the following probe oligonucleotides were made for nf-b - dse - fret ( f , 6fam ; d , dabcyl ; lowercase letters , single - stranded tail ; underline , nf-b binding site ) : 5-fag ttg agg gga ctt tcc cag gcg act cac tat agg cgg tgt ctc gct cgc-3 ( nf-01f ) , 5-agt tga ggg gac ttt ccc agg cga ctc act ata ggc acc aca cca ttc cc-3 ( nf-13 ) , 5-ggg aat ggt gtg gtg cct ata gtg agt cgc ctg gga aag tcc cct caa ctd-3 ( nf-14d ) , 5-gcg agc gag aca ccg cct ata gtg agt cgc ctg gga aag tcc cct caa ct-3 ( nf-02 ) . to prepare probes ,
0.5 m of each oligonucleotide was mixed in annealing buffer ( 10 mm hepes - naoh [ ph 7.9 ] , 50 mm kcl , 0.1 mm edta ) , denatured for 2 min at 95c , and then cooled gradually to allow annealing .
duplexes were stored at 4c and diluted with binding buffer ( 10 mm hepes  naoh [ ph 7.9 ] , 50 mm kcl , 0.1 mm edta , 2.5 mm dtt , 10% glycerol , 0.05% nonidet p40 ) before use .
all other chemicals were reagent grade or better and were obtained from wako ( tokyo , japan ) .
the gst - fusion recombinant human nf-b proteins ( p50 , p52 ) were expressed and purified(13 ) and glutathione was removed by dialysis . in general , dna - binding experiments were carried out in the following manner : 5 l nf - d1 ( consisting of nf-01f and nf-14d ) and 5 l protein in binding buffer were incubated in a 96-well ( half - area ) microplate for 30 min at room temperature .
next , 40 l nf - d2 ( consisting of nf-02 and nf-13 ) was added and allowed to incubate for another 60 min at room temperature .
fluorescence was measured at 535 nm after excitation at 485 nm using an envision microplate reader ( perkinelmer , waltham , ma , usa ) . in experiments with inhibitor , 0.5 l inhibitor , 5 l nf - d1 , and 4.5 l protein in binding buffer were incubated before addition of 40 l nf - d2 . to obtain an ic50 ,
the following equation was solved using the  solver  add - on in microsoft excel : where y is the observed fluorescence , x is the inhibitor concentration , a is the lowest fluorescence , b is the highest fluorescence , and c gives the largest absolute value of the slope of the curve .
s / b was obtained by the following formula : where a is the mean fluorescence of four wells in the absence of protein and p is the mean florescence of four wells in the presence of protein .
z-factors were obtained as follows : where a is the mean fluorescence value of four wells without protein , p is the mean florescence of four wells with protein , sda is the standard deviation of a , and sdp is the standard deviation of p.
the following probe oligonucleotides were made for nf-b - dse - fret ( f , 6fam ; d , dabcyl ; lowercase letters , single - stranded tail ; underline , nf-b binding site ) : 5-fag ttg agg gga ctt tcc cag gcg act cac tat agg cgg tgt ctc gct cgc-3 ( nf-01f ) , 5-agt tga ggg gac ttt ccc agg cga ctc act ata ggc acc aca cca ttc cc-3 ( nf-13 ) , 5-ggg aat ggt gtg gtg cct ata gtg agt cgc ctg gga aag tcc cct caa ctd-3 ( nf-14d ) , 5-gcg agc gag aca ccg cct ata gtg agt cgc ctg gga aag tcc cct caa ct-3 ( nf-02 ) . to prepare probes ,
0.5 m of each oligonucleotide was mixed in annealing buffer ( 10 mm hepes - naoh [ ph 7.9 ] , 50 mm kcl , 0.1 mm edta ) , denatured for 2 min at 95c , and then cooled gradually to allow annealing .
duplexes were stored at 4c and diluted with binding buffer ( 10 mm hepes  naoh [ ph 7.9 ] , 50 mm kcl , 0.1 mm edta , 2.5 mm dtt , 10% glycerol , 0.05% nonidet p40 ) before use .
all other chemicals were reagent grade or better and were obtained from wako ( tokyo , japan ) .
