Patent Application: US-54129506-A

Abstract:
this invention relates to nucleoside , nucleotide , and oligonucleotide analogs that incorporate non - standard nucleobase analogs , those that present a pattern of hydrogen bonds to a paired nucleobase analog in a complementary strand that is different from the pattern presented by adenine , guanine , cytosine , and thymine . most specifically , this invention discloses and claims processes for amplifying nucleic acid analogs containing non - standard nucleobases using polymerase chain reactions , and combinations of non - standard nucleobases , analogs of standard nucleotides , and enzymes that perform this amplification . most specifically , this invention is for the use of 2 - thiothymidine triphosphate instead of thymidine triphosphate in a six letter polymerase chain reaction that includes 2 ′- deoxyadenosine triphosphate , 2 ′- deoxyguanosine triphosphate , 2 ′- deoxycytidine triphosphate , 2 ′- deoxy - iso - guanosine triphosphate , and 2 ′- deoxy - iso - cytidine triphosphate , as well as their forms that contain side chain modifications . because of the size and hydrogen bonding properties of the sulfur unit in 2 - thiot , 2 - thiot does not mispair effectively with the minor tautomer of isog . this permits the pcr amplification of a six letter artificially expanded genetic information system , we examined the relative rates of misincorporation of 2 - thiottp and ttp opposite isog using affinity electrophoresis with a fidelity - per - round of ca . 98 %. the analogous pcr employing ttp has a fidelity - per - round of only ca . 93 %. therefore , this invention represents the first example of a six letter artificial genetic system that is amplifiable by a thermostable polymerase , and capable of darwinian evolution .

Description:
thiones ( the c ═ s unit , as in thiot ) do not serve well as hydrogen bond acceptors , either in solution or in watson - crick pairing [ lez67 ][ dar73 ][ vor74 ][ rac77 ]. for example , while a nucleobase pair between 2 - thiot and adenine contributes to duplex stability ( as measured by δg ) as well as a pair between t and a , a nucleobase pair between 2 - thiot and 2 - aminoadenine destabilizes the duplex by 0 . 8 kcal / mol ( corresponding to a 2 . 4 ° c . decrease in t m in a 20 nucleotide duplex ) [ kut96 ]. this destabilization has been attributed to the increased steric crowding within the c ═ s h — nh — contact in the minor groove of the double helix . the minor tautomer of isog , responsible for its mispairing with thymidine , delivers a ho - group to the minor groove in a position that allows the h of the ho - group to form a hydrogen bond with the minor groove c = o of standard thymidine . the inventive concept , therefore , was to replace the c = o at position 2 of standard thymine by a c ═ s thione unit , that is , by replacing thymidine by 2 - thiothymidine . this led to the prediction that the 2 - thiothymidine - isog ( minor tautomer ) nucleobase pair should be disfavored relative to the thymidine - isog ( minor tautomer ) nucleobase pair in a polymerase active site as well ( fig2 ). this invention discloses and claims processes for amplifying nucleic acid analogs containing non - standard nucleobases using polymerase chain reactions , and combinations of non - standard nucleobases , analogs of standard nucleotides , and enzymes that perform this amplification . most specifically , this invention is for the use of 2 - thiothymidine triphosphate ( 2 - thiottp ) instead of thymidine triphosphate in a six letter polymerase chain reaction that includes 2 ′- deoxyadenosine triphosphate , 2 ′- deoxyguanosine triphosphate , 2 ′- deoxycytidine triphosphate , 2 ′- deoxy - iso - guanosine triphosphate , and 2 ′- deoxy - iso - cytidine triphosphate 2 ′- deoxyadenosine triphosphate , as well as their modified forms . experimentally , we expected that any dna polymerase from evolutionary family a or family b will work . the presently preferred polymerases are from family a . the most preferred polymerase is the “ klenow ” fragment of taq polymerase . these polymerases do not discriminate well against the t - isog pair , should discriminate well against the 2 - thiot : isog pair . thus , we predicted that the pcr amplification of a six letter artificially expanded genetic information system of the instant invention should have a useful