The photocleavable cyclic oligonucleotide according to the invention is the one that possesses a base sequence having the hybridization ability toward DNA or RNA to targeted, and is further provided with the structure cyclized by a photocleavable group. Accordingly, the photocleavable cyclic oligonucleotide according to the invention, after having been introduced in vivo, is hardly susceptible to the nuclease decomposition reaction owing to its cyclic structure and thus it is capable of diffusing toward the predetermined sites in vivo with sufficient time. Moreover, by being irradiated with the light at an appropriate wavelength after a predetermined period of time, the photocleavable group as described above is cleaved photochemically, thus cutting the predetermined bond. This permits the oligonucleotide that was cyclic to be a linear oligonucleotide which expresses the function of an antisense oligonucleotide.

TECHNICAL FIELD 
This invention relates to oligonucleotide that has been cyclized by 
photocleavable groups. 
BACKGROUND ART 
Oligonucleotides are known to be very important, useful substances in the 
field of biology and medical science: as probes that recognize and detect 
specific sequences in the detection of nucleic acids; as primers for the 
use in PCR method which is an essential technique in genetic engineering; 
and further as antisense oligonucleotides for the use in the antisense 
method which is actively researched in recent years in the field of gene 
therapy. 
In order for the aforementioned antisense oligonucleotides to manifest 
their respective functions, they all need the ability of their base 
sequence moiety to hybridize with complementary base sequences--what is 
known as "hybridization ability". 
Further, it is required that the aforementioned antisense oligonucleotides 
possess not only the hybridization ability, but also the stability in 
vivo. For example, some attempts have been made concerning the control of 
genetic information by the use of antisense oligonucleotides. See, 
Zamecnick, Stephenson et al., Proc. Natl. Acad. Sci., U.S.A., 75, 280-284 
(1978). The oligonucleotides which are used for this purpose are normally 
derived from nature and many of them have only, extremely low resistance 
to nuclease. This presents a problem that they are susceptible to 
undesired decomposition reactions in vivo. Consequently, a variety of such 
modified oligonucleotides that remedy these drawbacks are actually being 
developed. 
One such example is an oligonucleotide with a phosphorothioate bond, which 
is referred to as "S-Oligo" (DeClercq et al., Science, 165, 1137-1139 
(1969)), and it can be easily synthesized on a DNA auto-synthesizer. It is 
known that this kind of oligonucleotides have substantial resistance to 
nuclease. See, Wickstrom et al., J. Biol. Biophys. Meth., 13, 97-102 
(1986). Another example is an oligonucleotide with a methylphosphonate 
bond, which is referred to as "MP-Oligo" (Miller et al., Biochemistry, 18, 
5134-5142 (1979)). Substitution of one of the oxygen atoms present in 
phosphoric ester bonds of DNA of the natural type with a methyl group 
provides the nucleotide with resistance to nuclease, and in addition, it 
eliminates a charge at the phosphoric acid moiety, thus substantially 
improving membrane permeability. 
However, the aforementioned S-Oligo is a racemic mixture including many 
isomers with their chiral centers at phosphoric ester bond moieties, and 
has a drawback in that it is provided with a low affinity to RNA or DNA. A 
further drawback is that it does not possess sufficient stability in vivo 
(i.e., resistance to nuclease). 
Also, the MP-Oligo is a racemic mixture like the S-Oligo, and has a 
drawback in that it is provided with a low affinity to RNA or DNA. A 
further drawback is that its water solubility is low because there is no 
charge at the phosphoric ester bond moieties. 
Furthermore, the still further drawback of the antisense oligonucleotides 
known in the art is that once they have been introduced in vivo, they can 
not be controlled with regard to the expression of their activities such 
as concentrations, sites, and time.

DISCLOSURE OF INVENTION 
This invention has been made in consideration of the drawbacks as described 
above that are inherent in the modified antisense oligonucleotides in the 
prior art. The present inventors have discovered antisense 
oligonucleotides having novel structures which possess sufficiently high 
affinities to RNA or DNA and in addition are provided with sufficient 
resistance to nuclease in vivo and which remedy the drawbacks, and have 
accomplished the invention. 
Specifically, a photocleavable cyclic oligonucleotide according to the 
invention is the one that hybridizes with DNA or RNA to be targeted, and 
is further provided with a structure cyclized by a photocleavable group. 
As used herein, the term, "photocleavable group" means a group having a 
moiety known as a photocaged reagent in the art, wherein specific bonds 
can be cleaved by irradiation at specific wavelengths. 
Accordingly, the photocleavable cyclic oligonucleotide according to the 
invention, after having been introduced in vivo, is not susceptible to 
nuclease decomposition reaction owing to its cyclic structure and is 
capable of diffusing toward the predetermined sites in vivo with 
sufficient time. Furthermore, the aforementioned photocleavable group 
decomposes photochemically, after a predetermined period of time, upon 
irradiation at an appropriate wavelength and the specific bond is cleaved 
to transform the oligonucleotide with a cyclic structure into a linear 
oligonucleotide, which enables the oligonucleotide to hybridize with RNA 
or DNA to be targeted. 
In addition, after the photocleavable cyclic oligonucleotide has diffused 
into a region where the target DNA or target RNA is present, it forms a 
complex by interacting with a partial base sequence of the target DNA or 
target RNA and, upon irradiation, decomposes photochemically to become 
linear, which can effectively bring out its activity as an antisense 
oligonucleotide. 
More specifically, this invention provides a cyclic oligonucleotide 
comprising at least one photocleavable group, wherein the oligonucleotide 
is intramolecularly bonded by the photocleavable group. 
Also, the invention provides the cyclic oligonucleotide as described above 
wherein the photocleavable group has the following structure: 
##STR1## 
Further, the invention provides the cyclic oligonucleotide as described 
above wherein the photocleavable group has the following structure: 
##STR2## 
Also, the invention provides the cyclic oligonucleotide as described above 
wherein the oligonucleotide comprises from 10 to 200 bases. 
Also, the invention provides the cyclic oligonucleotide as described above 
wherein the oligonucleotide comprises from 30 to 100 bases. 
Still further, the invention provides the cyclic oligonucleotide as 
described above wherein the oligonucleotide is provided with a first base 
sequence capable of hybridizing with at least a partial base sequence of a 
target nucleic acid upon cleavage of the photocleavable group. 
