mapping nucleobases 
 in dna or rna modified by alkylation or oxidation 
 often relies on the ability to specifically cleave the oligonucleotide 
 at the site of modification , followed by gel electrophoresis to develop 
 the map . a method established by maxam and gilbert
utilizes base - specific 
 alkylation followed by hot piperidine cleavage as the key steps in 
 sequencing the dna bases .
the success 
 of this method lies in the ability to specifically alkylate the dna 
 bases , as well as having high yields in the hot piperidine cleavage 
 step .
a similar base - specific labeling protocol followed by chemical 
 cleavage has been advanced for sequencing rna ; however , 
 the inherent alkaline instability of rna requires substitution of 
 piperidine at ph 11 with aniline at ph 4.5 .
these methodologies developed 
 a framework for identifying other labile sites of modification in 
 dna and rna , for example , those resulting from base oxidation .
site - specific oxidation of nucleobases , if left unrepaired , can 
 lead to mutations that are proposed to cause a wide range of diseases 
 such as alzheimer s disease , melanoma , prostate cancer , and 
 diabetes .
many research efforts have focused 
 on understanding the mechanisms and products that result from oxidation 
 of the nucleobases in dna oligomers to develop hypotheses that explain 
 cellular mutagenesis .
the nucleobase guanine stands out as a prominent 
 site of oxidation 
 because it has the lowest standard reduction potential . in dna ,
oxidation of 2-deoxyguanosine 
 ( dg ) is modulated by the context of the reaction ( i.e. , single - stranded , 
 duplex , or g - quadruplex contexts ) . moreover , in duplex 
 dna , the reactivity of dg toward oxidation shows a subtle sequence 
 context effect . locating site - specific 
 oxidation at dg nucleotides in dna oligomers is determined by hot 
 piperidine work up followed by gel electrophoresis to read the damaged 
 sites in comparison to a control ladder . another approach to identifying 
 damage sites
utilizes the base excision repair enzyme fpg for cleavage 
 at the lesion . in rna ,
oxidation of guanosine 
 ( rg ) has been studied , but to a lesser degree than dna oxidation . for the chemical cleavage methods to be successful at mapping oxidation 
 sites , it is critical to know the rates and extent to which scission 
 of the strand occur for different oxidation products of dg and rg .
oxidation of the guanine heterocycle yields classes of products 
 based on the extent of oxidation .
two - electron oxidation of dg yields 
 8-oxo-7,8-dihydro-2-deoxyguanosine ( dog ) and 5-carboxamido-5-formamido-2-iminohydantoin-2-deoxyribonucleoside 
 ( d2ih ) , while four - electron oxidation of dg yields two diastereomers of 
 spiroiminodihydantoin-2-deoxyribonucleoside ( dsp ) , two 
 diastereomers of 5-guanidinohydantoin-2-deoxyribonucleoside 
 ( dgh ) , and 2,5-diaminoimidazolone-2-deoxyribonucleoside ( diz ) 
 that readily hydrolyzes to 2,2,4-triamino-2h - oxazol-5-one-2-deoxyribonucleoside 
 ( dz ) .
the six - electron oxidation of dg yields dehydroguanidinohydantoin-2-deoxynucleoside 
 ( dgh ) , a compound of limited stability that ultimately 
 decomposes to a ribosyl - urea lesion .
another product that is formed during certain oxidations , but is 
 not formally oxidized , is 2,6-diamino-4-hydroxy-5-formamidopyrimidine-2-deoxyribonucleoside 
 ( fapydg ) , a ring - opened hydrolysis product of dg ( scheme 1 ) .
many of these lesions resulting 
 from dg oxidation have been observed in genomic samples of biological 
 origin , except d2ih and dgh , whose cellular existence is awaiting 
 confirmation . in cellular rna samples , rog is the only lesion that 
 has been characterized thus far .
detection 
 of these lesions in cellular samples typically occurs by nuclease 
 digestion that leads to complete loss of sequence information , followed 
 by mass spectrometry .
therefore , in vitro model studies with dna or rna 
 oligomers allow understanding the sequence context effects on lesion 
 formation .
the standard approach for identification of lesion sites 
 requires strand cleavage by piperidine ( dna ) or aniline ( rna ) followed 
 by gel electrophoresis ; another method utilizes esi - ms , but 
 this approach is limited to short oligomers with only one possible 
 site of modification .
oxidations conducted on dna 
 oligomers of known sequences have yielded 
 a wealth of information concerning the sequence context - dependent 
 reactivity of dg toward oxidants and the products . for interpretation of these data from the point of view of biology , 