the gst - fusion recombinant human nf-b proteins ( p50 , p52 ) were expressed and purified(13 ) and glutathione was removed by dialysis . in general , dna - binding experiments were carried out in the following manner : 5 l nf - d1 ( consisting of nf-01f and nf-14d ) and 5 l protein in binding buffer were incubated in a 96-well ( half - area ) microplate for 30 min at room temperature .
next , 40 l nf - d2 ( consisting of nf-02 and nf-13 ) was added and allowed to incubate for another 60 min at room temperature .
fluorescence was measured at 535 nm after excitation at 485 nm using an envision microplate reader ( perkinelmer , waltham , ma , usa ) . in experiments with inhibitor , 0.5 l inhibitor , 5 l nf - d1 , and 4.5 l protein in binding buffer were incubated before addition of 40 l nf - d2 . to obtain an ic50 ,
the following equation was solved using the  solver  add - on in microsoft excel : where y is the observed fluorescence , x is the inhibitor concentration , a is the lowest fluorescence , b is the highest fluorescence , and c gives the largest absolute value of the slope of the curve .
s / b was obtained by the following formula : where a is the mean fluorescence of four wells in the absence of protein and p is the mean florescence of four wells in the presence of protein .
z-factors were obtained as follows : where a is the mean fluorescence value of four wells without protein , p is the mean florescence of four wells with protein , sda is the standard deviation of a , and sdp is the standard deviation of p.
two partially double - stranded dna probes , named d1 and d2 , are used .
each probe has a double - stranded region containing a protein binding site and two single - stranded tails .
one strand of d1 is labeled with a fluorophore at the 5-end and the other is labeled with a quencher at the 3-end .
the fluorophore and quencher are placed at the same end of the double - stranded region ; therefore , the fluorescence of the fluorophore is quenched .
the tails of d1 are complementary to those of d2 , so that d1 hybridizes with d2 to form a four - way structure .
as the double - stranded regions of the two probes have identical sequences , the junction of the structure migrates spontaneously , followed by irreversible dissociation to give two fully double - stranded duplexes . in other words , strand exchange occurs between d1 and d2 . as a result
, the quencher - labeled strand of d1 is exchanged for its non - labeled counterpart in d2 ; therefore , fluorescence is restored .
dna - binding proteins bind to the duplex and block strand exchange , thereby suppressing the fluorescence elevation
( a ) in the absence of target protein , strand exchange between d1 and d2 will occur and fluorescence will be elevated .
( b ) in the presence of target protein , strand exchange will not occur and fluorescence will remain quenched . to illustrate the method , we attempted to detect the nf-b ( p50 ) interaction with dna .
nuclear factor-b plays a pivotal role in the coordinated transcription of multiple inflammatory genes and is a probable drug target.(1416 ) two probes , nf - d1 and nf - d2 , were prepared to test quantitative detection of p50 binding to dna .
their double - stranded regions are identical and include an nf-b binding sequence , d(gggactttcc ) .
these probes interact with each other through their single - stranded tails and are then involved in a strand exchange reaction .
each strand of nf - d1 was labeled with 6fam and dabcyl at the double - stranded terminus .
nf - d1 , various concentrations of recombinant p50 , and nf - d2 were mixed in a half - area 96-well microplate and changes in fluorescence were measured .
the fluorescence signal of nf - d1 increased rapidly within 30 min after addition of nf - d2 and was fivefold higher than that of nf - d1 alone at 60 min in the absence of p50 .
fluorescence elevation was suppressed in a p50 concentration - dependent manner by half at 40 nm p50 and almost completely at 320 nm p50 .
( a ) five microliters of 40 nm nf - d1 was mixed with 5 l of 0 nm ( open circles ) , 400 nm ( open squares ) , 800 nm ( closed squares ) , 1600 nm ( open triangles ) , or 3200 nm ( closed triangles ) p50 and incubated for 30 min .
then , 40 l of 10 nm nf - d2 was added and fluorescence was measured ; thus , the final concentrations of p50 were 0 , 40 , 80 , 160 , and 320 nm , respectively . closed circles with a dotted line represent nf - d1 alone .