Also, the invention provides the cyclic oligonucleotide as described above 
wherein the oligonucleotide is provided with a first base sequence capable 
of hybridizing with at least a partial base sequence of a target nucleic 
acid upon cleavage of the photocleavable group and with a second base 
sequence through which the oligonucleotide forms a complex with the target 
nucleic acid. 
BEST MODE FOR CARRYING OUT THE INVENTION 
The photocleavable cyclic oligonucleotide according to this invention is an 
oligonucleotide which has a structure cyclized by at least one 
photocleavable group. Further, the base sequence of the photocleavable 
cyclic oligonucleotide according to the invention has in its part a base 
sequence capable of hybridizing with a nucleic acid to be targeted (DNA, 
RNA or the like) in a complementary manner. In addition, the 
photocleavable cyclic oligonucleotide according to the invention has in 
its part a base sequence capable of forming a complex with the nucleic 
acid to be targeted. 
A preferred embodiment of the photocleavable cyclic oligonucleotides 
according to the invention is an oligonucleotide which has the structure 
schematically shown in FIG. 1 and which includes a specific base sequence 
cyclized by the photocleavable group: the base sequence is made linear 
after the photocleavage and is able to hybridize with the target nucleic 
acid as an antisense oligonucleotide, and it is represented as Base 
Sequence A in FIG. 1. As shown in FIG. 2, when the photocleavable cyclic 
oligonucleotide according to the invention is introduced into the region 
where the target nucleic acids are present, it is resistant to a variety 
of nuclease by virtue of its cyclic structure and can diffuse sufficiently 
close to the target nucleic acid without being subjected to hydrolysis. 
Moreover, by being irradiated with the light of desired intensity at 
desired sites and times, the photocleavable group of the photocleavable 
cyclic oligonucleotide according to the invention is cleaved to provide a 
linear oligonucleotide. Such linear oligonucleotide can hybridize with the 
specific base sequence of the target nucleic acid present in its vicinity, 
which allows it to exhibit the function of an anitisens-oligonucleotide. 
Simultaneously, the linear oligonucleotide that does not hybridize with 
the target nucleic acid is rapidly hydrolyzed by the nuclease present in 
the vicinity. 
As schematically shown in FIG. 3, another preferred structural example of 
the photocleavable cyclic oligonucleotides according to the invention is 
an oligonucleotide which has a specific base sequence cyclized by the 
photocleavable group and a base sequence capable of interacting with a 
target nucleic acid in the cyclized form: the former base sequence is made 
linear after the photocleavage and is able to hybridize with the target 
nucleic acid as an antisense oligonucleotide, and it is represented as 
Base Sequence A in FIG. 3; and the latter base sequence is represented as 
Base Sequence B in FIG. 3. As shown in FIG. 4, when the photocleavable 
cyclic oligonucleotide according to the invention is introduced into the 
region where the target nucleic acids are present, it is resistant to a 
variety of nuclease by virtue of its cyclic structure and can diffuse 
sufficiently close to the target nucleic acid without being subjected to 
hydrolysis, further allowing the formation of a complex by the interaction 
with a portion of the target nucleic acid (represented as Base Sequence S 
in FIG. 3). In this case, by being irradiated with the light of desired 
intensity at desired sites and times, the photocleavable group of the 
photocleavable cyclic oligonucleotide according to the invention is 
cleaved to provide a linear oligonucleotide. Such linear oligonucleotide 
can hybridize with the specific base sequence of the target nucleic acid 
present in its vicinity, which allows it to effectively exhibit the 
function of an anitisens-oligonucleotide. There is no particular 
limitation to the kind of Base Sequence B which can interact as described 
above, as well as to its base number, but it is preferred that Sequence B 
has a longer sequence than Base Sequence A. 
The structural characteristics of the photocleavable cyclic 
oligonucleotides according to the invention will be explained hereinafter. 
Photocleavable Groups 
The photocleavable cyclic oligonucleotide according to the invention is the 
one that has a photocleavable group within its molecule. Such 
photocleavable group is the one that is photochemically cleaved by 
irradiation to render its cyclic structure linear as a result, and thus 
manifests the activity of an antisense oligonucleotide. See FIGS. 1 and 3. 
Accordingly, such photocleavable group is the one that bonds the 5'-end and 
the 3'-end of a linear oligonucleotide with a base sequence which should 
function as an antisense oligonucleotide, to form a cyclic structure 
wherein at least one part of the bond is to be cleaved under irradiation. 
Therefore, the structures that can be used for this purpose are not 
particularly limited in this invention and they may be any functional 
groups having the aforementioned properties. For example, a functional 
group that is conventionally known as a photocaged reagent is one such 
kind which can preferably be used. In the invention, the functional group 
is more preferably the one that forms a phosphoric ester bond. 
Furthermore, it is required that the photocleavable group having the 
aforementioned functional group be equipped with a functional group to be 
taken in the molecule by the method using a standard DNA/RNA 
autosynthesizer (e.g., on the basis of the phosphoramidite method). For 
example, groups of the nitrobenzyl type with the following structures can 
preferably be used for the photocleavable group which has the 
aforementioned two functions. 
As shown in FIGS. 5 and 6, it is recognized that one of the phosphoric 
ester bonds of this structure is cleaved selectively when it is subjected 
to irradiation at an appropriate wavelength and the structure is split 
into a phosphoric acid portion and a portion with a nitrosophenyl 
derivative. 
Thus, in order to introduce into an arbitrary site of the oligonucleotide, 
the photocleavable group of the nitrobenzyl type shown in FIGS. 5 and 6 
using the phosphoramidite method, phosphoramidite reagents with the 
following structures can, for example, be used. 
These reagents can respectively be prepared according to the synthetic 
methods shown in FIGS. 7 and 8, for example. Specifically, a 
phosphoramidite reagent (1) 
1-(o-nitrophenyl)-2-dimethoxytrityloxy!ethoxy-N,N-diisopropylamino-2-cyan 
oethoxyphosphine) can be synthesized by the reaction between 
(3-chloro-N,N-diisopropylamino-(2-cyanoethoxy)phosphine (1a) and 
(1-O-dimethoxytrityl-2-(o-nitrophenyl)-1,2-ethanediol (1b). Similarly, 
(1') can be synthesized by the method shown in EXAMPLES. 
The phosphoramidite reagents thus obtained are usable without further 
purification as a reagent for DNA/RNA autosynthesizer employing the 
phosphoramidite method. 
