Patent Application: US-201414524227-A

Abstract:
this invention combines artificially expanded genetic information systems with self - avoiding molecular recognition systems , in processes that involve template - directed primer extension in highly multiplexed form in mixtures containing large numbers of primers . this process yields extension products , or in its pcr format , amplicons , that have aegis tags that can be cleanly captured in highly complex mixtures .

Description:
the goal of the instant invention is to provide primers that could be extended by dna polymerases when templated on a natural dna , and to provide primers that could support pcr ( which requires that a primer , after being extended , must also be accepted as a template by a dna polymerase ), where the products of these processes have an aegis tag . with both samrs and aegis , the instant invention teaches a distinction between the hydrogen bonding pattern of a samrs system and the heterocycle used to implement it . as is well known in the art , appendages may be attached the 5 - position of pyrimidines without interfering with the hydrogen bonding that supports duplex formation . indeed , 5 - position alkyl , allyl , and acetylenic substituents at those positions generally encourage duplex formation . likewise , substituents at this position may carry tags useful for capture ( such as biotin ) or detection ( such as fluorescent species ). the instant invention teaches that similar substituents can be attached at the “ 5 - equivalent ” position of the heterocycle that implements the samrs and aegis , noting that the iupac numbering of the heterocycle may assign a different numbering to the 5 - equivalent position of any given heterocycle . analogous substitutions may be placed at the 7 - equivalent position of a 7 - deazapurine analog that is a part of a samrs and aegis . further , the 7 - equivalent nitrogen may be replaced by a ch unit simply to prevent hoogsteen binding . likewise , while 2 ′- deoxyribose is the preferred backbone when it is desired to have the samrs component be recognized by natural dna polymerases , rna polymerases , and reverse transcriptases , tighter binding is obtained by placing the samrs - and aegis - enabling heterocycles on 2 ′- ome , 2 ′- o - alkyl , and / or 2 ′- o - allyl ribose , pna , or lna , which are all taught here as part of the instant invention ( such disclosure not being obvious without such a teaching ). the discussion of the inventive steps by which the presently preferred implementations of the samrs concept were developed is provided in u . s . patent application ser . no . 12 / 229 , 159 , of which this is a continuation - in - part . u . s . patent application ser . no . 12 / 229 , 159 is incorporated in its entirety by reference . the presently preferred implementations of the samrs heterocycles are in fig3 . as noted in u . s . patent application ser . no . 12 / 229 , 159 , reduction to practice discovered as an unexpected phenomenon that the melting temperatures of duplexes supported by only base pairs joined by two hydrogen bonds were abnormally low . thus , while the 2 - thiot : a pair was modestly more stable on average ( with the metric being a higher tm in a variety of contexts ) than the t : a pair , a fact well known in the literature , and the i : c pair was significantly less stable than the g : c pair ( a fact also well known ), duplexes joined by only 2 - thiot : a , 2 - ap : t , i : c , and 4et c : g pairs were significantly less stable than expected . this observation prompted the exploration of primers having the self - avoiding property at the 3 ′- end of the primer more than at the 5 ′- end of the primer , as it is overlap of the 3 ′- ends of primers in primer libraries that causes primer - primer interactions that defeat the pcr analysis . thus , this would direct one of ordinary skill in the art to place standard nucleobases at the 