the resin was washed with dmf , meoh , dcm , and dmf 
 following each cycle .
the penultimate compound 
 was cleaved , purified , and characterized as described above . instead 
 of evaporating the cleavage cocktail ,
the precipitate was lyophilized , redissolved 
 in water , and filtered prior to hplc purification .
the following gradient 
 ( time , % b ) was used at 4 ml / min : 0 , 0 ; 5 , 0 ; 90 , 16 . ret .
maldi - tof - ms [ m + h ] : calcd , 1414.8 ; obsd , 1416.1 .
a solution ( 5 l ) 
 of dna analyte ( 1 fmol ) , bpuc19 dna reference ( 0.1 fmol ) , probe ( 0.2 
 mm ) , ctdna ( 4 mg / l ) , and trishcl ( 20 mm , ph 9 ) was added with 
 k3fe(cn)6 ( 5 l , 2 mm ) and allowed to 
 stand for 10 min .
the reaction was quenched with carrier dna and detergent 
 ( 20 l of ctdna ( 50 mg / l ) , dtt ( 10 mm ) , and 0.05% tween-20 ) .
g / l in 0.05% tween-20 ) and pelleted on a magnetic 
 pcr plate .
the beads were washed with wash buffer ( 100 l , 8 
 with 10 mm dtt , 10 mm guanidinehcl , and 0.05% tween-20 ) .
pei - beads 
 were resuspended in fresh cleavage / elution buffer ( 50 l : 0.1 
 m dtt , 0.1 m trishcl , at ph 10 , and 2 m nacl ) , incubated for 5 min , 
 and pelleted magnetically for an additional 5 min .
magnetic streptavaidin beads 
 ( dynabead myone t1 ) were washed with 0.05% tween-20 ( 3 ) . a suspension 
 of the beads ( 25 l , 1
g / l ) was then incubated with pei - bead 
 eluent ( 25 l ) . after 30 min , the beads were magnetically pelleted 
 and washed sequentially with 1 , 0.5 , and 0.25 ttbs 
 ( 100 l  3 at each concentration ; 1 ttbs , 0.05% 
 tween 20 , 40 mm trishcl at ph 9 , and 1 m nacl ) .
beads were washed 
 a final time with and resuspended in 50 l of 0.05% tween-20 .
each qpcr solution 
 ( 50 l ) contained 10% sample ( 5 l ) , 0.2 mm dntp , primers 
 ( 0.2 m each , table 1 ) , taqman 
 probes ( 0.1 m each ) , neb onetaq hotstart buffer ( 1 ) , 
 and onetaq hotstart dna polymerase ( 2.5  10
each qpcr experiment was prepared with a single calibration series 
 of analyte and reference dnas ( 0 , 2.58.5 logcn ) , ideally from 
 the same working solution as the experimental samples .
the 96-well 
 plate ( biorad mlp 9601 ) was sealed with optical film ( abi ) .
qpcr was 
 performed on the icycler iq ( filter set 4 : fam-490 and texas red-575 ) 
 using the following program : ( 1 ) 95 c for 15 s , ( 2 ) 66 c 
 for 1 min with optical measurement , ( 3 ) repeated 12 50 , 
 and ( 4 ) held at 25 c .
the following taqman probes were used : 
 5-d(fam - ttg atg ctg tcc ccg gac ga - bhq1 ) for p53 and 5-d(cal - fluor- 
 ctg aga gtg cac cat atg cgg tgt g - bhq2 ) for the puc19 internal standard .
the threshold position was manually set 
 at the same position in the exponential phases of the two sets of 
 amplification curves ( one set for each fluorophore ) .
cq ( cycle at 
 which fluorescence from amplification exceeds the background fluorescence ) 
 data were recorded and processed using microsoft excel .
calibration 
 curves ( cq vs log cn ; cn = copy number ) were generated by linear regression 
 for both reference and analyte dnas .
the amount ( log cn ) of dna was 
 calculated by interpolation from the calibration curves . the tagging 
 yield ( % yraw )
was calculated by1 the normalized tagging yield ( % ynorm ) was obtained by2where 
 % yrawmax was the % yraw with 100% p53og . 