( b ) fluorescence at 60 min for a positive control completely exchanged product ( nf-01f + nf-02 , named nf-01f02 ) , an exchanged product ( nf - d1 + nf - d2 ) , and an unexchanged product ( nf - d1 alone ) with 0 or 320 nm p50 .
duplex nf-01f02 , which consists of nf-01f and nf-02 , was prepared as a positive control as a completely exchanged product .
varied concentrations of nf-1d ( 1 , 2 , and 4 nm ) were mixed with twofold or fourfold amounts of nf-2d in the presence ( 40 nm ) or absence of p50 . as shown in table 1 ,
the combination of 2 nm nf - d1 and 8 nm nf - d1 showed the highest z-factor ( 0.93 ) .
s / b values ( ratio in the absence to presence of p50 ) were 1.92.3 and were not influenced by the dna concentration .
therefore , we used 2 nm nf - d1 with 8 nm nf - d2 in the following assays .
optimization of probe concentration we examined the specificity of the assay by using double - stranded competitor dna .
nf - cpt2 , nf - cpt3 , nf - cpt4 , and nf - cpt5 are non - specific competitors with one to four nucleotides substituted in the nf-b binding site .
the restoration level decreased as the number of substituted nucleotides in non - specific competitors increased and nf - cpt5 , with four substituted nucleotides , had little effect on p50 binding .
for further evaluation of specificity , we prepared non - specific probes , nfs - d1 and nfs - d2 , with nucleotide substitutions as in nf - cpt5 .
hence , we concluded that p50 suppressed strand exchange in a sequence - specific manner .
( a ) 2.5 l of 2000 nm competitor was mixed with 2.5 l of 800 nm p50 and incubated for 30 min .
then , 5 l of 20 nm nf - d1 was added and incubated for a further 30 min .
finally , 40 l of 10 nm nf - d2 was added and fluorescence was measured : nf - d1 and nf - d2 without p50 ( white bar ) , without competitor ( black bar ) , and with competitors ( gray bars ) . horizontally and diagonally
striped bars indicate non - specific probes ( nfs - d1 + nfs - d2 ) without and with p50 , respectively .
( b ) sequences of the competitor upper strand are shown : underline , nuclear factor-b ( nf-b ) binding site ; bold type , substituted nucleotide in binding site .
error bars represent 3 sd ( n = 4 ) . a 10-point dose  response experiment with evans blue ( eb ) was also carried out .
one hundred m evans blue inhibits nf-b binding to dna by emsa and has been suggested to bind non - covalently to the p50 dna binding region by molecular modeling.(17 ) in dse - fret , 10 m eb showed little effect on p50 , but 30 m eb inhibited p50 completely ( fig .
the ic50 values of eb for p50 and p52 inhibition were 12.9 and 12.8 m , respectively .
we also showed that our method can be used for evaluation of an uncompetitive inhibitor , ( )-dhmeq , which binds covalently to a specific cys residue of rel family proteins to inhibit their dna binding.(18,19 ) we detected an inhibitory effect of ( )-dhmeq on p50 and p52 by dse - fret ( fig .
5 ) . as shown previously,(19 ) ( )-dhmeq was less potent against p52 ( ic50 62.5 m ) compared to p50 ( ic50 8.8 m ) .
half a microliter of various concentrations of eb was mixed with 4.5 l of 890 nm p50 ( a ) or p52 ( b ) and incubated for 30 min .
then , 5 l of 20 nm nf - d1 was added and incubated for a further 30 min .
finally , 40 l of 10 nm nf - d2 was added and fluorescence was measured .
horizontal axes show the concentration of eb in incubations with protein and nf - d1 .
half a microliter of various concentrations of ( )-dhmeq was mixed with 4.5 l of 440 nm p50 ( a ) or p52 ( b ) and incubated for 30 min .
then , 5 l of 20 nm nf - d1 was added and incubated for a further 30 min .
finally , 40 l of 10 nm nf - d2 was added and fluorescence was measured .
horizontal axes show the concentrations of ( )-dhmeq in incubations with protein and nf - d1 .
the dse - fret technique can detect protein  dna interaction quantitatively and specifically using a simple procedure ; just mix and measure .
it detected p50 binding with a high z-factor and a dose  response analysis was carried out with two types of inhibitor ( eb and ( )-dhmeq ) .