5 ′- end . as a consequence , rules were developed that give the presently preferred embodiments for the primer segments . the preferred 5 ′- end of the primer is a moiety commonly used in primers , including without limitation oh ( the 5 ′- oh group is free ), o - phosphate ( allowing the 5 ′- end to be ligatable ), o - oligonucleotide , — nh 2 , or a phosphate or an amino group linked to a biotin or a fluorescent tag . the 3 ′- terminal nucleotide preferably has one of the standard nucleotides , adenine ( or diaminopurine ), thymine , guanine , cytosine , or uracil , or one of the a *, t *, g *, and c * nucleobases , with the most preferred application being a standard nucleotide at the 3 ′- end . the samrs - containing segment next in from the 3 ′- end is preferably 4 to 6 nucleotides in length , and entirely composed of a *, t *, g *, and c * nucleotides , although a single standard nucleotide can be in segment . the next segments , proceeding away from the 3 ′- end , are presently preferred to be constructed exclusively from a , t , g , and c , although single samrs nucleotides in this region can function as well , preferably if they are thiothymidine or thiouracil . this segment is chosen to give a desired affinity to its complement , and is preferably 10 to 20 nucleotides long . in any case , the presently preferred sum of these two segments primers is at least 15 , so as to achieve useful affinity to a target oligonucleotide . these segments , including the 3 ′- nucleotide , the samrs - rich segment , and the samrs - poor segments , are designed to be substantially complementary to a portion of the sequence of a target oligonucleotides , to which it will hybridize in the claimed process . the preferred aegis tag contains nucleobases independently selected from the group consisting of a , t , g , c , k , x , v , j , s , b , z and p , wherein k , x , v , j , s , b , z and p are the nucleobases disclosed in fig1 or fig2 . the tag must contain at least one k , x , v , j , s , b , z and p , but more preferably it contains at least two , and is preferably 5 to 30 nucleotides long . mosquito - borne arboviruses must be detected in public health surveillance environments . this example combined the self avoiding molecular recognition system ( samrs ), which enables high levels of multiplexing , with an artificially expanded genetic information system ( aegis ), which enables very clean pcr amplification in nested pcr formats . luminex “ liquid microarrays ” were exploited for downstream multiplexed detection . this example showed this combination supporting single - tube pcr amplification assays to seek rna from 21 mosquito - borne rna viruses from the genera flavivirus , alphavirus , and orthobunyavirus . this assay differentiated between many closely - related viral targets , including dengue , west nile , japanese encephalitis , and the california serological group viruses . primers and capture probes containing artificial samrs and aegis nucleotides ( table 2 ) were synthesized on abi 394 and abi 3900 synthesizers in - house . primers and capture probes were designed to complement a majority of the strains from each of the target viruses . for the simulants , ssdna oligonucleotides ( amplimers , appendix a . supplementary data table 2 ) were chosen arbitrarily to represent a single strain . table 2 hybrid samres - aegis primers and aegis ( aptc ) probes used in this study . all reverse primers are 5 ′- biotinylated ; the probes are 5 ′- amino - c12 - modified . the aegis tags in the primers are underlined . oligos genome gb primers / probes sequences 5 ′- 3 ′ region accession no . forward wnm1 ctaptccpccapcpapc 163 - 181 nc - 009942 primer cgcgtgttgtccttg * a * t * t * g seq id no 1 reverse wnm1 cagpaagpggtpgptpg 312 - 293 nc - 009942 primer cacacctctccatcga * t * c * c * a seq id no 2 wn probe appttcacapcaattpctcc 259 - 278 nc - 009942 seq id no 3 forward je m1 ctaptccpccapcpapc 