Patent Application: US-37668895-A

Abstract:
a process for non - enzymatic convalent ligation of two oligonucleotides having the structure shown below : ## str1 ## the spontaneous reaction of these above oligonucleotides produces a thiophosphorylacetylamino linkage as shown below : ## str2 ## when carried out in the presence of a template , this reaction gives 90 % yields within 20 minutes at micromolar concentrations .

Description:
in accordance with the present invention there is provided a method of forming an oligonucleotide generally by the steps of disposing at least two oligonucleotides in aqueous solution wherein one of the oligonucleotides includes an α - haloacyl group and the other of the nucleotides includes a phosphothioate group and then covalently binding the oligonucleotides together through the α - haloacyl group and the phosphothioate groups spontaneously forming a thiophosphorylacetylamino group therebetween . this method exploits the fact that the coupling reaction described herein is very slow in very dilute aqueous solutions but is fast in the presence of a template polynucleotide . that is , the reaction is accelerated in the presence of a target polynucleotide that possesses the sequence section complementary to the probe oligomers . the present invention employs as a therapeutic agent two short oligomers ( for example , 8 to 20 - mers ) which will spontaneously link together covalently after binding at adjacent positions on the target polynucleotide . with this system , one will approach the binding affinity and recognition properties of a longer oligomer probe such as between 16 to 40 - mer , but retain the dependency and base pairing characteristics of the shorter probes ( 8 to 20 - mer ). in other words , the present invention provides the specificity of shorter polynucleotides while possessing the effect of longer polynucleotides . inherent in the present invention is the need and use of polynucleotides including reactive groups which will spontaneously react to form a covalent bond therebetween when the groups are in spacial proximity to each other . specifically , the present invention utilizes at least two oligonucleotides wherein one set of oligonucleotides includes the first reactive group and the second set of oligonucleotides include the second reactive group such that upon being brought in proximity to each other , the groups will spontaneously react to form a stable covalent bond . examples of such pairs of reactive groups are ester + hydrazide , rc ( o ) s - + haloalkyl and rch 2 s - + α - haloacyl . preferably , the present invention utilizes an α - haloacyl group , such as a bromoacetylamino group and a thiophosphoryl group , which form a thiophosphorylacetylamino bridge efficiently , selectively , and irreversibly in dilute aqueous media . as demonstrated below , the products are stable in water and hybridize well with complementary polynucleotides . at low oligomer concentrations , such as less than 1 μm , and in absence of a complementary template the reactions are very slow but can be carried out to high conversion within a few days by freezing the solution . the freezing techniques are described in detail below . coupling is quite fast ( greater than 90 % conversion in 20 minutes ) when carried out in solution in the presence of a complementary oligonucleotide that serves as a template , as shown below in the example section . selectivity is also a major concern in diagnostic applications of the present invention and generally in the use of oligonucleotides . the same features of the present invention that make the novel chemistry of the present invention attractive for therapeutic applications also make it attractive for diagnostic uses . for example , the present invention could be utilized in a diagnostic system as follows . referring to fig1 a is an oligomer consisting of , for example , a 10 - mer bearing a marker (*) in the chain and a bromoacetylamino group at the 3 &# 39 ;- terminus . b is another short oligomer with a thiophosphoryl group at the 5 &# 39 ; end . c is a target oligonucleotide sequence with a sequence complementary to a + b . if in dilute solution the coupling of a and b is sufficiently slow in absence of the template , relative to coupling in the presence of the template , only coupling on the template will be significant . this chemical ligation system could therefore be employed in amplification and detection analogously to the enzymatic ligation system ( ligase chain reaction , or lcr ). it has the potential to be superior since some non - specific coupling introduces a source of error in the enzymatic scheme . the fact that at very low concentrations of oligonucleotides ( that is , in the range of interest in diagnostic applications ) the rate of the chemical ligation in absence of template becomes extremely slow indicates that the non - template directed coupling could be unimportant in this case . as shown in fig2 the ligation indicated in equation 1 for oligomers 1 and 2 exploits the facile reaction of a phosphorothioate with an α - haloacyl derivative . specifically , compound 1 ( seq . i . d . 