moreover , the differential effect of ( )-dhmeq on p50 and p52 was well displayed .
yamamoto et al . found that ( )-dhmeq inhibited p50 more strongly than p52 in emsa analysis.(19 ) the same preference of the inhibitor was shown by dse - fret , with the ic50 for inhibition of p50 being sevenfold lower than that for p52 .
however , the amount of ( )-dhmeq required to inhibit the proteins differed between the two methods . a ( )-dhmeq concentration fourfold that of p50 ( 8020 m ) inhibited protein binding completely in emsa,(19 ) whereas a ( )-dhmeq concentration 44-fold that of p50 ( 8.80.2 m ) was required to inhibit half of the p50 binding in dse - fret .
in contrast , inhibition of ( )-dhmeq to p52 was shown only by dse - fret .
although we carried out the assay in a 96-well ( half area ) format , a 384-well format can be used with appropriate specialized equipment .
the probes used in dse - fret consist of a double - stranded moiety and two single - stranded tails .
inadequate intra- and intermolecular hybridization of tails interferes with appropriate formation of a four - way junction and markedly reduces the efficiency of strand exchange .
these tails were also confirmed to not have protein binding sequences using the transcription factor analysis tool tfsearch ( version 1.3 online , http://mbs.cbrc.jp/research/db/tfsearch.html ) .
the double - stranded moiety should have a sequence that allows protein binding , but is otherwise not restricted in sequence . taken together , these features make design of a probe for dse - fret as easy as that for emsa . to our knowledge , this is the first report to show that nf-b blocks spontaneous strand exchange .
several proteins , including histone octamer , p53 , trf1 , and trf2 , have also been found to block strand exchange.(1012 ) however , this blockage can be explained based on particular features of these proteins : thus , the histone octamer forms a large protein  dna complex and the other proteins have junction - binding activity .
panyutin and hsieh(20 ) found that spontaneous strand exchange is also blocked by a mismatch , which suggests that this process may be blocked easily by a small energy barrier . in this paper
nuclear factor-b is a desirable target for therapy in various cancers and inflammatory diseases.(21,22 ) in most cancer cells , nf-b is localized in the nucleus and is constitutively active .
therefore , anticancer drug targeting of nf-b may have great therapeutic value by inhibiting cell growth or increasing the sensitivity of conventional chemotherapy .
we believe that the dse - fret assay will be a powerful tool to isolate novel nf-b inhibitors that inhibit dna binding activity and cancer growth .
we also found that dna binding of transcription factors sp1 and ap1 ( c - jun ) can be detected by dse - fret ( data not shown ) .
therefore , dse - fret may be applicable to detection of many dna - binding proteins .
 nuclear factor-b ( nf-b ) is a key regulator of cancer progression and the inflammatory effects of disease . to identify inhibitors of dna binding to nf-b 
 , we developed a new homogeneous method for detection of sequence - specific dna - binding proteins . 
 this method , which we refer to as dse - fret , is based on two phenomena : protein - dependent blocking of spontaneous dna strand exchange ( dse ) between partially double - stranded dna probes , and fluorescence resonance energy transfer ( fret ) . 
 if a probe labeled with a fluorophore and quencher is mixed with a non - labeled probe in the absence of a target protein , strand exchange occurs between the probes and results in fluorescence elevation . 
 in contrast , blocking of strand exchange by a target protein results in lower fluorescence intensity . 
 recombinant human nf-b ( p50 ) suppressed the fluorescence elevation of a specific probe in a concentration - dependent manner , but had no effect on a non - specific probe . 
 competitors bearing a nf-b binding site restored fluorescence , and the degree of restoration was inversely correlated with the number of nucleotide substitutions within the nf-b binding site of the competitor . 
 evaluation of two nf-b inhibitors , evans blue and dehydroxymethylepoxyquinomicin ( [ ]-dhmeq ) , was carried out using p50 and p52 ( another form of nf-b ) , and ic50 values were obtained . 
 the dse - fret technique also detected the differential effect of ( )-dhmeq on p50 and p52 inhibition . 
 these data indicate that dse - fret can be used for high throughput screening of anticancer drugs targeted to dna - binding proteins . 