the oxidation reactions are conducted under low product conversion 
 ( i.e. , single - hit chemistry ) .
typically , 
 the reaction yield is determined by hot piperidine cleavage of the 
 oligomers using a standard protocol ( 0.21 m piperidine , 30 
 min , 90 c ) ; however , the yields 
 and rates for cleavage of the consistently observed dg oxidation products 
 have not been established under identical reaction conditions .
ligation - mediated pcr is an approach for determining lesion sites 
 in genomic samples that relies on the ability to site - specifically 
 cleave the lesion for ligation on a primer sequence used to identify 
 the original lesion location . in this 

method , chemical cleavage is one approach for effecting strand scission 
 at the lesion site . in rna , cleavage at sites of base modification 
 sites employs aniline following a standard procedure ( 0.21 
 m , ph 4.5 , 60 c , 20 min ) . as for 
 dna ,
strand cleavage efficiencies are not known for rg oxidation products 
 in rna oligomers . because rates of chemical strand scission for dg 
 and rg lesions have not been established , the extent of an oxidation 
 reaction on dna or rna polymers is inaccurately determined . in the 
 present study ,
the rates of cleavage for og , sp , gh , 2ih , and z were 
 determined in dna oligomers treated with piperidine , and in rna oligomers 
 treated with aniline under standard reaction conditions .
these data 
 determined that lesion cleavage rates using typical reaction conditions 
 are quite variable , and some are not significantly cleaved , even at 
 long incubation times .
these data will be paramount for researchers 
 that oxidize oligomers of dna or rna with known sequences and then 
 interpret the reaction yields from a biological prospective , for which 
 oxidation events on these polymers are a rare occurrence .
the dg oxidation 
 products dog , ( s)-dsp , ( r)-dsp , and a mixture of the dgh diastereomers were synthesized in the 18-mer 
 dna oligomer 5-tca tgg gtc xtc ggt ata-3 
 ( where x = lesion site ) .
the dna oligomer was synthesized 
 via solid - phase synthesis by the dna / peptide core facility at the 
 university of utah with a dog phosphoramidite ( glen research , sterling , 
 va ) at the position of x following standard methods . 

analytical ion - exchange hplc - purified samples provided the dog - containing 
 dna oligomer for the piperidine studies .
syntheses 
 of the dsp diastereomers and dgh diastereomers were conducted following 
 literature protocols .
the dsp diastereomers 
 were individually purified while the diastereomers of dgh were studied 
 as a mixture because they readily interconvert .
syntheses of dz and the ( r)- and ( s)-d2ih diastereomers were 
 achieved in a 15-mer dna oligomer containing a single dg site ( 5-aat 
 cca cga cac ctc-3 ) following literature methods . all product oligomers were purified using an analytical ion - exchange 
 hplc column that resolves diastereomeric products ( figure s1 ) .
the dna oligomer products were characterized by 
 esi - ms ( dog : calcd 5560.6 , found 5560.8 ; mixture of the dsp diastereomers : 
 calcd 5576.6 , found 5576.4 ; mixture of the dgh diastereomers : calcd 
 5550.6 , found 5550.2 ) , or maldi - ms was used to characterize a mixture 
 of the d2ih diastereomers as well as for dz ( d2ih : calcd 4498.8 , found 
 4498.2 ; dz : calcd 4445.8 , found 4445.3 ) .
the dz - containing oligomer 
 was found to have limited stability due to further decomposition of 
 dz to guanidinoformimine-2-deoxyribonucleoside ( dgf ) .
the dgf - containing strand was characterized 
 by maldi - ms ( dgf : calcd 4398.8 ; found 4398.2 ) .
because of the limited 
 stability of dz in the dna oligomer leading to dgf , the dz sample 
 was studied immediately after purification and a 2 h dialysis to remove 
 the purification salts .
the rg - oxidation products rog , ( r)-rsp , ( s)-rsp , rz , ( r)-r2ih , ( s)-r2ih , and rgh diastereomers were synthesized 
 in the 7-mer rna oligomer 5uuuguuu-3. 
 selective conversion of the single rg to the rsp diastereomers , rgh 
 diastereomers , r2ih diastereomers , and dz was achieved via literature 
 protocols .
these strands were then purified 
 by the same method as stated for the lesions in the dna oligomers 
 ( figure s2 ) .
the 7-mer containing rog was 
 synthesized using the phosphoramidite of rog ( chem genes , wilmington , 
 ma ) following the manufacturer s protocol .
the rna oligomer 
 products were characterized by maldi - ms ( rog : calcd 2136.3 , found 
 2136.7 ; ( s)-rsp and ( r)-rsp diastereomer 
 mixture : calcd 2152.3 , found 2153.0 ; rgh diastereomer mixture : calcd 
 2126.3 , found 2126.1 ; rz : calcd 2099.3 , found 2099.4 ; ( r)-r2ih and ( s)-r2ih diastereomer mixture : calcd 
 2154.4 , found 2154.3 ) . to monitor the cleavage reactions , 