10612 - 10628 nc_001437 primer gaccaacgtcagg * c * c * a * c seq id no 4 reverse je m1 cagpaagpggtpgptpg 10769 - 10748 nc_001437 primer gggtctcctctaacctct * a * g * t * c seq id no 5 je probe cacppcccaapcctcptcta 10705 - 10724 nc_001437 seq id no 6 forward sle ctaptccpccapcpapc 10561 - 10577 nc_007580 m1 primer tggcacgtaggct * g * g * a * g seq id no 7 reverse slem1 cagpaagpggtpgptpg 10634 - 10614 nc_007580 primer cagacagcacctttagc * a * t * g * c seq id no 8 sle probe capaccapaaatpccacct 10591 - 10610 nc_007580 seq id no 9 forward yf m3 ctaptccpccapcpapc 25 - 44 nc_002031 primer gtgcattggtctgcaa * a * t * c * g seq id no 10 reverse yf m3 cagpaagpggtpgptpg 164 - 146 nc_002031 primer ccatattgacgccca * g * g * g * t seq id no 11 yf probe papcpattapcapapaactpac 91 - 112 nc_002031 seq id no 12 forward d1 ctaptccpccapcpapc 105 - 127 fj639679 . 1 primer gtctttcaatatgctgaaa * c * g * c * g seq id no 13 forward d2 ctaptccpccapcpapc 10433 - 10452 eu482570 . 1 primer gaggccacaaaccatg * g * a * a * g seq id no 14 forward d3 ctaptccpccapcpapc 103 - 128 eu482596 . 1 primer gtctatcaatatgctgaaa * c * g * c * g seq id no 15 forward d4 ctaptccpccapcpapc 10363 - 10379 gq199883 . 1 primer atgcgccacggaa * g * c * t * g seq id no 16 reverse d ( 1 , 3 ) cagpaagpggtpgptpg 174 - 152 ( d1 ) fj639679 . 1 primer tgagaatctcttcgccaac * t * g * t * g seq id no 17 reverse d ( 2 , 4 ) cagpaagpggtpgptpg 10497 - 10479 eu482570 . 1 primer ggaggggtctcctct * a * a * c * c ( d2 ) seq id no 18 d1 probe cpapaaaccpcptptcaact 128 - 147 fj639679 . 1 seq id no 19 d2 probe cpcatppcptaptppactap 10457 - 10476 eu482570 . 1 seq id no 20 d3 probe apaaaccptptptcaactpp 131 - 151 eu482596 . 1 seq id no 21 d4 probe pcptppcatattppactapc 10383 - 10402 gq199883 . 1 seq id no 22 forward mve ctaptccpccapcpapc 535 - 551 nc_000943 primer tgatcgccattcc * a * a * c * c seq id no 23 reverse mve cagpaagpggtpgptpg 614 - 594 nc_000943 primer ggtgtcatcacacataa * a * t * c * c seq id no 24 mve probe ptcppattcpapccattpac 571 - 590 nc_000943 seq id no 25 forward - rocio ctaptccpccapcpapc 1883 - 1903 ay632542 primer caagaacccagttgaca * c * a * g * g seq id no 26 reverse - rocio cagpaagpggtpgptpg 2036 - 2015 ay632542 primer gggaacaaatggattgac * c * g * t * c seq id no 27 rocio probe papaacctacatpatctcactcc 1977 - 1999 ay632542 seq id no 28 forward - ctaptccpccapcpapc 11034 - 11057 nc_003899 eeem1 primer ctgagagcggatcatttaca * t * t * c * c seq id no 29 reverse - cagpaagpggtpgptpg 11133 - 11111 nc_003899 eeem1 primer caatctcctttgcaggtaa * c * t * g * c seq id no 30 eee probe pcttttaapctpcapptctpc 11084 - 11104 nc_003899 seq id no 31 forward - ctaptccpccapcpapc 4339 - 4360 nc_001449 veem1 primer cagtagcgattccactgt * t * g * t * c seq id no 32 reverse - cagpaagpggtpgptpg 4485 - 4462 nc_001449 veem1 primer gagtcatttcccatttcttg * t * c * c * c seq id no 33 vee probe pctpacapctttapacaccac 4415 - 4435 nc_001449 seq id no 34 forward - ctaptccpccapcpapc 345 - 366 nc_003908 weem1 primer caagaacatagcctctaa * g * g * c * g seq id no 35 reverse - cagpaagpggtpgptpg 482 - 460 nc_003908 weem1 primer gcgtacacatcttggtata * c * t * g * c seq id no 36 wee probe tptatpcacacapacpccac 418 - 437 nc_003908 seq id no 37 forward - ce ctaptccpccapcpapc 675 - 694 u12800 primer cggcatgattgcaaag * a * g * t * c seq id no 38 reverse - ce cagpaagpggtpgptpg 792 - 770 u12800 primer cggagcttatggcaacttt * a * t * c * c seq id no 39 ce probe ptttpapcpacactpctapaac 731 - 752 u12800 seq id no 40 forward jtc ctaptccpccapcpapc 283 - 304 ef681804 primer caacgatcttaccatcca * t * c * g * g seq id no 41 reverse jtc cagpaagpggtpgptpg 435 - 412 ef681804 primer ccattgttccaatgaatgcc * a * t * t * g seq id no 42 jtc probe1 capapapaactcataappapcac 365 - 387 ef681804 seq id no 43 jtc probe2 pcaccatcataaatccaattpcapa 384 - 408 ef681804 seq id no 44 forward lac ctaptccpccapcpapc 577 - 597 nc_004110 primer cacagagtcaagcaagg * c * a * t * g seq id no 45 reverse lac cagpaagpggtpgptpg 736 - 715 