Cyclic Oligonucleotides 
The base sequence for the cyclic oligonucleotide according to this 
invention is not limited. While the molecule is cleaved photochemically at 
its site that is a photocleavable group which is to be introduced into 
this oligonucletide and provides a linear oligonucleotide, the base 
sequence can arbitrarily be selected such that once it has been cleaved in 
a manner set forth, it is able to hybridize with RNA or DNA to be targeted 
(target nucleic acids) and to function as an antisense-oligonucleotide. 
Furthermore, the cyclic oligonucleotide contains a base sequence capable of 
interacting with the target nucleic acid and forming a complex while 
retaining a cyclic structure. Such base sequence is the one that is 
complementary to a partial base sequence of the target nucleic acid and 
may contain the number of bases which enables interaction such as 
hybridization. The kind and number of such base sequence can appropriately 
be selected, but the number is preferably from 10 to 200 bases, more 
preferably from 30 to 100 bases. In cases where the base number is within 
the aforementioned range, the base sequence contains an oligonucleotide 
portion with 15-30 bases which normally can be used as an antisense 
oligonucleotide and, if necessary, it becomes possible to synthesize a 
cyclic photocleavable oligonucleotide having a base portion capable of 
interacting with the target nucleic acid. 
Further, although not particularly limited thereto, the bonding by the 
action of ligase is preferably used to cyclize the linear oligonucleotide 
obtained as described above (which may or may not contain the 
photocleavable group). To this end, the 5'-end is phosphorylated and then 
the cyclization is enabled by conducting a conventional ligase reaction 
using an appropriate template. In this instance, the preparation of an 
oligonucleotide that serves as the template for the ligase reaction can 
readily be carried out using a standard autosynthesizer or the like. 
Although there is also no particular limitation to the ligase reaction, the 
use of the template oligonucleotide bonded with biotin makes 
aftertreatment easy. 
FIG. 9 illustrates an embodiment of the preparation of the cyclic 
oligonucleotides according to the invention. 
Also, in the synthesis of a short oligonucleotide, other methods are 
employable, for example, where an oligonucleotide with its 5'-end 
phosphorylated and a template are provided to form a triple-strand and it 
is then cyclized with BrCN-imidazole. See, Prakash, G. and Kool, E. T., J. 
Chem. Soc., Chem. Commun., (1991) 1161-1163. 
In addition, it is possible to synthesize the cyclic oilgonucleotides 
according to the solid phase method. See, Napoli, L. D., Montesarchio, D., 
Piccialli, G., Satacroce, C., Mayol, L., Galenone, A., Messere, A., 
Gazetta Chimica Italiana 121 (1991) 505-508. 
Photocleavage Reaction 
There is no particular limitation to the photocleavage reaction of the 
cyclic oligonucleotide having a photocleavable group according to the 
invention. For example, in the case of a functional group of the 
nitrobenzyl type, conditions such as the wavelength and intensity of the 
light to be irradiated are well known in the art and those conditions are 
usable without any significant changes. 
Furthermore, the whole sample may be irradiated with irradiation light, or 
a portion of the sample may be narrowly irradiated. When the specific site 
of a portion of the sample is narrowly irradiated, the antisense activity 
only manifests at the specific site as illustrated in FIG. 2 or FIG. 4. 
EXAMPLES 
Although this invention is concretely illustrated by way of examples, it is 
not limited to the following examples insofar as it does not depart from 
its essence. 
Synthesis of 3-Chloro-N,N-diisopropylamino-(2-cyanoethoxy)phosphine (1a) 
3-Dichloro-(2-cyanoethoxy)phoshine 27 g was dissolved in 80 ml of anhydrous 
diethyl ether under the nitrogen atmosphere in a reactor fitted with a 
nitrogen gas inlet tube, magnetic stirrer, and dropping funnel and it was 
cooled to -15.degree. C. To this was slowly added in dropwise 31 g of 
diisopropylamine dissolved in 30 ml of anhydrous diethyl ether under 
sufficient stirring. After stirring for 18 hours, diisopropylamine 
hydrochloride which had precipitated was filtered off. 
After removal of the solvent under reduced pressure, the crude product was 
obtained as a light yellowish transparent oily substance. This was 
separated and purified by distillation under reduced pressure 
(108-115.degree. C./0.1 mmHg) (22 g; the purity was greater than 95% based 
on gaschromatography). Structural analysis by .sup.1 HNMR (JEOL JNM-PMX60, 
in deuteriochloroform): .delta. 3.6-4.2 (4H, m, ethylene of the 
cyanoethoxy group); 2.9 (2H, t, methine of the isopropyl group); 1.3 (12H, 
d, methyl of the isopropyl group). 
Dimethoxytritylation of o-Nitrophenyl-1,2-ethanediol 
A reactor in which a 100 ml flask had been fitted with a magnetic stirrer 
and dropping funnel was charged with 2.5 g of o-nitrophenyl-1,2-ethanediol 
and 50 ml of anhydrous pyridine. To this was added 4.6 g of 
dimethoxytrityl chloride under cooling at 5.degree. C. After allowing to 
react for 18 hours under stirring, a majority of pyridine was removed 
under reduced pressure and 100 ml of ethyl acetate and 100 ml of saturated 
brine were added to the residue. The organic solvent layer was washed with 
an aqueous saturated sodium bicarbonate solution and saturated brine, 
dried over sodium sulfate, and the solvent was removed under reduced 
pressure to give 6.7 g of a red oily substance. The resulting crude 
product was purified with a silica gel column chromatograph (chloroform 
eluent containing 0.5% triethylamine) and 6.0 g of the desired product, 
1-O-dimethoxytrityl-2-(o-nitrophenyl)-1,2-ethanediol) (1b) was obtained. 
Structural analysis by .sup.1 HNMR (JEOL JNM-PMX60, in 
deuteriochloroform): .delta. 6.9-8.1 (17H, m, phenyl of the 
dimethoxytrityl, phenyl of the nitrophenyl); 4.0 (6H, s, methoxy of the 
dimethoxyphenyl); 3.8 (1H, t, methine); 3.4 (2H, d, methylene). 