1 ) in fig2 has a 3 &# 39 ;-( bromoacetylamino )- 3 &# 39 ;- deoxythymidine unit at the 3 &# 39 ;- terminus . for preparation of compound 1 , 15 μl of 0 . 4m aqueous n - succinimidyl bromoacetate ( obtained from calbiochem ) was added to 4 . 9 a 260 units of the 3 &# 39 ;- aminodeoxyribooligonucleotide precursor , acacccaatt - nh 2 . the method of preparation is described by gryaznov et al ., 1992 6 . the reaction was carried out in 10 μl of 0 . 2m sodium borate buffer at room temperature . after 35 minutes , the mixture was diluted with 0 . 5 ml of water , desalted by gel filtration on a nap - 5 column ( produced by pharmacia ), and purified by rp hplc high pressure liquid chromatography and again desalted , giving 4 a 260 units of compound 1 . the elusion times are as follows : rp hplc , 17 . 4 minutes ; ie hplc , 17 . 4 minutes . the ie hplc carried out above and all similar procedures carried out below was carried out on a dionex omni pak na100 4 × 250 mm column at ph 12 ( 10 mm sodium hydroxide ) with a 2 % per minute gradient of 1 . 0m sodium chloride in 10m sodium hydroxide . for rp hplc , a hypersil ods column ( 4 . 6 × 200 mm ) was used with a 1 % per minute gradient of acetonitrile in 0 . 03m triethylammonium acetate buffer at ph 7 . 0 . compound 2 ( seq . i . d . 2 ) was synthesized on a 1 μmole scale on a milligen / biosearch cyclone dna synthesizer using lcaa cpg supported 5 &# 39 ;- dimethoxytrityl - n - isobutyryldeoxyguanosine . standard cyanoethyl phosphoramidite chemistry was used . when chain elongation was complete , the terminal 5 &# 39 ;- hydroxyl group was phosphitilated ( 5 minutes ) with 150 μl of a 0 . 1m solution of &# 34 ; phosphate on ™&# 34 ; reagent ( from cruachem ) in acetonitrile and 150 μl of 0 . 5m tetrazole in acetonitrile . the resulting phosphite was sulfurized by treatment with a 5 % solution of sulfur in pyridine / carbon disulfide ( 1 : 1 , v / v , 45 minutes at room temperature ). after cleavage of the dmt group ( 3 % dca in dichloromethane , 1 . 5 minutes ) the supported polymer was worked up as in the case of compound 1 . reaction of a thiophosphoryloligonucleotide with a haloacetylaminoaromatic derivative in dms and water has been employed in preparing dye - oligonucleotide conjugates 7 . depending upon the use of the invention and the desired kinetics , coupling of the oligonucleotides can be carried out in either aqueous solution , in a frozen state in ice , or in an aqueous solution in the presence of template , as discussed above and as exemplified below . in an initial experiment , 1 . 0 ml of a solution ( ph 7 . 05 , 15 mm phosphate , 85 mm nacl ) containing compounds 1 ( 0 . 39 a 260 units , 4 μm ) and 2 ( 0 . 41 a 260 units , 4 μm ) was prepared and kept at 0 ° c . for 5 days . the solution was warmed to 50 ° c . for 2 . 5 hours , and finally frozen and stored at - 18 ° c . for an additional 5 days . analysis by ie hplc of samples after 2 hours and 48 hours showed formation of a slower eluting product , oligomer 3 ( fig2 ), in yields of about 25 % and 80 %, respectively . no significant change was observed after the additional 3 days at 0 ° c . or warming at 50 ° c . however , the reaction did proceed further in the frozen state , affording a high conversion to compound 3 ( seq . i . d . 3 ) within 5 days as shown in fig3 . the enhanced extent of reaction in the ice matrix may be attributed to the high local concentration of the oligonucleotide reactants within the cavities in the ice . other reactions have been similarly carried out in an ice matrix 8 . in light of this result , an equimolar mixture of compounds 1 and 2 ( 2 μm each ) in the same buffer was directly frozen and held at - 18 ° c . the hplc profiles obtained from samples after 5 hours and daily thereafter show progression to give a high yield of 3 in 5 days , fig4 showing representative data . data for coupling compounds 1 and 2 in solution in the presence of a complementary oligonucleotide template ( ccattttcagaattgggtgt , compound 4 , seq . i . d . 4 ) are presented in fig5 . the system was the same as in the first experiment except template 4 was also present ( 4 μm ). in this case the reaction proceeded to & gt ; 90 % completion within 20 minutes and was essentially complete within 2 hours . the structure assigned to compound 3 is supported by the properties of a model compound ( t -- nhc ( o ) ch 2 -- sp ( o )( o - ) o -- t , prepared in solution on a larger scale than used for compound 3 ), by the mobility of compound 3 on gel electrophoresis ( rm 0 . 58 , compared to rm 0 . 89 , 0 . 95 , and 0 . 61 for compounds 1 , 2 , and 4 , respectively ), and by the stability of the complex formed with the complementary oligonucleotide , 4 . retention time , rp hplc 10 . 5 minutes ; fab + mass spectrum , m + h + 620 , m + na + 642 ; 31 p nmr , β in d 2 o , 18 . 7 ppm , prior references have disclosed characteristics for the alkylthiophosphate group . 9 rm values are relative to bromophenol blue in a 20 % polyacrylamide / 5 % bis acrylamide gel . the tm value , 56 ° c . in 0 1m nacl , approaches that of the complex formed from the corresponding all - phosphodiester 20 - mer and compound 4 ( 60 ° c .) 