
				  regulation of chemical or physical properties of oligodeoxynucleotides ( odns ) is important for the development of future gene diagnostics and therapy [ 1 , 2 ] .
because the function of odns is based on their conformation and hybridization properties with their complementary dna or rna , various attempts have been made to manipulate these basic characteristics using chemical modification and external stimuli [ 311 ] .
these include the binding of a metal ion to mismatched [ 3 , 4 ] or modified nucleobases [ 5 , 6 ] , interaction of boron compounds with modified riboses [ 7 , 8 ] , and photochemical methods that use nitrobenzene [ 9 , 10 ] or azobenzene   functionalities on odns .
high - energy ionizing radiation is an attractive stimulus for controlling the activity of biomaterials , because the radiation reaction can be controlled spatially and temporally without any additives [ 12 , 13 ] . in particular , x - ray has potential because it has high live - body permeability , and thus , has been extensively used for medical treatment and diagnosis . in this paper , we describe the current state of research on controlling the function of odns by x - irradiation .
this paper includes our recent research on the development of artificial odns possessing a 2-oxoalkyl group   or disulfide bonds [ 15 , 16 ] , whose properties and conformation can be regulated by x - irradiation .
we applied their characteristics to regulation of hybridization , radiation - activated dnazyme , regulation of the polymerase reaction , and conformation change of odns and decoy molecules for inhibition of protein - dna interactions .   when diluted aqueous solutions are irradiated , practically all of the absorbed energy is deposited in water molecules , and the observed chemical changes are brought about indirectly by the molecular and , in particular , the radical products of water radiolysis .
it is well known that ionization and excitation of water molecules by ionizing radiation occur and generate electronically excited states ( h2o * ) , radical cations ( h2o ) , and dry electrons ( edry ) . the excited water molecules h2o * dissociate in a homolytic manner to hydrogen atoms ( h ) and hydroxyl radicals ( oh ) , while h2o deprotonates to oh , and edry is solvated to eaq . eventually , eaq , oh , and h are generated as the major active species with g values of 280 , 280 , and 60 nmol / j , respectively , in the radiolysis of water ( figure 1 ) [ 17 , 18 ] . among the active species , we draw attention to the high reactivity of hydrated electrons and hydrogen atoms , which reduce a wide range of molecules under hypoxic conditions .
we designed artificial odns that have high affinity for these reducing species and evaluated their reaction characteristics upon x - irradiation .
previously , we designed and developed several types of radiation - activated prodrugs , which consisted of a cytotoxic agent and a substituent with high electron affinity [ 13 , 1922 ] .
the nontoxic or less - toxic prodrugs underwent one - electron reduction by eaq to release the active agent to exhibit its inherent cytotoxicity . during the process of developing radiation - activated prodrugs , we identified a series of 2-oxoalkyl groups [ 1921 ] that act as efficiently removable substituents by x - ray treatment under hypoxic conditions in aqueous solution ( figure 2 ) .
an activation mechanism has been proposed that the 2-oxoalkyl group undergoes one - electron reduction by eaq to form the corresponding  * anion radical , followed by thermal activation into the  * anion radical , which is readily hydrolyzed to release the 2-oxoalkyl group .
we have applied these characteristics of the 2-oxoalkyl group to develop several prodrugs of antitumor agents such as 5-fluorouracil and 2-deoxy-5-fluorouridine ( 5-fdurd ) .
these contexts of previous research prompted us to apply 2-oxoalkyl substituents to radiolytic regulation of dna duplex formation .
we synthesized odns caged by a 2-oxopropyl group at a given thymine n(3 ) position ( dt ) , which has a characteristic structure similar to the previous radiolytic reduction - activated 5-fdurd prodrug ( figure 3 ) .
one - electron reduction of odns possessing dt , initiated by hypoxic x - irradiation , occurred to remove exclusively the 2-oxopropyl group and produce the corresponding unmodified odns .