Synthesis of 
1-(o-Nitrophenyl)-2-dimethoxytrityloxy!ethoxy-N,N-diisopropylamino-2-cyan 
oethoxyphosphine (1) 
1-O-Dimethoxytrityl-2-(o-nitrophenyl)-1,2-ethanediol thus obtained above 
6.0 g and 3.1 g of triethylamine was dissolved in 50 ml of anhydrous 
dichloromethane and to the solution was added dropwise 3.6 g of 
3-chloro-N,N-diisopropylamino-(2-cyanoethoxy)phosphine dissolved in 10 ml 
of anhydrous dichloromethane under cooling at 5.degree. C. After allowing 
to react for 1 hour, 150 ml of ethyl acetate was added to the reaction 
solution and it was washed with saturated brine three times. The crude 
product which was obtained after removal of the solvent was purified with 
a silica gel column chromatograph (hexane/ethyl acetate 2:1 eluent 
containing 0.5% triethylamine) and 8.9 g of the desired product (purity of 
98% as determined by reverse phase HPLC) was obtained. Structural analysis 
by .sup.1 HNMR (JEOL JNM-PMX60, in deuteriochloroform): .delta. 6.8-8.1 
(17H, m, phenyl of the dimethoxytrityl, phenyl of the nitrophenyl); 4.0 
(1H, t, methine); 3.7 (6H,s, methyl of the dimethoxyphenyl); 3.3-3.6 (6H, 
m, methylene of the cyanoethoxy group, methylene of the 
nitrophenylethanediol); 2.3 (2H, t, methine of the isopropyl group); 1.2 
(12H, d, methyl of the isopropyl group). Molecular weight: (TOF-MS): 
669.30; Calcd.: 669.76. 
tert-Butyldimethylsilylation of o-Nitrophenyl-1,2-ethanediol 
A reactor fitted with a magnetic stirrer was charged with 100 ml of 
dichloromethane, 10 ml of trimethylamine, 5 mg of dimethylaminopyridine, 
and 10.0 g of o-nitrophenyl-1,2-ethanediol under the nitrogen atmosphere 
in a dark room and the reagents were mixed. The resulting solution was 
cooled to 5.degree. C. tert-Butyldimethylsilylchloride 9.8 g was then 
added in several portions to the solution and it was stirred for about 3 
hours. The end point of the reaction was monitored by TLC and the reaction 
was determined to be complete when the disappearance of the starting 
materials was observed. The purity of the reaction product was confirmed 
on a TLC and the product was used in the next step without further 
purification. 
Dimethoxytritylation of 
(1-O-tert-Butyldimethylsilyl-2-(o-nitrophenyl)-1,2-ethanediol) 
Trimethylamine 10 ml was added to the aforementioned reaction solution as 
obtained and then 18.6 g of dimethoxytritylchloride was added in several 
portions at room temperature. As such, stirring continued overnight and 
the completion of the reaction was confirmed by TLC. The solvent was 
removed under reduced pressure and to the resulting residue was added 200 
ml of ethyl acetate. The solution was further washed thoroughly with water 
and saturated brine and the solvent was removed under reduced pressure to 
give 21 g of an oily substance. The thus obtained residue was purified by 
silica gel column chromatography using chloroform as an eluent and 22.0 g 
of an oily substance was obtained. Structural analysis by .sup.1 HNMR 
(JEOL JNM-PMX60, in deuteriochloroform as .delta. (ppm)): 6.6-7.8 (17H, m, 
phenyl of the dimethoxytrityl group, phenyl of the nitrophenyl); 5.5 (1H, 
t, methine); 3.9 (2H,d, methylene); 3.8 (6H, s, methyl of the 
dimethoxymethy group); 1.0 (9H, s, tert-butyl of the 
tert-butyldimethylsilyl group); 0.1 (6H, s, methyl of the 
tert-butyldimethylsilyl group). 
Elimination of tert-butyldimethylsilyl group of 
1-O-tert-Butyldimethylsilyl-2-O-dimethoxytrityl-2-(o-nitrophenyl)-1,2-etha 
nediol 
A reactor fitted with a magnetic stirrer was charged with 21 g of 
1-O-tert-butyldimethylsiliyl-2-dimethoxytrityl-2-(o-nitrophenyl)-1,2-ethan 
ediol and 100 ml of tetrahedrofuran at room temperature and to the solution 
was added 2.6 g of tetrabutylammonium fluoride in several portions under 
vigorous stirring. As such, stirring continued for about 1 hour and after 
the completion of the reaction was confirmed by TLC, the reaction solution 
was concentrated to terminate the reaction. Ethyl acetate was added to the 
residue and the solution was washed with water and saturated brine. After 
drying, the solvent was removed under reduced pressure to give 18 g of an 
oily residue. This residue was purified with a silica gel column 
chromatograph employing chloroform as an eluent, affording 16.0 g of an 
oily product. 
2-Dimethoxytrityloxy-2-(o-nitrophenyl)!-ethoxy-N,N-diisopropylamino-2-cyan 
oethoxyphosphine (1') 
A reactor fitted with a magnetic stirrer was charged with 100 ml of 
dichloromethane, 7.0 g of triethylamine, 16.0 g of 
1-dimethoxytrityl-1-(o-nitrophenyl)-1,2-ethanediol in a dark room and to 
this was added in dropwise a solution of 
3-chloro-N,N-diisopropylamino-2-cyanoethoxyphosphine 8.5 g in 
dichloromethane at room temperature. The reaction proceeded rapidly and 
was completed in about 30 minutes. The solvent was removed under reduced 
pressure, and the residue was dissolved in ethyl acetate. The solution was 
washed with water and saturated brine, and then dried. The solvent was 
removed under reduced pressure and 19 g of the crude product was obtained. 
The product was purified with a silica gel column flash chromatograph 
employing hexane/ethyl acetate (2:1) as an eluent to give 11 g of the 
product (1'). Structural analysis by .sup.1 HNMR (JEOL JNM-PMX60, in 
deuteriochloroform as .delta. (ppm)): 6.6-7.9 (17H, m, phenyl of the 
dimethoxytrityl group, phenyl of the nitrophenyl); 5.4 (1H, t, methine); 
3.9 (2H,d, methylene); 3.7 (6H, s, methyl of the dimethoxytrityl group); 
3.3-3.6 (4H, m, methylene of the cyanoethoxy group); 2.3 (2H, t, methine 
of the isopropyl group); 1.3 (12H, d, methyl of the isopropyl group). Mass 
spectrometry (SHIMAZU/KRATOS KOMT MALDI IV): 669.51; Calcd: 669.76. 