nc_004110 primer ggcctccttttccccatt * t * a * a * g seq id no 46 lac probe patptcacapaappttpcapc 663 - 683 nc_004110 seq id no 47 forward ks ctaptccpccapcpapc 376 - 396 u12801 primer gtgaggacgagtcacaa * a * a * g * g seq id no 48 reverse ks cagpaagpggtpgptpg 476 - 453 u12801 primer gagatagatttctacaccgt * t * g * c * c seq id no 49 ks probe patcaapapcactptcatcaatcc 401 - 424 u12801 seq id no 50 forward ssh ctaptccpccapcpapc 687 - 707 j02390 primer ccaagagcctgaaggaa * g * t * a * g seq id no 51 reverse ssh cagpaagpggtpgptpg 793 - 772 j02390 primer ccttacttatgggagcct * g * a * t * g seq id no 52 ssh probe pacactpccapatcattcttpc 740 - 761 j02390 seq id no 53 forward 2 ca ctaptccpccapcpapc 112 - 132 ( sa ) u47139 common primer cggtgcaaatggatttg * a * t * c * c seq id no 54 forward 2 ca ctaptccpccapcpapc 111 - 131 ( sn ) u47140 common primer cggtgcaaatggatttg * a * t * c * c seq id no 54 reverse 1 ca cagpaagpggtpgptpg 235 - 216 ( sa ) u47139 common primer gagagcagctttggct * t * t * t * g seq id no 55 reverse 1 ca cagpaagpggtpgptpg 234 - 215 ( sn ) u47140 common primer gagagcagctttggct * t * t * t * g seq id no 55 sa probe cpatcaptttptcttcapttappatc 174 - 199 u47139 seq id no 56 sn probe cttacapccpttapaatcttcttcc 181 - 205 u47140 seq id no 57 forward mel ctaptccpccapcpapc 659 - 679 u12802 primer ctgaaggatgtagagca * g * c * t * g seq id no 58 reverse mel cagpaagpggtpgptpg 777 - 755 u12802 primer gccgaattcattagaggac * c * a * t * c seq id no 59 mel probe capaapttcpptpttapacttcc 725 - 747 u12802 seq id no 60 pcr targeting rna virus simulants was set up in 1 × jumpstart reaction buffer ( 10 mm tris - hcl , ph 8 . 3 ; 50 mm kcl ; 1 . 5 mm mgcl 2 ; 0 . 001 % ( w / v ) gelatin ) ( sigma - aldrich , st . louis , mo .). the other components of the reaction mixture were ( in a total volume of 100 μl ): 2 . 5 ng / μl dna oligo ; 0 . 4 mm dntps ; 0 . 4 μm each , forward t7 primer and reverse target - specific primer ; jumpstart taq dna polymerase ( 2 units , sigma ), nuclease - free ddh 2 o ( added to create a final volume of 100 μl ). after the initial denaturation at 95 ° c . for 2 minutes , 35 cycles of amplification were performed ( 94 ° c . for 30 seconds , 55 ° c . for 30 seconds , and 72 ° c . for 1 minute ). a final extension cycle was run at 72 ° c . for 5 minutes . each pcr product ( in 100 μl ) was ethanol - precipitated and dissolved in nuclease - free dd h 2 o ( 12 μl ). the resulting pcr products were sequenced in both directions ( university of florida , icbr ) and 5 pmol ( about 2 μl ) of the concentrated pcr product was used as a t7 - dna template to make rna simulants . for simulant production , a t7 rna polymerase - dependent transcription reaction mixture ( 20 μl ) was set up in a 1 × transcription buffer ( 40 mm tris , ph7 . 8 , 20 mm nacl , 18 mm mgcl 2 , 2 mm spermidine hcl , 10 mm dtt ). the reaction mixture contained atp , ctp , gtp , and utp ( 75 mm stock concentration 2 μl each ), dna template ( 2 . 5 - 5 pmol , purified and concentrated pcr product ); t7 rna polymerase ( 2 μl of a 200 u / μl to give 20 u / μl final concentration ). reaction mixtures were incubated at 37 ° c . for 8 - 12 hours . turbo dnase was then added ( 2 u per reaction mixture , life technologies ) to remove dna template . the mixtures were then incubated at 37 ° c . for 15 - 20 minutes . rna products were isolated by phenol - chloroform extraction and dissolved in nuclease - free water ( 20 μl ). rna products were resolved by 3 % tbe agarose - gel electrophoresis and quantitated by their uv absorbance at 260 nm . the purity of rnas was evaluated from their a260 / a280 ratio . for pure rna , a ratio of 1 . 8 - 2 . 