the lesion - containing dna oligomers 
 were 5-labeled with p for visualization by gel 
 electrophoresis followed by phosphorimager autoradiography .
piperidine cleavage reactions for lesion - containing 
 dna oligomers were conducted by mixing 100 pmol of unlabeled strand 
 with 20  000 cpm of p - labeled strand in 50 l 
 of ddh2o . for each lesion ,
nine tubes were prepared for 
 taking time points at 0 , 5 , 10 , 20 , 30 , 45 , 60 , 90 , and 120 min . to 
 these samples
were added 50 l of freshly prepared aqueous piperidine 
 ( 2 m ) and -mercaptoethanol ( bme , 0.5 m ) to give a final concentration 
 of 1 m dna with 1 m piperidine and 0.25 m bme .
next , the samples 
 were incubated at 90 c , and at each time point , a sample was 
 removed from the heat source .
the samples were lyophilized to dryness 
 and resuspended in 10 l of gel loading dye ( 0.25% xylene cyanol , 
 0.25% bromophenol blue in an aqueous 30% v / v glycerol solution ) . to 
 a 20% denaturing polyacrylamide gel electrophoresis ( page ) , 5 l 
 of sample in loading dye was added .
the samples were electrophoresed 
 at 45 w for 2 h , after which the gel was loaded into a phosphor screen 
 and visualized by phosphorimager autoradiography after an 18 h exposure . 

one reaction was also monitored by ion - exchange hplc on 
 a sample that was not p labeled that allowed observation 
 of all species in solution during the reaction .
the method utilized 
 was the same as the one used for product purification . to monitor 
 the cleavage reactions ,
the lesion - containing rna oligomers 
 were 5-labeled with p for visualization by gel 
 electrophoresis followed by phosphorimager autoradiography .
aniline cleavage reactions for lesion - containing 
 rna oligomers were conducted by mixing 100 pmol of unlabeled strand 
 with 20  000 cpm of p - labeled strand in 50 l 
 of ddh2o . for each lesion ,
seven tubes were prepared for 
 taking time points at 0 , 10 , 20 , 30 , 40 , 60 , and 90 min . to these 
 samples
was added 50 l of freshly prepared aniline ( ph 4.5 , 
 2 m ) to furnish a final concentration of 1 m rna and 1 m aniline . 

the aniline at ph 4.5 was prepared by taking a solution of reagent 
 grade aniline and adjusting the ph to 4.5 with glacial acetic acid 
 to obtain a 2 m stock solution .
next , the samples were incubated at 
 60 c in the dark , and at each time point , a sample was removed 
 from the heat source .
after completion of the reactions , the samples 
 were processed in an identical fashion as previously described for 
 the lesion - containing dna oligomers .
scheme 2a shows the proposed mechanism for 
 piperidine - induced strand scission at the site of a dna lesion . in the first step ,
this 
 intermediate increases the acidity of h2 that accelerates 
 -elimination of the 3-phosphate terminus in the second 
 step . in the third step , -elimination of
the 5-phosphate 
 terminus occurs to yield complete cleavage at the site of the lesion . 
 the correct kinetic rate equation for this complex reaction should 
 take into account all intermediate reactions ; however , in practice , 
 piperidine cleavage reactions are followed by page , in which only 
 the intact strand and the cleaved strand are observed . by monitoring 
 loss of the starting material and the appearance of the product ,
the 
 rate equation can be simplified to a pseudo - first - order rate equation . 