the amount of 8-oxodguo detected 
 was calculated by3or by4 dna solutions ( 10 l , 500 pm p53-g , and 
 50 pm bpuc19 ) in tes 
 buffer ( 20 mm tris hcl , ph 7.5 , 2 mm edta , 0.2 m nacl ) in a 96-well 
 titer plate were treated for 50 min at 37 c with an equal volume 
 of oxidizing agents ( fecl2 , h2o2 , 
 and ascorbic acid ) the oxidizing agents ranged in concentration from 
 50 m fecl2 , 400 m h2o2 , and 4 mm ascorbic acid to 1.5 m fecl2 , 12.5 m 
 h2o2 , and 0.125 mm ascorbic acid and were changed 
 in 2-fold increments .
two control reactions were carried out , one 
 with p53-og and one with p53-g as analytes .
the control reactions 
 contained h2o ( 10 l ) in place of the oxidizing reagents . 

the reactions were quenched with quencher q ( 10 l of 50 mg / l 
 calf thymus dna and 30 mm l - methionine ) and purified by silica 
 spin column chromatography ( qiagen qiaquik pcr purification kit ) , 
 resulting in a final solution ( 50 l ) of dna buffered in 10 
 mm tris hcl at ph 7.5 .
8-oxodguo was quantified from an aliquot of 
 the dna sample ( 4 l ) mixed with 14 ( 1 l , 
 0.5 mm ) , following the general procedure for tagging and qpcr described 
 above .
we previously 
 developed a method for quantifying 8-oxodguo in dna 
 that took advantage of its lower oxidation potential than native nucleotides 
 and many dna lesions , in combination with nucleophilic trapping of 
 a reactive oxidization product of the lesion .
multiple aspects of the previously reported method for detecting 
 8-oxodguo were modified in the current work .
the horseradish peroxidase 
 amplification was replaced with quantitative pcr , procedures for removing 
 excess probe were examined , and new probes were synthesized .
the overall 
 procedure ( scheme 2 ) involved tagging , removal 
 of the excess probe , binding tagged dna to streptavidin - coated magnetic 
 beads , removal of untagged dna ( nonbiotinylated dna ) , and finally 
 pcr amplification of the bead bound dna .
the ideal probe will selectively 
 form a covalent bond to 8-oxodguo in a large excess of dg and be readily 
 removed from dna when it is noncovalently bound .
we postulated that 
 incomplete removal of noncovalently bound 1 contributed 
 to the background signal and prevented us from reaching a lower limit 
 of detection .
consequently , we sought to design probes that would 
 provide good yields of tagged 8-oxodguo but be readily removed .
the triethylene glycol group was 
 expected to increase the probe s water solubility , facilitating 
 its removal from dna .
in addition , the longer linker separating the 
 dna tagging and reporting domains could enhance probe function .
probe 2 was synthesized by coupling the biotinylated triethylene 
 glycol ( 7 , scheme 3 ) with the 
 fmoc - protected spermine derivative ( 3 ) .
the carboxylated 
 spermine derivative ( 3 ) was prepared via a recently improved 
 procedure , whereas 7 was obtained from the commercially 
 available diamine .
diamine 4 was dimethoxytritylated 
 prior to biotinylation to facilitate purification and handling of 
 the advanced intermediates due to the polar nature of biotin and the 
 lack of a convenient way of visualizing the compounds upon thin layer 
 chromatography analysis .
the fmoc - protected probe was purified by 
 column chromatography and deprotected with cyclohexylamine .
probe 2 was purified from this reaction by extracting it into water 
 and washing away less polar entities via extractions .
the functionality 
 of 2 was generally established by analyzing its tagging 
 of an independently synthesized oligonucleotide containing 8-oxodguo 
 ( 5-p-8 ) . the general tagging and 
 washing procedure employed
however , instead 
 of using qpcr , tagging was quantified using liquid scintillation counting 
 by measuring the amount of p in the washing solutions 
 and on the bead . at ph 8.0 , 2 ( 0.1 mm ) yielded > 60% 
 tagged 
 8-oxodguo ( data not shown ) . while an amine 
 is necessary to trap the oxidized 8-oxodguo , we 
 considered the possibility that the polycationic probes ( e.g. , 1 and 2 ) bind too avidly to dna , resulting in 
 higher backgrounds . consequently , we tested the biotinylated intermediate 
 ( 7 , scheme 3 ) and synthesized 9 , which has the same net + 1 charge as 7 .
probe 9 was synthesized by solid - phase peptide synthesis starting 
 from wang resin that was preloaded with -fmoc--alloc 
 lysine ( 10 ) .
the orthogonally protected resin enabled 
 us to couple the very polar ( and poorly soluble ) biotin component 
 last , just prior to peptide cleavage from the solid support . while 
 the synthesis of 9 only required coupling boc - lysine 
 prior to cleavage of the alloc group ( pd(0 ) ) and reaction with biotin , 
 this strategy proved useful for synthesizing several other probes 
 ( see below ) .
although noncovalently bound 7 and 9 may be easier to remove from the dna , neither one efficiently 
 tagged dna containing 8-oxodguo . using 9 as 
 a prototype , we synthesized a series of 
 related probes ( 1113 ) containing 
 between 1 and 3 arginine residues , followed by a lysine at the amino 
 terminus .
in addition , following pd(0 ) cleavage of the alloc group , the liberated 
 -amino group was coupled to aminocaproic acid prior to conjugating 
 biotin .
this assembly method increased the distance between the tagging 
 and capture ( biotin ) domains of the probes .
peptide 13 , containing 3 arginine residues , possessed the same overall positive 
 charge as 1 .
the tagging abilities of the arginine probes 
 ( 1113 ) were crudely evaluated with 
 an independently synthesized oligonucleotide containing 5-p-8 and liquid scintillation counting , as described 
 above for 2 .
although the tagging efficiency of 11 was 3-fold greater at the higher concentration used , a much 
 smaller difference was observed with the more highly positively charged 
 probes ( 12 and 13 ) .
the concentration of 
 k3fe(cn)6 ( 1 and 10 mm ) also had little effect 
 on tagging yield .
overall , the probe containing the same overall charge 
 as 1 ( 13 ) provided the highest tagging efficiency . tagging efficiency of 1113 for p-8 as a function of probe and oxidant concentrations . 