Synthesis of Linear oligonucleotides 
5'-pCGCAAGCTTC-X-GCCAAGCGCGCAATTAACCCCTCAAACCGC-3' (2), 
5'-pCGCAAGCTTCGCCAAGCGCGCAATTAACCCCTCAAACCGC-3' (3), 
5'-biotin-GAAGCTTGCGGCGGTTTGAG-3' (4), 
5'-pGCCAAGCGCGCAATTAACCCCTCAAACCGC-3' (5), 
5'-pGCCAAGCGCGCAATTAACCCCTCAAACCGCCGCAAGCTTC-3' (6), 
5'-pCGCAAGCTTCp-3' (7), 
and 5'-pCGCAAGCTTC-Y-GCCAAGCGCGCAATTAACCCCTCAAACCGC-3' (8) 
were synthesized on an Applied Biosystems, Model 394 using the conventional 
phosphoramidite method. With respect to (2), the reagent (1) synthesized 
as described above (which is referred to as the group "X" in the formula) 
was used, and the reagent (1') synthesized as described above (which is 
referred to as the group "Y" in the formula) was used for (8). 
A phosphoric acid group was further appended to the 5'-end of 
Oligonucleotides (2), (3), (5), (6), and (7) on the DNA autosynthesizer 
using the phosphoramidite method. Biotin was further appended to the 
5'-end of Oligonucleotide (4) on the DNA autosynthesizer using the 
phosphoramidite method. 
The resulting oligonucleotides were treated with ammonia (30% NH.sub.4 OH 1 
hour at room temperature, and 55.degree. C., 8 hours) to carry out after 
treatments such as removal of protecting groups and further after they 
were roughly purified with Oligo-PakSP (available from Milipore Inc.) 
(only with respect to (4)), the oligonucleotides were desalted with a 
NAP-25 column (available from Pharmacia Inc.) and concentrated under 
centrifugation. 
The purification of the oligonucleotides were respectively carried out by 
HPLC using an ion exchange column or by reverse phase HPLC (LC-10A 
available from Shimazu Manufacturing Co. Ltd.) In a similar manner, a 
70-mer oligonucleotide 
5'-pCGCAAGCTTCGCCCGCACCGATCGC-X-GCCAAGCGCGCAATTAACCCCCTTCCCAACAGTTGCTCAAAC 
CGC-3' was synthesized with a DNA autosynhtesizer and purified. 
Preparation of Cyclic oligonucleotides 
Linear Oligonucleotide (2), (3), or (8) 10 .mu.l (100 pmol/.mu..mu.l), 10 
.mu.l of (4) as the template for cyclization, 8930 .mu.l of ultrapurified 
water, and 1 ml of T4 DNA Ligase buffer (available from Takara Shuzo Co. 
Ltd.) were mixed and allowed to stand under the shielded light at 
27.degree. C. for 30 minutes. Subsequently, the mixture was allowed to 
stand at 16.degree. C. for several minutes under the shielded light and 
with the addition of 50 .mu.l of T4 DNA Ligase (available from Takara 
Shuzo Co. Ltd, 350 Unit/.mu.l), the mixture was allowed to stand for 4 
hours. After concentration under centrifugation, the mixture was desalted 
with a NAP-25 column, then concentrated, centrifuged again and the 
cyclization template was removed therefrom with magnetic beads that were 
coated with avidin (DYNABEADS M280 Streptavidine available from DYNAL). 
Purification was conducted by fractionating on a HPLC and the desired 
fractions were collected and desalted. The resulting cyclic 
oligonucleotides are referred to as (2'), (3'), and (8'), respectively. 
The HPLC conditions used are as follows: 
Column: TOSOH TSKgel DNA-NPR 4.6 mm .phi..times.7.5 mm; 
Flow Rate: 1.0 ml/min; 
Column Oven Temperature: 37.degree. C.; 
Buffer A: 20 mM Tris-HCl, pH 9; 
Buffer B: 1.0M NaCl in Buffer A 
Gradients: A/B (%), from 60/40 to 40/60 over 30 minutes. 
The resulting cyclic oligonucleotides were analyzed by electrophoresis on a 
20% polyacrylamide gel (hereinafter referred to as "PAGE") and the 
mobility of the linear 40-mer oligonucleotide was found to be different 
from that of the cyclic 40-mer cyclic oligonucleotide. It was confirmed 
that the cyclic oligonucleotide had slightly greater mobility than the 
linear oligonucleotide. 
The 70-mer linear oligonucleotide synthesized as described above was also 
cyclized by manipulations similar to those used above to afford cyclic 
oligonucleotides. Analysis by PAGE affirmed that the mobility of the 
linear 70-mer oligonucleotide was different from that of the cyclic 70-mer 
oligonucleotide. 
Nuclease-resistance Tests for Cyclic oligonucleotides 
The resistance tests against nuclease were conducted on linear 
oligonucleotide (2), which was purified and which did not contain a 
photocleavable group, and cyclic oligonucleotide (2') (hereinafter the 
linear nucleotide is referred to as "Linear", and the cyclic 
oligonucleotide as "Circular"). 
The nuclease enzymes used were: (1) four kinds of exonuclease, namely 
Exonuclease III, Exonuclease V, Exonuclease VII, and .lambda. Exonuclease; 
and (2) three kinds of endonuclease, namely S1 Nuclease, Mung Bean 
Nuclease, and BAL 31 Nuclease. 
The tests were conducted by following the procedure as described below 
(See, FIG. 10): 
(1) Exonuclease Resistance Test 
Each approximately 15 pmol of oligonucleotides (both Linear and Circular) 
was added to 15 .mu.l of an exonuclease reaction solution containing: 
Exonuclease III, 50 mM Tris-HCl (pH 8.0), 5 mM MgCl.sub.2, and 10 mM 
2-mercaptoethanol; Exonuclease V, 66.7 mM glycine-NaOH (pH 9.4), 30 mM 
MgCl.sub.2, 8.3 mM 2-mercaptoethanol, and 0.5 mM ATP; Exonuclease VII, 50 
mM Tris-HCl (pH 7.9), 50 mM calcium phosphate, (pH 7.6), 8.3 mM EDTA, and 
10 mM 2-mercaptoethanol; and .lambda. Exonuclease, 67 mM glycine-KOH (pH 
9.4), 2.5 mM MgCl.sub.2, and 0.05% BSA. To this mixture was added the 
enzymes (Exonuclease III 90 U; Exonuclease V 3.8 U; Exonuclease VII 5 U; 
and .lambda. Exonuclease 2.5 U). After the respective reaction solutions 
had been incubated for 30 minutes at 37.degree. C. in a thermostatic bath, 
they were run on a 20% PAGE (20 mM, 40 minutes, staining with ethidium 
bromide (EtBr) for 30 minutes) and the degree of decomposition of Linear 
or Circular was investigated. 