the assumptions in this simplified approach are that the piperidine 
 concentration does not change , as is validated by the large excess 
 of piperidine used to effect strand scission ( 10-fold 
 excess ) .
the second simplifying assumption was the use of the steady - state 
 approximation , in which the concentrations of the reaction intermediates 
 were assumed to reach a steady state , and their concentrations were 
 limited by the rate of the first reaction step .
verification of this 
 assumption was applied to a test reaction that monitored the piperidine 
 cleavage of a ( r)-dsp - containing dna oligomer by 
 hplc .
this approach allowed inspection of all species in solution , 
 which is not possible by page . in this analysis , the intact strand , 
 cleavage products ( 5 and 3 fragments ) , and intermediates 

the intermediates were not characterized due to their 
 instability . in support of the steady - state approximation ,
the intermediate 
 peak ratios did not significantly change during the 90 min reaction 
 profile , while the starting material decreased and the product strands 
 increased ( figure s3 ) . to monitor cleavage 
 reactions for all lesions , the traditional approach of monitoring 
 these reactions by page
was selected , and a pseudo - first - order reaction 
 rate equation was applied for mathematically modeling .
the rate - limiting 
 step was assumed to be formation of the schiff - base intermediate . 

thus , plots of the ln [ intact strand ] vs reaction time provided linear 
 data points that were described by eq 1 , where 
 the slope of the line provides the reaction rate constant.1 monitoring the time - course cleavage for the guanine oxidation 
 products 
 studied in a dna oligomer by page gave a wide distribution of reaction 
 course profiles ( figure 1a and figure s4 ) . for example , the dog lesion was not 
 significantly cleaved ( < 1% ) by hot piperidine during the 2 h reaction 
 ( figure 1a ) .
next , the dsp diastereomers were 
 found to have undergone slow hot piperidine strand scission ( figure 1a ) , with rate constants for the isomers to be nearly 
 identical values of ( 9.5  1.5 )  10 and ( 9.5  1.2 )  10 min for the r and s isomers , respectively 
 ( table 1 ) .
for dgh , strand scission by hot 
 piperidine was faster , with a cleavage rate constant of ( 22  
 3 )  10 min ( table 1 ) , while the dz lesions were cleaved with a rate 
 constant of ( 51  5 )  10 min ( table 1 ) .
finally , the diastereomers of 
 d2ih cleaved the quickest with rate constants of ( 100  10 )  
 10 and ( 110  11 )  10 min for the r and s isomers , respectively ( table 1 ) .
these studies 
 demonstrate that the dg oxidation products , other than dog , cleave 
 with hot piperidine at variable rates , and near - quantitative reaction 
 yields can be obtained if the reactions are allowed to progress long 
 enough .
the pseudo - first - order kinetic rate constants allow 
 determination 
 of the half - life ( t1/2 ) of each lesion 
 under the reaction conditions . these values will allow researchers 
 to calculate the reaction time required to achieve the yield they 
 would desire . the dog lesion in dna
can not be found by piperidine 
 cleavage reactions , and this point is further discussed below . as 
 for the other dg lesions in dna , the t1/2 values were 73 min for the dsp diastereomers , 32 min for the dgh 
 diastereomers , 14 min for dz ,
6.8 min for ( r)-d2ih , 
 and 6.0 min for ( s)-d2ih ( table 1 ) . on the basis of these results , all dg - lesions , besides 
 dog , can be cleaved in high yield as long as the reactions are allowed 
 to progress for a sufficiently long time period .
plots of ln[intact strand ] 
 vs time for dg oxidation products in 
 dna ( a ) and rg oxidation products in rna ( b ) oligomers .
( a ) time - dependent 
 cleavage yields for the dna lesions , dog , ( r)-dsp , 
 ( s)-dsp , dgh , dz , ( r)-d2ih , and 
 ( s)-d2ih are plotted .
the reaction yields were monitored 
 on 5-p - labeled strands that were separated by 
 page and quantified by phosphorimager autoradiography .
cleavage reactions 
 for these damaged dna oligomers were conducted with fresh , aqueous 
 piperidine ( 1 m ) and bme ( 0.25 m ) at 90 c .
( b ) time - dependent 
 cleavage yield for the rna lesions , rog , ( r)-rsp , 
 ( s)-rsp , rgh , rz , ( r)-r2ih , and 
 ( s)-r2ih are plotted .
the reactions were monitored 
 in the same fashion as those for the dna oligomers .
cleavage reactions 
 for the damaged rna oligomers were conducted with fresh , aqueous aniline 
 ( ph 4.5 ) at 60 c in the dark .
scheme 2b shows the 
 proposed mechanism for 
 aniline - induced strand scission at the site of a rna lesion .
this intermediate increases the 
 acidity of h2 that accelerates -elimination of the 
 3-phosphate terminus .
finally , -elimination of the 
 5-phosphate terminus occurs to yield complete cleavage at 
 the site of the lesion .
because of the similarity in the cleavage 
 mechanisms , and the fact that the same concentration of aniline was 
 used to cleave rna lesions as was used for piperidine to cleave dna 
 lesions , the same assumptions were used to fit the data to determine 
 the pseudo - first - order reaction rate constants ( eq 1 ) .
the rates of aniline - mediated cleavage ( 1 m , ph 4.5 , 
 60 c ) for all rg lesions were assayed by following strand cleavage 
 via page analysis . under these reaction conditions , rog showed < 1% 
 cleavage after a 90 min reaction ( figure 1b ) . 