the unmodified 59  mer odns were formed from caged odns with g values of 100140  nmol / j for consumption of caged odns and 50110  nmol / j for formation of unmodified odns . on the other hand , the radiolytic removal of the 2-oxopropyl group from caged odns under aerobic conditions was markedly inefficient , in contrast to the prompt formation of unmodified odns under hypoxic conditions . compared with the noncaged odn 1a ( tm = 48.9c ) , the caged odn 1 ( tm = 37c ) showed a dramatic decrease in melting temperature ( tm ) , indicating that the 2-oxopropyl group was effective in the destabilizing the duplex . to characterize the hybridization properties of caged odns , we also subjected a hypoxically irradiated odn 1 containing dt at the center of the swa i recognition site to enzymatic digestion . after the hypoxic irradiation of odn 1 and subsequent addition of an equimolar amount of odn 2 , which is complementary to odn 1 , treatment with swa
we observed strand cleavage of the resulting duplex at the corresponding restriction site , whereas no strand cleavage occurred when nonirradiated duplex was treated with enzyme .
thus , the ability for ordinary duplex formation was restored by hypoxic x - irradiation of caged odns .
artificial dna derivatives that can regulate their recognition properties and functions with the aid of external triggers are useful for a variety of applications . as an example , we tried to make dnazyme , the function of which can be regulated by hypoxic x - irradiation .
we conducted a modification of dnazyme 17e which cleaves both rna and dna / rna chimeric substrates in the presence of transition metal ions such as zn .
we designed dnazyme 17e possessing dt ( odn 3 ) at the crotch of the loop region and characterized its cleavage ability using dna / rna chimeric substrate ( odn 4 ) , in which the fluorophore and quencher were incorporated into the 5- and 3-ends , respectively .
the fluorescence emission of the fluorophore was suppressed intramolecularly by the quencher , while the cleavage of odn 4 led to recovery of emission .
thus , we could monitor the strand cleavage of odn 4 by fluorescence emission . caged 33  mer dnazyme , odn 3 , which was synthesized by automated dna synthesis ,
was preirradiated and then incubated in the presence of dna / rna for 30  min at 25c . as shown in figure 4 , we observed weak fluorescence emission , when odn 3 without irradiation was incubated with odn 4 , indicating that the cleavage activity of dnazyme 17e was efficiently reduced by incorporation of dt into the strand .
it is striking that the fluorescence intensity from odn 4 increased with increasing radiation dose up to 40  gy for caged dnazyme .
these results lead to the conclusion that hypoxic x - irradiation removed the 2-oxopropyl group on the caged dnazyme , thereby resulting in the formation of active dnazyme 17e which can cleave the dna / rna chimeric substrate .  
dna elongation of primer odn 5 by the klenow fragment of dna polymerase i was regulated by caged odn 7 .  
when 10  mer caged odn 7 without x - irradiation was added to the mixture of primer odn 5 , template odn 6 and enzyme , dna polymerization occurred to form a 37  mer odn , which was complementary to odn 6 with a yield of 96% .
this result indicates that caged odn 7 did not act as a suppressor of enzymatic reaction as odn 7 could not hybridize with template odn 6 because of steric hindrance of the 2-oxoalkyl group .
in contrast , x - irradiation resulted in removal of the 2-oxoalkyl group from odn 7 , and thereby addition of x - irradiated odn 7 prevented dna elongation by duplex formation with template odn 6 .
disulfides , thiols , and their corresponding radicals have been shown to induce unique reactions , including formation or rupture of covalent bonding under reduction conditions .
for example , the disulfide radical anion ( rssr ) generated by one - electron reduction of a disulfide is converted into a sulfide anion ( rs ) and a thiyl radical ( rs ) [ 25 , 26 ] .
sulfide anions tend to undergo disulfide exchange reactions in the presence of disulfide compounds , while thiyl radicals abstract a hydrogen atom to form thiols [ 2729 ] .
in addition , hydrogen atoms are assumed to reduce disulfide to form a radical anion .
these reaction characteristics motivated us to investigate radiolytic reduction of odns possessing disulfide bonds [ 15 , 16 ] .
after the hypoxic irradiation , two odns ( odn 9 and odn 10 ) were formed as reaction products via an intramolecular strand - crossing reaction concomitantly with the regeneration of odn 8 .
we subsequently applied these unique reaction characteristics to template - directed strand crossing .   in the presence of a complementary odn ( odn 13 )
, two odns possessing a disulfide bond ( odn 11 and odn 12 ) produced a specified odn ( odn 14 ) via interstrand crossing upon hypoxic irradiation .