(2) Endonuclease Resistance Test 
Each approximately 150 pmol of oligonucleotides (both Linear and Circular) 
was added to 150 .mu.l of an endonuclease reaction solution containing: S1 
Nuclease, 30 mM sodium acetate (pH 4.6), 280 mM NaCl, and 1 mM ZnSO.sub.4 
; Mung Bean Nuclease, 30 mM sodium acetate (pH 5.0), 100 mM NaCl, and 5% 
glycerol; BAL 31 Nuclease, 20 mM Tris-HCl (pH 8.0), 600 mM NaCl, 12 mM 
MgCl.sub.2, and 1 mM EDTA. To this mixture was added the enzymes (S1 
Nuclease 0.5 U; Mung Bean Nuclease 1 U; and BAL 31 Nuclease 0.2 U). The 
reaction solution was incubated at 37.degree. C. (S1 and Mung Bean) or at 
30.degree. C. (BAL 31). Once the reaction started, a 50 .mu.l aliquot was 
taken after periods of 0, 2, and 5 minutes, respectively and it was 
transferred to a fresh tube containing 5 .mu.l of 0.5M EDTA to completely 
terminate the enzyme reaction. 
After the completion of the reaction, the reaction solution was analyzed by 
HPLC (TOSOH TSK gel DNA-NPR, Solvent A: 20 mM Tris-HCl (pH 9.0),and 
Solvent B: 1M NaCl with a gradient of A/B being from 100/0 to 40/60 over 
60 minutes in Solvent A) and the degree of decomposition of Linear or 
Circular was investigated. The changes observed through HPLC analysis are 
summarized in Table 1. 
TABLE 1 
______________________________________ 
Area of the 
Total of the 
Ratio of the 
Decline of the 
Time Parent peak 
peak area parent peak 
parent peak 
(min) 
(mV .times. sec) 
(40-52 min) 
(40-52 min) 
(%) 
______________________________________ 
S1 Nuclease 
Linear- 
0 195.07 201.68 96.72% -- 
2 127.82 178.83 71.47% 26.11% 
5 66.04 208.51 31.67% 67.26% 
Circular- 
0 137.77 138.56 99.43% -- 
2 79.72 81.91 97.33% 2.11% 
5 49.96 54.44 91.77% 7.70% 
Mung Bean Nuclease 
Linear- 
0 275.52 281.17 97.99% -- 
2 205.09 258.38 79.38% 18.99% 
5 145.43 287.89 50.52% 48.45% 
Circular- 
0 183.06 190.16 96.27% -- 
2 144.50 153.76 93.98% 2.38% 
5 96.17 107.98 89.06% 7.49% 
BAL31 Nuclease 
Linear- 
0 54.97 87.62 62.74% -- 
2 12.93 122.95 10.52% 83.24% 
5 4.44 186.08 2.38% 96.20% 
Circular- 
0 102.89 107.65 95.57% -- 
2 60.76 67.32 90.25% 5.57% 
5 23.94 30.34 78.91% 17.44% 
______________________________________ 
Summarizing the above results, the following general 
trend was recognized: 
Exonuclease Circular 
Linear 
______________________________________ 
Exonuclease III - (+) 
Exonuclease V - + 
Exonuclease VII - + 
.lambda.Exonuclease 
- + 
______________________________________ 
endonuclease 
______________________________________ 
S1 Nuclease 
complete decomposition 
complete decomposition 
before 15 minutes past 
in 5 minutes 
Mung Bean 
survives even after 
complete decomposition 
Nuclease 30 minutes in 30 minutes 
BAL31 complete decomposition in 
complete decomposition 
Nuclease 10 minutes in 5 minutes 
______________________________________ 
The enzymes that were tested for their resistance activity against 
exonuclease did not decompose the cyclic oligonucleotides. In other words, 
the bands on PAGE showed no changes, and this clearly indicates that the 
cyclic oligonucleotides are resistant to these exonuclease enzymes. 
Also, all the endonuclease enzymes that were tested for their resistance 
activity against endonuclease showed differences in the degree of 
decomposition between Linear and Circular. Accordingly, it is evident that 
cyclization of the oligonucleotides provides them with the resistance 
activity against endonuclease. 
Irradiation of Linear and Cyclic Oligonucleotides Containing Photocleavable 
Groups 
Irradiation experiments were conducted on the following oligonucleotides: 
Linear 40-mer Oligonucleotide (2) containing the photocleavable group X; 
30-mer oligonucleotide (5) wherein the 5'-end generated upon irradiation of 
(2) was phosphorylated; 
Cyclic 40-mer oligonucleotide (2') containing the photocleavable group X; 
and 
40-mer Oligonucleotide (6) wherein the 5'-end generated upon irradiation of 
(2') was phosphorylated. 
Here, since Oligonucleotide (2) comprises a 10-mer oligonucleotide and a 
30-mer oligonucleotide both of which are bonded by the photocleavable 
group X, the irradiation causes (2) to split into the 10-mer and 30-mer. 
With respect to the sites cleaved by irradiation, a nitrosophenyl group 
remains at the 3'-end of the 10-mer product and a phosphoric acid group 
remains at the 5'-end of the 30-mer product. 
Furthermore, when cyclic oligonucleotide (2') is subjected to irradiation, 
it is cut at the site cleaved photochemically to form a single linear 
40-mer oligonucleotide. A nitrosophenyl group remains bonding to the 
5'-end of this linear 40-mer oligonucleotide. After Oligonucleotide (2) or 
(2') was dissolved in a TE buffer solution at a concentration of 50 
pmol/10 .mu.l and irradiated with a xenon lamp (USHIO, 300 W, no filter, 
at room temperature) for 30 minutes, the solution was analyzed on a 20% 
PAGE. 
As shown in FIG. 11, these results demonstrate that the product (Lane 2) 
from irradiation of Oligonucleotide (2) (Lane 1) appears at almost the 
same position as Oligonucleotide (5) (Lane 3) and that the product (Lane 
5) from irradiation of oligonucleotide (2') (Lane 4) appears at the almost 
the same position as Oligonucleotide (6) (Lane 6). 
Further, irradiation experiments were conducted on the following 
oligonucleotides: 
Linear 40-mer Oligonucleotide (8) containing the photocleavable group Y; 
30-mer Oligonucleotide (5) wherein the 5'-end generated upon irradiation of 
Oligonucleotide (2) was phosphorylated; 
Cyclic 40-mer Oligonucleotide (8') containing the photocleavable group X; 
and 
40-mer Oligonucleotide (6) wherein the 5'-end generated upon irradiation of 
Oligonucleotide (8') was phosphorylated. 