yields were determined using streptavidin - coated magnetic beads via 
 liquid scintillation counting . finally , to minimize the effect of probe bound noncovalently 
 to 
 target dna , we synthesized a peptide ( 14 ) in which the 
 highly positively charged dna binding domain was separated from biotin 
 ( used to capture tagged dna ) and the tagging domain by a cleavable 
 disulfide linkage .
we rationalized that while a positively charged 
 dna binding domain might assist delivering the tagging agent , it would 
 also hinder removing it after reaction .
the cleavable disulfide facilitates 
 removing biotin that is not covalently bound to the dna and reduces 
 the background .
synthesis was carried out on the wang resin containing -fmoc--alloc 
 lysine ( 10 ) .
a s - monomethoxytrityl protected 
 cysteine , which served as the disulfide precursor was incorporated , 
 followed by the aminocaproic acid spacer and the lysine employed as 
 the tagging component .
the biotin group was introduced as described 
 above prior to revealing the cysteine thiol that was elaborated further 
 to the disulfide .
the thiol was condensed with cystamine by disulfide 
 exchange , and the resulting primary amine was used to introduce the 
 triarginine dna binding domain .
all of the peptide probes were purified 
 by reverse - phase hplc following cleavage from the solid phase support 
 and characterized by mass spectrometry ( see supporting 
 information ) .
large excesses of probes 
 relative to dna are used in the reactions to maximize the tagging , 
 which involve trapping a reactive species . excess probe
considering that the probes are 
 typically employed at 100 m , a large majority must be removed . 

several techniques to minimize the excess probe after tagging were 
 investigated , including size - exclusion spin column chromatography , 
 silica - spin column chromatography , nacl / etoh precipitation and washing , 
 and polyethylene imine - coated magnetic bead ( pei - beads ) binding and 
 washing .
the former two were extremely limited due to cost , low - throughput , 
 and labor intensiveness .
precipitation was time - consuming and was 
 inefficient at separating the probe(s ) from dna .
in contrast , purification 
 by pei - beads was fast , facile , and , barring high - ionic strength media , 
 flexible with conditions for probe removal .
a variety of washing solutions 
 ( 10 mm ) containing dtt or positively 
 charged small molecules ( guanidine , lysine , arginine , and spermidine ) 
 were assayed for removing excess probe .
their effectiveness was screened 
 using p-15 ( 10 pmol ) and probe 14 ( 1 nmol ) .
pei - beads were added and washed with the additives . when 
 excess probe was present , p-15 could not 
 bind to the streptavidin - coated beads . in contrast , when 14 was removed using any of the wash solutions , biotinylated dna ( p-15 ) bound the beads as effectively as that 
 when 14 had not been added .
given the ease of preparation , 
 both guanidine and dtt were maintained in subsequent experiments . 