radiolytic reduction of odns possessing a disulfide bond resulted in the easy preparation of a specified odn ( figure 6 ) . as well as
these unique reaction characteristics , the disulfide bond plays an important role in the construction of the secondary or tertiary structure of dna architecture [ 3032 ] .
disulfide bond formation and dissociation are significantly affected by the redox state , and therefore the higher - order structure can be controlled by redox reactions .
we next conducted the radiolytic reduction of hairpin - type odns possessing two disulfide bonds in order to establish guides for the design of radiation - associated systems to regulate the dna higher - order structure .
we carried out the radiolytic reduction of 20  mer hairpin - type odn 15 by the x - radiolysis of an argon - purged aqueous solution in the usual manner .
as shown in figure 7 , the corresponding cyclized odns , which were identified by hplc analysis and esi - tof mass measurements , were formed with g values of 100  nmol / j for the formation of cyclized odns .
we also confirmed that the reaction efficiency decreased substantially upon increasing the odn chain length , whereas it was improved by elevating the reaction temperature .   in a separate experiment
, we characterized the effect of reactive species generated by radiolysis of water on the cyclization of odns possessing disulfide bonds . when hairpin - type odn 15 was x - irradiated in an aqueous solution containing oh - scavenging 2-methyl-2-propanol purged with nitrous oxide ( n2o ) gas , which efficiently captures eaq , cyclization was moderately suppressed . on the other hand ,
the formation of cyclized odns was dramatically suppressed in the presence of 2-propanol , which captures both oh and h. these results strongly underline the importance of h as well as eaq for the activation and strand - crossing reaction of disulfide bonds .  
end - capped dna has recently attracted much attention in the field of gene therapy because of its high stability under biological conditions [ 3337 ] .
further attempts have been made to apply radiolytically cyclized odn to a decoy strategy directly targeting transcription factors .
we prepared a 28  mer hairpin - type odn 19 possessing two disulfide bonds and characterized its radiolytic reaction , stability , and binding properties against nf-b .
similar to the results described above , odn 19 was efficiently cyclized by hypoxic x - irradiation .
the cyclized odn 20 was almost - totally resistant to enzymatic digestion by snake venom phosphodiesterase , while prompt digestion was observed for hairpin - type odn 19 .
we next demonstrated that radiolytically cyclized odn 20 interacts with nf-b as a decoy by in vitro competition assay .
an electrophoretic mobility shift assay revealed that addition of cyclized odn 20 effectively prevented the binding of nf-b with a control duplex ( odn 17/odn 18 ) in a sequence - selective manner .
thus , the radiolytic formation of cyclized odns is promising for a decoy strategy that controls gene expression by inhibition of specific transcription - regulation proteins .
artificial oligonucleotides whose hybridization properties , conformation , and functions can be regulated by external stimuli are applicable to a wide range of gene research areas . from this perspective
, we have summarized our recent advances in the development of caged oligonucleotides activated by x - irradiation .
radiolytic reduction by reactive species such as hydrated electrons or hydrogen atoms caused removal of the 2-oxoalkyl group on base units and exchange reaction of the disulfide bond .
the hybridization properties and conformation of odns possessing these substituents are modulated by x - irradiation , and thereby they show promise as radiation - activated dnazyme as modulators of the dna polymerase reaction and as decoy molecules . however , these artificial odns are premature for practical use because high radiation doses are required to activate the caged odns . because high doses of x - irradiation may cause damage to cellular dna , it is difficult to apply the present system to in vivo experiments as it stands .
one of the key strategies to overcome this problem is further chemical modification of radiation - activated substituents that have a high affinity for hydrated electrons and hydrogen atoms .
our current interest focuses on the construction of improved caged dna systems that are highly sensitive to small doses of x - irradiation and their application to in vivo gene regulation .
 a number of advances have been made in the development of modified oligodeoxynucleotides ( odns ) , and chemical or physical properties of which are controlled by external stimuli . 
 these intelligent odns are promising for the next generation of gene diagnostics and therapy . 
 this paper focuses on the molecular design of artificial odns that are activated by x - irradiation and their applications to regulation of hybridization properties , conformation change , radiation - activated dnazyme , and decoy molecules . 
1. Introduction
2. Design and Reaction Characteristics of   2-Oxoalkyl Caged Oligodeoxynucleotides
3. Radiolytic Reduction of Oligodeoxynucleotides Possessing  Disulfide Bonds
4. Conclusion