the diastereomers of rsp gave slightly more cleavage than rog with 
 < 10% strand scission .
the fitting equations to these data determined 
 the cleavage rate constants for the r and s diastereomers of rsp to be ( 1.2  0.2 )  10 and ( 1.4  0.2 )  10 min , respectively ( table 1 ) .
next , the diastereomers of rgh were found to give more strand 
 scission under these conditions ( figure 1b ) . 
 for rgh , the aniline cleavage rate constant was ( 6.9  0.5 )  
 10 min ( table 1 ) .
the rz reaction preceded more quickly giving a reaction 
 rate constant of ( 51  6 )  10 min ( table 1 ) .
finally , the r2ih 
 diastereomers cleaved the fastest ( figure 1b ) with the r and s isomers having 
 reaction rate constants of ( 61  7 )  10 and ( 63  7 )  10 min , respectively ( table 1 ) .
these studies demonstrate 
 that site specific cleavage of rg oxidation lesions in rna were slower 
 than the analogous cleavage reactions in dna with piperidine . the pseudo - first - order reaction rate constants for cleavage of 
 rg lesions in rna allow calculation of the t1/2 values for these lesions by aniline . on the basis of these 
 rate constants ,
the t1/2 for the rsp diastereomers 
 were calculated to be 580 and 500 min for the r and s isomers , respectively ( table 1 ) . 