we recognized that these experiments do not guarantee that the probe 
 is completely removed and that any excess probe that is even noncovalently 
 bound to dna would contribute to the background signal .
however , we 
 were unable to directly measure low levels of probe remaining bound 
 to the bead .
it was conceivable that one could measure the bead - bound 
 probe by radiolabeling the latter , but this was deemed impractical . 

after eight rounds of washes , dna was eluted using high ionic strength 
 buffer containing dtt to reduce the disulfide ( 0.1 m trishcl , 
 ph 10 , 2 m nacl , 0.1% tween-20 , and 0.1 m dtt ) . after 5 min ,
the suspension 
 was clarified by magnetic pelletization for 5 min . the eluted dna 

was then bound to streptavidin beads ( 15 pmol binding capacity ) and 
 quantified by liquid scintillation counting .
all probes contained biotin as 
 a means for capturing tagged dna and separating tagged and untagged 
 material from one another .
these beads contain a hydrophobic surface , which was 
 desirable , as it should bind more weakly to the charged molecules 
 ( dna and probes ) employed in these studies .
a 916 bp pcr fragment 
 obtained from a plasmid containing the p53 gene was used as analyte 
 dna . the pcr product containing 8-oxodguo ( p53-og )
an otherwise identical 
 pcr product was prepared without 8-oxodguo ( p53-g ) using a forward 
 primer that did not contain the lesion .
( the pcr fragment without 
 8-oxodguo was used for optimizing conditions for removing untagged 
 dna . ) a 1.2 kb biotinylated pcr fragment of puc19 ( bpuc19 ) was used 
 as an internal standard .
wash buffers varying in ph and ionic strength were screened , 
 and tbs buffer ( 10 mm tris , ph 9 , and 1 m nacl ) was found to be the 
 best .
qpcr was chosen because of its large dynamic range , as well as its 
 compatibility with sample preparation methods that shorten the time 
 required to carry out the procedure . the previous procedure required 
 adsorbing the ( tagged and untagged ) dna to the surface of the microtiter 
 plate well , followed by binding the horseradish peroxidase 
employing magnetic 
 beads facilitates the removal of untagged ( nonbiotinylated ) dna by 
 washing .
removal of untagged dna is one crucial parameter for minimizing 
 the limit of detection because untagged dna will be amplified equally 
 as untagged during pcr .
however , greater reproducibility was achieved by adding an undamaged 
 dna ( a 1.2 kb pcr fragment from puc19 ) to the mixture prior to tagging 
 as internal standard .
the internal standard and target dnas were quantified 
 by multiplex qpcr using taqman probes containing different fluorophores 
 for each nucleic acid substrate .
having optimized the qpcr process 
 and individual assay steps ( scheme 2 ) , the 
 performance of probes 2 , 9 , and 1114 were compared ( table 1 ) .
the background with 
 no oxidant ( k3fe(cn)6 ) was also measured using 
 p53-g .
in general , the data were consistent with the more crude measurements 
 described above for 1113 .
lower 
 charged probes ( e.g. , 9 and 11 ) captured 
 the 8-oxodguo containing dna less efficiently .
however , the more highly 
 charged probes yielded a higher background signal , as evidenced by 
 the tagging of p53-g .
cleavable probe 14 provided the 
 highest overall tagging yield and greatest selectivity ( 2,000-fold ) . 