10 and differs significantly from values for complexes formed from compounds 1 or 2 with compound 4 ( 37 ° c . and 31 ° c .). in addition , the internucleotide -- nh ( co ) ch 2 sp ( o )( o - )-- link was found to be cleaved selectively on oxidation with ki 3 9 ( fig5 ). more specifically , the duplex containing compounds 3 and 4 ( 0 . 3 a 260 units each ) in 100 μl of water was treated with 100 μl of 0 . 2 m aq . ki 3 for 15 minutes at 50 ° c . then 10 μl of 1 m aq . dtt was added to the solution . after 5 minutes the mixture was desalted on a nap - 5 column and analyzed by ie hplc . the above experimentation provides evidence that the present invention presents novel chemistry which provides a convenient means for selectively and irreversibly coupling oligonucleotides in aqueous solution in the range of 4 μm oligomer concentration or greater . the products have been shown to be stable in neutral solution and for a few hours even at ph 12 at room temperature . at concentrations below 1 μm , the rate in the liquid phase become extremely slow . however , the reactions can be carried to near completion in the frozen state . the rate of coupling is markedly accelerated by the presence of a complementary oligonucleotide template . these properties provide a potential in the design of chemical amplification systems and in situ ligation in antisense application as well as in building complex structures from oligonucleotide blocks based on known chemistry . the invention has been described in an illustrative manner , and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation . obviously many modifications and variations of the present invention are possible in light of the above teachings . 1 . ( a ) bischofberger , n . and wagner , r . w . &# 34 ; antisense approaches to antiviral agents &# 34 ; virology , 3 , 57 - 66 ( 1992 ). ( b ) uhlmann , e . and peyman , a . &# 34 ; antisense oligonucleotides : a new therapeutic principle &# 34 ; chemical reviews , 90 , 543 - 584 ( 1990 ). 2 . proceedings , international conference on nucleic acid medical applications , cancun , mexico , jan 26 - 30 , 1993 ; p . o . p . ts &# 39 ; o and p . s . miller , organizers , john hopkins university , baltimore , md . 3 . proceedings , international conference on nucleic acid medical applications , cancun , mexico , january , 1993 , pg . 60 . 4 . woolf , t . m ., melton , d . a ., and jennings , d . g . b . proc . natl . acad . sci . usa 89 , 7305 - 7309 ( 1992 ). 5 . ( a ) naylor , r . ; gilham , p . t . biochemistry 1966 . 5 , 2722 - 2728 . ( b ) sokolova , n . i . : ashirbekova , d . t . ; dolinnaya , n . g . ; shabarova , z . a . febs letters 1988 , 232 , 153 - 155 . 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( a ) thuong , n . t . ; chassignol , m . terrahedron lett . 1987 , 28 , 4157 - 4160 . ( b ) francois , j . c . ; saison - behmoaras , t . ; barbier , c . ; chassignol , m . ; thoung , n . t . ; helene , c . proc . natl . acad . sci . usa 1989 , 86 , 9702 - 9706 . 8 . ( a ) beukers , r . ; ylstra , j . ; berends , w . rec . tray . chim . 1958 , 77 , 729 - 732 . ( b ) letsinger , r . l . ; ramsay , o . b . ; mccain , j . h . j . am . chem . soc . 1965 , 87 , 2945 - 2953 . 9 . mag , m . ; luking , s . ; engels , j . w . nucleic acids res . 1991 , 19 , 1437 - 1441 . 10 . letsinger , r . l . ; zhang , g . ; sun , d . k . ; ikeuchi , t . ; sarin , p . s . proc . natl . acad , sci . usa 1989 , 86 , 6553 - 6556 . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 4 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 11 base pairs ( b ) type : nucleic acid ( c ) strandedness : both ( d ) topology : both ( ii ) molecule type : dna ( genomic )( ix ) feature : ( a ) name / key : misc . sub .-- difference ( b ) location : replace ( 1 .. 11 , &# 34 ;&# 34 ;)( d ) other information : / note =&# 34 ; n is a bromoacetylaminogroup &# 34 ;( xi ) sequence description : seq id no : 1 : acacccaattn11 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 11 base pairs ( b ) type : nucleic acid ( c ) strandedness : both ( d ) topology : both ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : misc . sub .-- difference ( b ) location : replace ( 1 .. 2 , &# 34 ;&# 34 ;)( d ) other information : / note =&# 34 ; n is a thiophosphorylgroup &# 34 ; ( xi ) sequence description : seq id no : 2 : nctgaaaatgg11 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 22 base pairs ( b ) type : nucleic acid ( c ) strandedness : both ( d ) topology : both ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : misc . sub .-- difference ( b ) location : replace ( 11 .. 12 , &# 34 ;&# 34 ;)( d ) other information : / note =&# 34 ; nn is athiophosphorylacetylamino group &# 34 ;( xi ) sequence description : seq id no : 3 : acacccaattnnctgaaaatgg22 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 20 base pairs ( b ) type : nucleic acid ( c ) strandedness : both ( d ) topology : both ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : misc . sub .-- feature ( b ) location : 1 .. 20 ( d ) other information : / note =&# 34 ; complementary to seq . 3 without nn &# 34 ;( xi ) sequence description : seq id no : 4 : ccattttcagaattgggtgt20