1 is expected . the absence of template dna in the rna samples was confirmed by conventional pcr with platinum taq dna polymerase ( life technologies ) and the ethidium - bromide gel . samples were aliquoted and kept at − 80 ° c . monoplex pcrs were first performed using each target rna simulant separately to assess the efficacy of the primers in pcr cycling , as well as to determine the sensitivity of the assay . reactions were then optimized under multiplexed conditions to minimize cross - amplification or cross - hybridization resulting from possible sequence similarity between targets . these were carried out in 1 × reaction mix ( life technologies ) with rna simulant ( 4 ng / μl ) in a final volume of 20 μl accordingly to the invitrogen protocol for the superscript one - step rt - pcr with platinum taq ( life technologies ). the reaction mixture contained 0 . 2 mm of dztp ; 0 . 025 μm each of 21 pairs forward and reverse hybrid samrs - aegis target - specific primers ; 0 . 25 μm external aegis forward and reverse - biotinylated primers ; 2 . 5 units rt / platinum taq enzyme mix . additional 1 . 5 mm mgso 4 were added to the rt - pcr buffer . cycling conditions were : one cycle of the cdna synthesis and pre - denaturation ( 53 ° c . for 30 minutes and 94 ° c . for 2 minutes ), 55 cycles of pcr ( 94 ° c . for 15 seconds , 53 ° c . for 30 seconds , and 70 ° c . for 30 seconds ) and final extension at 72 ° c . for 5 minute . a “ no - target ” pcr negative control was included with each assay run . to favor incorporation of biotin - labelled reverse primers to maximize hybridization sensitivity , the second pcr was performed with only reverse biotinylated primer ( reverse primer extension reaction , rper ). to destroy excess primers and deactivate dntps prior to rper , exosap - it enzyme mixture ( 2 μl , affymetrix , cleveland , ohio usa ) were added to aliquots ( 5 μl ) of standard or samrs - aegis nested pcr . reaction mixtures were incubated at 37 ° c . for 30 minutes and the enzyme mixture was destroyed by heating at 80 ° c . for 20 minutes . treated pcr products were added directly to the reverse primer extension reaction . briefly , a rper ( 20 μl ) was set up in 1 × thermopol buffer ( 20 mm tris - hcl , 10 mm ( nh 4 ) 2 so 4 , 10 mm kcl , 2 mm mgso 4 , 0 . 1 % triton x - 100 , ph 8 . 8 at 25 ° c .) with 3 μl of each exosap - treated rt - pcr product , 5 ′- biotinylated external ( common ) reverse aegis primer ( 0 . 2 μm ), and vent ( exo -) dna polymerase ( 1 unit neb ). without conversion ( an “ extension ” reaction ), dntps ( final 0 . 2 mm each ) were added . for the dz incorporation into the final amplicon (“ conversion ”), nucleoside triphosphates ( datp , dttp , dgtp , and dztp , final concentration 0 . 2 mm of each ) were added . the “ extension ” and “ conversion ” reaction mixtures were incubated in s biorad ( dna engine ) peltier thermal cycler at 95 ° c . for 1 min , followed by 20 cycles ( 94 ° c . for 20 seconds , 55 ° c . for 30 seconds , 72 ° c . for 30 seconds ). a final incubation was run at 72 ° c . for 1 minute . reaction mixtures were then held at 4 ° c . and quenched with 4 mm edta . for standard rt - pcr products , a set of 21 reverse target - specific primers ( 0 . 2 μm each ) was added to the “ extension ” or “ conversion ” reactions . the other reaction components were the same as above . capture probes modified with an amino - c12 linker at the 5 ′- end were coupled to luminex microplex carboxylated micro - spheres (“ beads ”) by a carbodiimide - based procedure according to the manufacturer &# 39 ; s protocol . briefly , for each combination of probe and bead set ( table 5 ), 2 . 5 million luminex beads were resuspended in 0 . 1 m mes buffer ( morpholine ethane sulfonic acid , 50 μl , ph 4 . 5 ) with probe ( 4 μl of 0 . 1 mm stock to give 0 . 4 nanomole final concentration ), and treated twice with 1 - ethyl - 3 -[ 3 - dimethylamino - propyl ]- carbodiimide hydrochloride ( edc , 5 μl of a 10 mg / ml solution , thermo scientific / pierce , rockford , ill .) at room temperature for 30 min , rinsed in tween 20 ( 0 . 02 % aqueous solution ), then rinsed with a sodium dodecylsulfate solution ( 0 . 1 %), and resuspended in tris - edta buffer ( ph 8 . 