although the tagging yield for 14 was only 40% , 
 this is less significant than the selectivity .
increasing the tagging 
 yield to 100% would only improve the sensitivity 2.5-fold , 
 assuming that the background reaction remained the same . finally , 

the limit of detection using 14 was established using 
 a mixture of p53-g and p53-og totaling 0.9 fmol ( 1 ng ) .
the 
 signal ( % yraw ) was distinguishable from 
 that for the background ( p53-g only , 2.9  0.7 ) when the sample 
 contained as little as 14 amol of p53-og ( 6.5  1.0 ) in a sample 
 containing 0.9 fmol dna strands .
this translates to 14 amol 8-oxodguo 
 per 1.65 pmol nt based upon 916 bp per p53-og molecule or < 12 8-oxodguo/10 nt .
the amount of 8-oxodguo formed 
 in the p53-pcr fragment upon treatment 
 with fecl2 , h2o2 , and ascorbate was 
 measured using 14 and the above - described process .
the 
 8-oxodguo dependence on the level of oxidative stress was determined 
 over a range of ferrous ion concentrations and compared to the amount 
 determined by cadet using lc / ms to analyze dna exposed to the same 
 conditions ( figure 2 ) .
the 
 agreement in the amount of 8-oxodguo formed as measured by the two 
 methods was excellent over the range of fecl2 between 0.8 
 and 12.5 m ( figure 2 ) .
however , the 
 methods yielded very different results at 25 m fecl2 , where we measured 477  113 8-oxodguo nt/10 nt , 
 but one would expect a value of 250 using cadet s lc / ms 
 method . nonetheless , the two methods agree well with one another over 
 a 15-fold range .
quantity of 8-oxodguo formed from fe oxidation 
 of 
 p53-g as a function of oxidant level .
the data are compared to the 
 levels previously established under the same oxidation conditions 
 using lc / ms analysis of enzyme digested dna .
data plotted are the 
 average of 3 independent measurements , and error bars represent the 
 sd of these measurements .
probe 14 enables us to detect 
 as little as 14 amol 
 of 8-oxodguo in 34 h. typical experiments used 1 ng of dna 
 substrate , and the quantitative analysis agrees well with data obtained 
 using enzyme digestion and lc / ms analysis .
the amount of dna substrate used in the assay is considerably less 
 than that in a typical experiment using lc / ms .
furthermore , the small quantity of substrate needed will 
 be useful for analyzing valuable nucleic acids that are in short supply , 
 such as telomeric dna .
the sensitivity of the experiment is enhanced 
 by the use of qpcr , which can in principle be used to detect very 
 small numbers of molecules , provided the background tagging reaction 
 can be reduced further .
furthermore , the qpcr method only requires 
 that a portion of the target dna sequence be known .
because dna is not digested during 
 the analysis , the method is potentially useful for analyzing for 8-oxodguo 
 at specific sites by taking advantage of known methods for recognizing 
 specific nucleic acid sequences .
the advent of a pei - bead washing 
 procedure and a probe ( 14 ) that allows us to reductively 
 cleave the highly positively charged dna binding domain from the reporter 
 group reduces the likelihood that the background signal is due to 
 the noncovalently bound probe .
nonspecific tagging of dg by the probe(s ) 
 under the oxidative conditions of the reaction is a possible source 
 of background signal .
future improvements 
 will address this potential problem by examining the oxidation conditions 
 in the tagging reaction .
given the large dynamic range of qpcr and 
 its sensitivity , reducing the background signal will decrease the 
 method s limit of detection and increase its value to the scientific 
 community .
 8-oxo-7,8-dihydro-2-deoxyguanosine 
 ( 8-oxodguo ) is a commonly 
 formed dna lesion that is useful as a biomarker for oxidative stress . 
 
 although methods for selective quantification of 8-oxodguo exist , 
 there is room for additional methods that are sensitive and utilize 
 instrumentation that is widely available . 
 we previously took advantage 
 of the reported reactivity of 8-oxodguo to develop a method for detecting 
 the lesion by selectively covalently tagging it with a molecule equipped 
 with a biotin label that can be used subsequently with a reporting 
 method ( xuel . , and greenbergm . m. ( 2007 ) 
 j. am . 
 chem . 
 soc.129 , 701017497789 ) . 
 we now report a method that can 
 detect as little as 14 amol of 8-oxodguo by tagging dna with a reagent 
 containing a disulfide that reduces background due to nonspecific 
 binding . 
 the reagent also contains biotin that enables capturing target 
 dna on streptavidin - coated magnetic beads . 
 the captured dna is quantified 
 using quantitative pcr . 
 the method is validated by comparing the amount 
 of 8-oxodguo detected as a function of fe2+/h2o2/ascorbate - dose to that reported previously using mass 
 spectrometry . 