Here, since Oligonucleotide (8) comprises a 10-mer oligonucleotide and a 
30-mer oligonucleotide both of which are bonded by the photocleavable 
group Y, the irradiation causes oligonucleotide (8) to split into the 
10-mer and 30-mer. With respect to the sites cleaved photochemically, a 
nitrosophenyl group remains at the 3'-end of the 10-mer product and a 
phosphoric acid group remains at the 5'-end of the 30-mer product. 
Furthermore, when cyclic Oligonucleotide (8) is subjected to irradiation, 
it is cut at the site cleaved photochemically to form a single linear 
40-mer oligonucleotide. A nitrosophenyl group remains bonding to the 
5'-end of this linear 40-mer oligonucleotide. After Oligonucleotide (8) or 
(8') was dissolved in a TE buffer solution at a concentration of 50 
pmol/10 .mu.l and irradiated with a xenon lamp (USHIO, 300 W, no filter, 
room temperature) for 30 minutes, the solution was analyzed on a 20% PAGE. 
As shown in FIG. 12, these results demonstrate that the product (Lane 2) 
from irradiation of oligonucleotide (8) (Lane 1) appears at almost the 
same position as Oligonucleotide (5) (Lane 3) and that the product (Lane 
5) from irradiation of Oligonucleotide (8') (Lane 4) appears at the almost 
the same position as Oligonucleotide (6) (Lane 6). 
Consequently, it is evident that the irradiation causes the cleavage of the 
linear and cyclic oligonucleotides at their respective sites where the 
photoclevable groups are present (See, FIGS. 11 and 12). 
The results of HPLC analysis on linear oligonucleotide (2) before and after 
irradiation are shown in FIGS. 13 and 14. The HPLC conditions used are as 
follows: 
Column: TOSOH TSKgel DNA-NPR 4.6 mm .phi..times.7.5 cm; 
Flow Rate: 0.75 ml/min; 
Column Oven Temperature: 37.degree. C.; 
Buffer A: 20 mM Tris-HCl, pH 9.0; 
Buffer B: 1.0M NaClO.sub.4 in Buffer A; 
Gradients: A/B (%), from 95/5 to 65/35 over 45 minutes; and 
Detection: 260 nm UV. 
This indicates that while Oligonucleotide (2) itself displayed a single 
peak with the retention time of 25.621 minutes, after irradiation it 
displayed two peaks with the retention times of 14.744 minutes and 24.427 
minutes, respectively. The peak with the retention time of 24.427 minutes 
coincided with the peak (24.382 minutes) of Oligonucleotide (5). 
Accordingly, the above results demonstrate that Oligonucleotide (2) was 
cleaved photochemically as shown in FIGS. 15 and 16. Likewise, 
Oligonucleotide (8) was cleaved photochemically as shown in FIG. 16. 
The oligonucleotides synthesized in this invention are as follows: NO. 1, 
Length: 41, Type: nucleic acid, Strandedness: single stranded, Topology: 
linear, Molecule Type: DNA, Sequence Description: pCGCAAGCTTC NGCCAAGCGC 
GCAATTAACC CCTCAAACCG C (wherein "pC" represents cytosine the 5'-end of 
which is phosphorylated and "N" represents an unspecified nucleotide); NO. 
2, Length: 40, Type: nucleic acid, Strandedness: single stranded, 
Topology: linear, Molecule Type: DNA, Sequence Description: PCGCAAGCTTC 
GCCAAGCGCG CAATTAACCC CTCAAACCGC; NO. 3 (with a cyclic structure), Length: 
40, Type: nucleic acid, Strandedness: single stranded, Topology: cyclic, 
Molecule Type: DNA, Sequence Description: CGCAAGCTTC GCCAAGCGCG CAATTAACCC 
CTCAAACCGC; NO. 4, Length: 20, Type: nucleic acid, Strandedness: single 
stranded, Topology: linear, Molecule Type: DNA, Sequence Description: 
BiotinGAAGCTTGCG GCGGTTTGAG (wherein "BiotinG" represents biotinylated 
guanine); NO. 5, Length: 30, Type: nucleic acid, Strandedness: single 
stranded, Topology: linear, Molecule Type: DNA, Sequence Description: 
pGCCAAGCGCG CAATTAACCC CTCAAACCGC (wherein "pG" represents guanine the 
5'-end of which is phosphorylated); NO. 6, Length: 40, Type: nucleic acid, 
Strandedness: single stranded, Topology: linear, Molecule Type: DNA, 
Sequence Description: pGCCAAGCGCG CAATTAACCC CTCAAACCGC CGCAAGCTTC 
(wherein "pG" represents guanine the 5'-end of which is phosphorylated); 
NO. 7, Length: 10, Type: nucleic acid, Strandedness: single stranded, 
Topology: linear, Molecule Type: DNA, Sequence Description: pCGCAAGCTTCp 
(wherein "pC" represents cytosine the 5'-end of which is phosphorylated 
and "Cp" represents cytosine the 3'-end of which is phosphorylated); NO. 
8, Length: 71, Type: nucleic acid, Strandedness: single stranded, 
Topology: linear, Molecule Type: DNA, Feature: location: 1; method for 
determining the feature: E; other information: 5'-end phosphorylated, 
Sequence Description: CGCAAGCTTC GCCCGCACCG ATCGCNGCCA AGCGCGCAAT 
TAACCCCCTT CCCAACAGTT GCTCAAACCG C (wherein "N" represents an unspecified 
nucleotide); and NO. 9, Length: 71, Type: nucleic acid, Strandedness: 
single stranded, Topology: cyclic Molecule Type: DNA, Sequence 
Description: CGCAAGCTTC GCCCGCACCG ATCGCNGCCA AGCGCGCAAT TAACCCCCTT 
CCCAACAGTT GCTCAAACCG C (wherein "N" represents an unspecified 
nucleotide). 
Interaction between Cyclic Oligonucleotide and Linear Oligonucleotide 
A single stranded 40-mer oligonucleotide (target) having a complementary 
sequence and the sequence of: 5'-GCGGT TTGAG GGGTT AATTG CGCGC TTGGC GAAGC 
TTGCG-3' was used to prepare a cyclic 40-mer oligonucleotide resulting 
from the cyclization of the base sequence. 