these lifetimes are too long to be usable for detection of rsp lesions 
 in rna , and therefore , support rsp as not being cleavable by aniline 
 in any practical applications . for the other rg lesions , the t1/2 values were determined to be 100 min for 
 rgh , 14 min for rz , and 11 min for the diastereomers of r2ih .
in the current studies , dog was not piperidine labile , 
 an observation that has stirred some debate in the literature .
the bme added during the reaction is hypothesized 
 to quench unwanted oxidation of dog .
after a 2 h incubation ( 1 m piperidine , 90 c ) , 10% of 
 the dog - containing strand cleaved based on page analysis , thus supporting 
 the hypothesis that dog can be oxidized during the cleavage reaction , 
 albeit slowly .
analysis of a test reaction by hplc confirmed dog to 
 slowly oxidize to dsp under the hot piperidine reaction conditions 
 ( figure s5 ) , an expected result because 
 at high ph oxidation of dog yields dsp . because dog is poorly cleaved by hot piperidine ,
one approach for 
 sequencing this lesion via chemical cleavage has been developed by 
 our laboratory . in this method ,
dog is 
 further oxidized to dgh with a selective and mild one - electron oxidant , 
 such as na2ircl6 , and the corresponding secondary 
 hydantoin lesion is much more labile to piperidine ( figures 1a and table 1 ) .
the hydantoins dsp and dgh were both found 
 to be labile to piperidine , but with considerably different rate constants 
 ( dsp = 9.5  10 min ; 
 dgh = 22  10 min ; table 1 ) .
further , the r and s isomers of dsp were studied individually and shown to give similar 
 cleavage rates with hot piperidine ( figure 1a and table 1 ) .
the standard piperidine reaction 
 time is 30 min that will give 25% and 48% strand scission for the 
 diastereomers of dsp and dgh , respectively ( figure 2a ) ; the incomplete strand scission currently observed for 
 these lesions after a 30 min piperidine reaction is consistent with 
 previous literature reports . to effect strand scission 
 in a yield >
90% for the hydantoins , the piperidine reaction times 
 must be > 242 min and > 106 min for dsp and dgh , respectively 
 ( figure 3a ) .
as for dz after a 30 min reaction , 
 the cleavage 
 yield would be 78% , an observation that is nearly identical to literature 
 reports ( 80% cleavage after 30 min ) .
furthermore , 
 extension of the reaction time to > 46 min will lead to > 90% 
 cleavage 
 at dz sites ( figure 3a ) .
( figure 2a ) , and the standard 30 min piperidine cleavage 
 reaction time will sufficiently identify d2ih sites ( figure 3a ) .
only one sequence context was studied for each 
 g lesion in dna , and sequence context effects on reaction rates may 
 exist ; however , it is anticipated that these effects will be minimal 
 at ph 11 and 90 c .
other reagents that cleave dna lesions at 
 lower ph have been utilized , such as n , n-dimethylethylenediamine ( 0.1 m ) at ph 7.4 and 37 , 65 , or 
 90 c .
studies were conducted to 
 determine if this reagent could cleave dog , dsp , or dgh under the 
 reported conditions ( 0.1 m , ph 7.4 , and 65 c ) .
these reactions 
 were monitored by hplc and found that n , n-dimethylethylenediamine was incapable of cleaving all three 
 lesions at reaction times < 2 h ( figure s6 ) .
cleavage efficiency 
 for dg lesions ( a ) and rg lesions ( b ) following 
 the standard chemical cleavage protocols .
( a ) the dg lesion - containing 
 oligomers were incubated following the standard cleavage recipe that 
 includes fresh , aqueous 1 m piperidine and 0.25 m bme at 90 c 
 for 30 min .
( b ) the rg lesion - containing oligomers were incubated 
 following the standard cleavage recipe that includes fresh , aqueous 
 1 m aniline ( ph 4.5 ) at 60 c for 20 min in the dark .
chemical cleavage of rg lesions in rna was determined 
 to have lower 
 rate constants for all lesions compared to dg lesions in dna . for 
 example
, the reaction rate for aniline cleavage of the rsp diastereomers 
 was very slow , and almost undetectable ; these reaction rate constants 
 determined that after a standard 20 min aniline reaction time , 3% 
 of the rsp diastereomers would cleave ( figure 2b ) .
the aniline reaction time would need to be increased to 1900 
 and 1700 min for the r and s diastereomers 
 of rsp , respectively , to effect > 90% strand scission ( figure 3b ) .
these reaction times are not practically useful , 
 effectively causing the rsp isomers to be resistant to aniline treatment . 
 in comparison
, the rgh isomers will cleave 13% after the standard 
 aniline reaction time ( figure 2b ) , and the 
 reaction time would need to be extended to > 330 min to effect > 90% 
 strand scission yield at these sites ( figure 3b ) .
next , rz would cleave by 64% under the standard reaction time 
 ( figure 2b ) , and extension of the cleavage 
 reaction to > 47 min will facilitate > 90% cleavage at these sites 
 ( figure 3b ) .
finally , 71% of the r2ih diastereomers 
 will 
 cleave under the standard reaction time ( figure 2b ) ; for these lesions , extending the reaction time to > 37 min 
 will 
 lead to > 90% strand scission at these sites ( figure 3b ) .
in contrast , rog did not give detectable amounts 
 of strand scission 
 with aniline , even after a 90 min reaction .
the only rg lesion detected in vivo so far is rog , an observation that validates rg 
 oxidation occurrence in the cell .
this is particularly 
 striking because there exists 3 times more rna in a cell than 
 dna , and rna takes on a variety of secondary structures that expose 
 the bases to solvent .
these exposed bases will be much more prone 
 to oxidation than those in duplex structures , and a major rg oxidation product will likely be rsp .
however , the use of rna oligomers and chemical cleavage agents will 
 not provide an accurate measure of the sites and yields for the rg 
 oxidation product rog , and likely rsp .
therefore , to study the formation 
 of these lesions in rna will require a different assay .
either a polymerase 
 stop assay can be performed , as has been previously shown , or rog can be further oxidized with a mild oxidant 
 ( na2ircl6 ) to yield a hydantoin ( e.g. , rgh ) 
 that is labile to aniline , an approach that has also been previously 
 proposed .
these studies in rna were only 
 conducted in one sequence context ; however , at ph 4.5 and 65 c , 
 it is anticipated that sequence context effects will be minimal .
minimum 
 reaction time to achieve > 90% cleavage at dg lesions in 
 dna oligomers ( a ) and at rg lesions in rna oligomers ( b ) .
the chemical 
 cleavage of lesion - bearing dna oligomers was conducted with freshly 
 prepared , aqueous piperidine ( 1 m ) and bme ( 0.25 m ) at 90 c , 
 and the lesion - bearing rna oligomers was conducted with freshly prepared , 
 aqueous aniline ( ph 4.5 ) at 60 c in the dark .
the reaction times 
 were computed from the strand scission rate constants values for these 
 lesions provided in table 1 . * these lesions 