fidelity , of at least 98 % per round . oligonucleotides ( table 1 ) were synthesized by integrated dna technologies ( coralville , iowa ) and purified by polyacrylamide gel electrophoresis ( 10 - 20 %). t - 1 5 ′- gtc ttc gtg tca cg ( isog ) cca tag tga gtc gta tta cgc - 3 ′ t - 2 5 ′- gcg aat taa ccc tca cta aag tac g ( isog ) t cgt cta tag tga gtc gta tta cgc - 3 ′ t - 3 5 ′- gcg aat taa ccc tca cta aag tac gat cgt cta tag tga gtc gta tta cgc - 3 ′ the “ klenow ” fragment of taq polymerase ( titanuim ™ taq ) was purchased from bd biosciences ( mountain view , calif .). as titanium ™ taq is a “ hot start ” enzyme , the enzyme was heated to 95 ° c . for 2 minutes , followed by rapid cooling to ambient temperature prior to any primer extension reactions . similarly , all polymerase chain reactions included an initial 2 minute 95 ° c . denaturation cycle . in a typical primer extension reaction ( 25 μl total volume ), 5 ′- 32 p labeled primer ( p - 1 , 25 pmol ) and template ( t - 1 , 30 pmol ) were mixed with buffer ( 10 mm bis - trispropane - hcl ph 9 . 1 , 5 mm mgcl 2 , 40 mm potassium acetate , 0 . 1 mg / ml bovine serum albumin ), heated ( 95 ° c ., 5 min ), and cooled to room temperature over 1 hour . polymerase ( 1 unit ) was added , and the mixture again heated ( 72 ° c ., 10 sec ). each pcr was initiated by adding the appropriate dntps ( 100 μm final concentration ). aliquots ( 2 μl ) were removed from the mixture at time intervals , diluted into a page loading / quench buffer ( 2 μl , 20 mm edta in formamide ), heated ( 95 ° c ., 5 min ) and resolved by electrophoresis using a 20 % page ( 7 m urea ). the gel was analyzed by phosphorimager . to enhance reproducibility , master mixes of the primer / template in buffer were prepared in large scale ( 100 μl ). single turnover primer extension reactions were performed by annealing 5 ′- 32 p labeled primer ( p - 2f , 1 pmol ) and template ( t - 2 or t - 3 , 1 pmol ) in the appropriate buffer described above . polymerase ( 1 pmol ) was added , and the mixture was heated ( 72 ° c ., 10 sec ). the reaction was initiated by adding isoctp ( 100 μm ) and either 2 - thiottp ( 100 μm ) or ttp ( 100 μm ) in the presence of unlabeled trap dna ( p - 2f , 100 pmol , t - 2 , 100 pmol ). the reaction was quenched ( 20 mm edta in hconh 2 ) after 20 sec , and the samples resolved by 20 % page ( 7 m urea ) containing p - acrylamidophenylmercury chloride ( apm , 1 μg / ml ). this permitted the separation of oligonucleotides containing thiothymidine ( which ran slower ) from those that did not . apm was synthesized as described [ igl88 ]. for each 6 - letter nucleotide system investigated , seven parallel pcr mixtures were cycled ( 30 rounds , 95 ° c . for 45 sec . then 45 ° c . for 45 sec . then 72 ° c . for 1 . 5 min ) with the same amounts of primers p - 2f ( 32 p labeled ) and p - 2r ( 1 pmol ; 6 × 10 11 molecules ) and varying concentrations of template t2 . these were obtained by 10 fold serial dilutions ( from 6 × 10 4 - 6 × 10 10 molecules per reaction ). as each 10 fold dilution in template was equivalent to ca . 3 . 3 rounds of amplification , the fidelity of the isoc : isog replication could be monitored on a round - by - round basis , with each amplicon requiring a different number of exponential amplifications to consume the primers ( table 2 .) following amplification , the mixtures were treated with an equal volume of acetic acid ( 0 . 1 mm ), and heated ( 95 ° c ., 30 min ), a procedure that depyrimidinylates the iso - cytidines that have been incorporated . the tubes were then opened , and the solvents removed by evaporation at atmospheric pressure . two volumes of nh 4 oh ( 0 . 1 mm ) were added and incubation continued ( 95 ° c ., 5 min ). this cleaves the product dna strands at sites where isoc had been present . the nh 4 oh was allowed to evaporate , and the mixtures diluted in 2 volumes of gel loading buffer ( 98 % formamide , 10 mm edta , 1 mg / ml bromophenol blue , 1 mg / ml xylene cyanol ff ) and analyzed by denaturing page ( 17 %). quantitation of the band generated by cleavage at isoc vs . full - length product ( not containing isoc ) provided a measure of the fidelity of isoc and isog replication . amplicons testing the substitution of 2 - thiottp for ttp in a pcr were generated as above , using primers p - 2f and p - 2r ( 1 pmol each ), template t - 3 ( 6 × 10 4 molecules ), and either all four natural dntps ( 100 μm each ) or by substituting 2 - thiottp for ttp . to estimate the fidelity per round of the pcrs , the percent of product containing isoc , as determined by the cleavage assay , was plotted versus the number of doublings required to consume all of the added primer . the number of product molecules generated in a perfect pcr is equal to n = n 2 r ( equation 1 ), where n equals the number of template molecules , n equals the number of product molecules , and r equals the number of rounds of perfect doubling required to use all primer molecules . similarly , the number of product molecules containing isoc is equal to n ic = n ( 1 + f ) r ( equation 2 ), where f is the fidelity per round . the percentage of the pcr product containing the isoc : isog base pair is equal to n / n ic , which simplifies to ( ½ + f / 2 ) r . data from the pcr amplifications were graphed and fit to the equation y = 100 ( 1 + f ) r using the program kaleidagraph version 3 . 5 ; synergy software , reading , pa . ), where x is the number of doublings ( i . e . pcr rounds ) as calculated in table 2 ; y is the percent cleaved product from each reaction . table 2 . pcr amplicons . each amplicon was cycled for 30 pcr rounds . each reaction had the same amounts of primer , but different amounts of template , and therefore different ratios of primer to template . the number of perfect doublings required to convert all primer to product is dependent on the primer to template ratio ( amplification ), and is equal to the number of rounds of pcr ( under ideal conditions ) needed to consume the primer . doublings = log 2 (# primer molecules /# template molecules ). template molecules 6 × 10 10 6 × 10 9 6 × 10 8 6 × 10 7 6 × 10 6 6 × 10 5 6 × 10 4 primer molecules 6 × 10 11 6 × 10 11 6 × 10 11 6 × 10 11 6 × 10 11 6 × 10 11 6 × 10 11 amplification 10 10 2 10 3 10 4 10 5 10 6 10 7 doublings 3 . 32 6 . 64 9 . 97 13 . 29 16 . 61 19 . 93 23 . 25 ( pcr rounds ) running - start primer extension reactions were performed with klentaq to determine the ability of the enzyme to incorporate either ttp or 2 - thiottp opposite isog . for each reaction , the polymerase was challenged to misincorporate the respective triphosphate opposite the isog residue at position 26 in the template ( t - 1 ), 3 nucleotides downstream of the primer ( pf - 1 ) terminus . reactions were run in parallel , one containing dgtp and ttp , one containing dgtp and 2 - thiottp , one containing dgtp and isoctp ( positive control ), and one containing only dgtp ( negative control ). aliquots of each reaction were quenched at various times and analyzed by page on a 20 % polyacrylamide gel . in running - start primer extension reactions ( fig3 ), klentaq polymerase incorporated all 3 dntps tested ( isoctp , ttp , 2 - thiottp ) opposite an isog in the template , with isoctp incorporated most efficiently , followed by ttp and 2 - thiottp . it is noteworthy that after one and three minutes of incubation , the polymerase incorporated approximately 2 fold more isoctp than ttp opposite isog . this result illustrates the known nonspecificity of polymerases challenged with a template containing isog . most polymerases also incorporate t as well as isoc opposite isog , either via a wobble base pair or , more likely , opposite the minor tautomer of isog that is complementary ( in the watson - crick hydrogen bonding sense ) to t [ rob98 ]. these data also show that 2 - thiottp was misincorporated very little opposite isog in the 0 . 5 and 1 min incubations . after 3 min , misincorporation gave rise to a more obvious band ( fig3 ). this establishes that 2 - thiottp is misincorporated opposite isog to a much lesser extent than is ttp . this is especially true at incubation times relevant for a typical pcr elongation step ( for dna ≦ 2 kb ) of between 45 and 90 seconds . in two parallel reactions , one containing equal concentrations of 2 - thiottp and ttp , and one containing equal concentrations of 2 - thiottp and isoctp , klentaq polymerase was challenged to choose a nucleotide to incorporate opposite isog . affinity electrophoresis on a polyacrylamide gel ( 20 %) containing p - acrylamidophenylmercury chloride ( apm , 10 μg / ml ) was used to separate those products extended with a 2 - thiot from those extended with a non sulfur - containing