0 ) to 5 . 0 . this was performed accordingly to the “ no wash ” luminex protocol ( http :// www . luminexcorp . com / support / supportresources /). in a pilot experiment “ wash ” and “ no wash ” luminex protocols were compared ; no differences were found between two protocols when applied to our target - specific probes &# 39 ; design . briefly , aliquots ( 5 μl ) of each extension or conversion reaction were transferred to 96 - well plates ( 96 - well pcr thermo polystyrene plates ; costar ). hybridization buffer ( 25 μl of 2 × tm 0 . 4 m nacl ; 0 . 2 m tris , 0 . 16 triton x - 100 , ph 8 . 0 ) containing 100 of each target - specific probe - coupled microsphere set per μl or totally 2 , 500 target - specific probe - coupled each microsphere types . microspheres were vortexed and sonicated for 20 seconds . the total volume was adjusted to 50 μl by 20 μl of ddh 2 0 . 25 μl of ddh 2 0 were added to each background well ( negative control ). hybridization was performed at 55 ° c . accordingly to the direct hybridization protocol ( dha ) provided by luminex : 95 ° c . for 5 min , cool to 55 ° c . at a speed of 0 . 1 ° c ./ second , 15 mm at the hybridization t 55 ° c . tm buffer ( 25 μl of 1 ×) containing streptavidin - r - phycoerythrin ( 2 μg , pjrs14 , prozyme ) were added to the each hybridization mixture , which was then incubated at 56 ° c . for 5 min . hybridization reactions were carried out in triplicate , and “ no - target ” controls were run in replicates of 6 . beads were analyzed for internal bead color and r - phycoerythrin reporter fluorescence using a luminex 200 analyzer ( luminex xmap technology , luminex corporation ) and the xponent software solutions . the median reporter fluorescence intensity ( mfi ) was computed for each bead type in the sample . the instrument &# 39 ; s gate setting was established before the samples were run , and was maintained throughout the course of the study . twenty - one mosquito - borne arboviruses were selected ( table 1 ) to assemble and develop xmap luminex assays diagnostic panel based on pcr amplification and using the artificial samrs - aegis technology [ yan10 ; yan13 ]. viral simulant rnas were produced in vitro by transcription of the appropriate templates using t7 rna polymerase . in pilot experiments , superscript one - step rt - pcr with platinum taq ( life technology ) was found to be more sensitive and robust than other enzyme combinations tested , and thus able to support the nested pcr amplification with external primers containing the nonstandard p nucleotide , which pairs with the z nucleotide . the target - specific standard or hybrid samrs - aegis forward and reverse primer pairs designed for the panel were tested first by monoplexed one - step rt - pcr with viral rna simulants . each monoplexed rt - pcr produced the expected amplicons , which were visualized by the ethidium - bromide staining following electrophoresis . multiplexed rt - pcr conditions were established in a series of preliminary experiments ( data not shown ). finally , multiplexed nested rt - pcrs were executed with each viral target using 21 pairs of specific samrs - aegis primers and aegis external primers ( table 2 ). pcr amplicons generated under optimized conditions were visualized on ethidium bromide gel as clearly resolved bands of the expected sizes ranging from 59 to 160 base pairs . the pcr negative control showed non - substantial level of primer - dimerization . amplicons containing only standard nucleotides were also produced by multiplexed one - step rt - pcrs and analyzed by agarose - gel electrophoresis . the 21 - fold multiplexed reactions were primed with full set of target - specific primers , each at the final concentration of 0 . 4 the standard pcr amplicons were visualized on the ethidium bromide gel as clearly resolved bands of the expected sizes . 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