36 .mu.l of the aforementioned target oligonucleotide (100 .mu.M) and 36 
.mu.l of the aforementioned cyclic oligonucleotide (100 .mu.M) were mixed 
in a total 150 .mu.l of a solution comprising 12 .mu.l of 10xBuffer (0.1M 
NaCl, 10 mM phosphate buffer, pH 7.0), 30 .mu.l of formamide, and 36 .mu.l 
of ultrapurified water. The resulting solution was subjected to annealing 
under the following conditions: for 5 minutes at 95.degree. C., then for 
20 minutes at 50.degree. C., and subsequently, at room temperature). 
The thus obtained solution was measured for any change in absorbance at 260 
nm with temperature variations (raising from 30.degree. C. to 85.degree. 
C. at the rate of 30.degree. C. per hour). 
In comparison, a similar measurement was made using a linear, 
single-stranded, 40-mer oligonucleotide with the base sequence that is 
complementary to the aforementioned target oligonucleotide. 
Temperature-dependence of absorbance was observed both for the cyclic 
oligonucleotide and single-stranded oligonucleotide when they were mixed 
with the target oligonucleotide under the conditions as described above. 
Here, the inflection point of the temperature-dependence curve of 
absorbance was taken as a melting point of the nucleotide by following the 
conventional method of measurement for the melting point of a nucleic acid 
(i.e., the temperature at which its double-strand changes to a single 
strand). Thus the melting point obtained was 61.degree. C. in the case of 
the cyclic oligonucleotide, whereas it was 67.degree. C. in the case of 
the single-stranded oligonucleotide which was used as a comparison. 
These results suggest that the aforementioned 40-mer cyclic oligonucleotide 
strongly interacts with the target oligonucleotide as does the 
single-stranded oligonucleotide, and that its product has a structure 
similar to the formation of a normal double strand. 
The formation of a stable complex arising from the interaction between the 
40-mer cyclic oligonucleotide and the single-stranded oligonucleotide was 
ascertained by separation using an anion exchange HPLC. The HPLC 
conditions used are as follows: 
Column: TOSOH TSKgel DNA-NPR 4.6 mm .phi..times.7.5 cm; 
Flow Rate: 0.8 ml/min; 
Column Oven Temperature: 37.degree. C.; 
Buffer A: 20 mM Tris-HCl, pH 9.0; 
Buffer B: 1.0M NaCl in Buffer A; 
Gradients: A/B (%), from 80/20 to 20/80 over 10 minutes; and 
Detection: 260 nm UV. 
FIG. 17 clearly shows a peak (30 minutes) illustrative of the presence of 
the complex between the cyclic oligonucleotide and the target 
oligonucleotide at a position different from either of the target 
oligonucleotide (24.5 minutes) and the cyclic oligonucleotide (24.3 
minutes). In addition, FIG. 18 shows a peak (25.3 minutes) resulting from 
a hybrid product of the target oligonucleotide and the cyclic 
oligonucleotide at a position different from either of the target 
oligonucleotide (24.5 minutes) and the single-stranded oligonucleotide 
(24.1 minutes). It is suggested that the difference in retention time 
reflects a difference between the ionic character due to a structure in 
solution resulting from the complex between the target oligonucleotide and 
the cyclic oligonucleotide and the ionic character due to a structure 
resulting from the hybrid product (with the formation of a complete 
double-strand) between the target oligonucleotide and the cyclic 
oligonucleotide. 
INDUSTRIAL APPLICABILITY 
The photocleavable cyclic oligonucleotide according to this invention, 
after having been introduced in vivo, is hardly susceptible to the 
nuclease decomposition reaction owing to its cyclic structure and thus it 
is capable of diffusing toward the predetermined sites in vivo with 
sufficient time. Moreover, by being irradiated with the light at an 
appropriate wavelength after a predetermined period of time, the 
photocleavable group as described above is cleaved photochemically, thus 
cutting the predetermined bond. This permits the oligonucleotide that was 
cyclic to be a linear oligonucleotide which is then able to hybridize with 
DNA or RNA to be targeted. 
Accordingly, when the photocleavable cyclic oligonucleotide having the 
structure according the invention is introduced in vivo and, after a 
period of time sufficient to diffuse toward arbitrary sites (e.g., a 
specific cell or site), the specific sites are irradiated, and an 
antisense oligonucleotide is expressed only when and where the irradiation 
was carried out: this enables gene control. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 11 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 41 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
#5'-end phosphorylated; "N" represents a 
group bon - #ding 1 or 2-(o-nitrophenyl) 
#and phosphoric esterdiol 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
# 41 GCGC GCAATTAACC CCTCAAACCG C 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
#5'-end phosphorylatedFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
# 40 CGCG CAATTAACCC CTCAAACCGC 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: cyclic 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
# 40 CGCG CAATTAACCC CTCAAACCGC 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 20 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
#5'-end biotinylatedINFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 20 TGAG 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 30 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
#5'-end phosphorylatedFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
# 30 ACCC CTCAAACCGC 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
#5'-end phosphorylatedFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
# 40 ACCC CTCAAACCGC CGCAAGCTTC 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 10 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
#5'-end of the first cytosine and 3'-end 
#tenth cytosine phosphorylated 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
# 10 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 71 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
#5'-end phosphorylated; "N" represents a 
group bon - #ding 1 or 2-(o-nitrophenyl) 
#and phosphoric esterdiol 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- CGCAAGCTTC GCCCGCACCG ATCGCNGCCA AGCGCGCAAT TAACCCCCTT CC - #CAACAGTT 
60 
# 71 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 41 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: cyclic 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
#"N" represents a group bonding 1 or 
2-(o-nitroph - #enyl) ethanediol and 
#ester phosphoric 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
# 41 GCGC GCAATTAACC CCTCAAACCG C 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 70 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: cyclic 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- CGCAAGCTTC GCCCGCACCG ATCGCGCCAA GCGCGCAATT AACCCCCTTC CC - #AACAGTTG 
60 
# 70 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 71 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: cyclic 
- (ii) MOLECULE TYPE: DNA 
- (iii) HYPOTHETICAL: NO 
- (iv) ANTI-SENSE: NO 
- (ix) FEATURE: 
#"N" represents a group bonding 1 or 
2-(o-nitroph - #enyl) ethanediol and 
#ester phosphoric 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- CGCAAGCTTC GCCCGCACCG ATCGCNGCCA AGCGCGCAAT TAACCCCCTT CC - #CAACAGTT 
60 
# 71 
__________________________________________________________________________