the 
 current research determined the chemical cleavage rate constants 
 for many lesions derived from oxidation of the nucleobase guanine 
 in dna and rna oligomers . in these studies , the base damages og , sp , 
 gh , z , and 2ih were site - specifically synthesized in dna and rna oligomers . 

these strands were then subjected to the standard chemical cleavage 
 reagents for dna ( 1 m piperidine , ph 11 , 90 c ) or rna ( 1 m aniline , 
 ph 4.5 , 60 c ) , and the pseudo - first - order rate constants for 
 stand scission were determined . in dna
, dog was not cleaved , and the 
 rate for the dsp diastereomers was found to be slow ( table 1 ) .
the rates of strand scission for dz , dgh , and 
 d2ih were faster , and only d2ih will cleave in a > 95% yield after 
 the standard reaction time of 30 min ( figure 2a ) .
the current results highlight that most lesions may require long 
 reaction times to obtain high strand scission yields ( figure 3a ) .
these results taken together identify a great 
 variability in the cleavage yield for the dg lesions .
the alternative 
 approach for identifying lesion sites in dna utilizes a base excision 
 repair enzyme , such as fpg ; however , fpg cleavage also suffers from 
 lesion - dependent cleavage kinetic differences .
in addition , 
 in rna strands , all lesions were found to cleave at a slower rate 
 than their analogous dna counterparts ( table 1 ) .
more specifically , rog and rsp gave insignificant cleavage with 
 aniline at long reaction times ( figure 1b ) , 
 while rgh and rz could be cleaved at very long reaction times ( figure 3b ) .
finally , these results identify og and sp lesions 
 in dna and rna strands to be the most stable ( table 1 ) , and they support a hypothesis that these lesions in the 
 genome or transcriptome would be much longer lived than the other 
 guanine base lesions .
 the nucleobase guanine in dna ( dg ) 
 and rna ( rg ) has the lowest 
 standard reduction potential of the bases , rendering it a major site 
 of oxidative damage in these polymers . 
 mapping the sites at which 
 oxidation occurs in an oligomer via chemical reagents utilizes hot 
 piperidine for cleaving oxidized dna and aniline ( ph 4.5 ) for cleaving 
 oxidized rna . in the present studies , a series of time - dependent cleavages 
 of dna and rna strands containing various guanine lesions were examined 
 to determine the strand scission rate constants . 
 the guanine base 
 lesions 8-oxo-7,8-dihydroguanine ( og ) , spiroiminodihydantoin 
 ( sp ) , 5-guanidinohydantoin ( gh ) , 2,2,4-triamino-2h - oxazol-5-one ( z ) , and 5-carboxamido-5-formamido-2-iminohydantoin 
 ( 2ih ) were evaluated in piperidine - treated dna and aniline - treated 
 rna . 
 these data identified wide variability in the chemical lability 
 of the lesions studied in both dna and rna . 
 further , the rate constants 
 for cleaving lesions in rna were generally found to be significantly 
 smaller than for lesions in dna . 
 the og nucleotides were poorly cleaved 
 in dna and rna ; sp nucleotides were slowly cleaved in dna and did 
 not cleave significantly in rna ; gh and z nucleotides cleaved in both 
 dna and rna at intermediate rates ; and 2ih oligonucleotides cleaved 
 relatively quickly in both dna and rna . 
 the data are compared and 
 contrasted with respect to future experimental design . 