dntp ( isoctp , or ttp ) [ igl88 ]. the gel was analyzed via radioimaging . fig4 shows the results of the direct competition experiments , wherein the dna containing 2 - thiot migrates at a slower rate than a typical oligonucleotide due to the interaction of its thiol with the mercury in the apm . it is observed that when placed in direct competition for incorporation , the polymerase incorporates either isoctp or ttp opposite isog , with less than 1 % of the extended product resulting from incorporation of 2 - thiottp . also notable is the observation that tag polymerase prefers 2 - thiottp over ttp as a substrate for incorporation opposite adenosine . this unexpected result was not observed for family b polymerases ( data not shown ). after showing that 2 - thiottp is misincorporated less frequently than ttp opposite isog residues , we then established that 2 - thiottp works in a pcr amplification . for this purpose , replicate polymerase chain reactions with klentaq polymerase were performed using the pcr replicon consisting of primers p - 2f and p - 2r and template t - 3 . three reactions were run in parallel , one containing the four natural dntps ( positive control ), one containing dctp , dgtp , datp , and 2 - thiottp , and one without ttp ( negative control ). each amplification was cycled for 30 rounds , and the products were analyzed by electrophoresis on a 2 % agarose gel . as seen in fig5 , the pcrs with ttp and 2 - thiottp generated comparable amounts of product . this result shows that 2 - thiottp is not only a satisfactory substrate for a polymerase , but can , in fact , be used as a substitute for ttp with little affect on the yield of products . to analyze the products of a polymerase chain reaction with isoc , isog , thiot , a , g , and c , amplification of the nonstandard base pair , we used the acid cleavage method of johnson et al . [ joh04 ]. this method exploits the facile depyrimidinylation of isocytidine upon incubation in acid under conditions where the cleavage of the glycosyl bonds of the standard nucleotides is slow . the resulting a basic site is then cleaved with base , and the products are analyzed by page . the relative amount of isoc that was remaining in a full length pcr product is estimated by the intensity of the cleavage band at the position where the isoc is expected , and normalized by the amount of full length product . these are crude estimates , as some cleavage occurs at other sites as well . fig6 shows the disappearance of isoc in the pcr product as a function of rounds of pcr for both reactions containing ttp and those substituting 2 - thiottp . this diagram shows that isoc is lost from the pcr product much more rapidly when ttp is used than when 2 - thiottp is used . the fidelity per round was obtained by fitting the data to the theoretical curve y =( ½ + f / 2 ) x ( see data analysis section for details ), where f is the fidelity per round . a pcr containing 2 - thiottp displays a fidelity - per - round of 98 %. in contrast , the fidelity - per - round for pcr containing ttp is only 93 %, under the conditions reported by johnson et al . ( 2 ). this example shows that substituting 2 - thiottp for ttp in a pcr sequence significantly increases the fidelity in a pcr amplification of an oligonucleotide containing the isoc - isog base pair . this represents the first chemistry - enzymology combination that has both sufficient fidelity and thermostability for practical application as a 6 - letter thermocycling pcr . direct competition experiments coupled with mercuric gel separations , as exploited here , should be generally useful in the future to assess the fidelity of incorporation of different non - standard nucleotides , when sulfur - containing nucleosides are involved . these experiments allow rapid estimation of the relative kinetic properties for competing dntps [ goo93 ][ blo93 ][ cre95 ]. this technique is preferable to the standard single nucleotide addition ( primer extension ) reactions or the scintillation proximity assay [ lut99 ] previously used to distinguish those nucleotide triphosphates incorporated opposite a particular non - natural nucleoside from those that are not . this technique can also be used to optimize reaction parameters such as relative dntp concentrations , buffers , and elongation time . 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