Patent Application: US-201515127617-A

Abstract:
disclosed are t7 rna polymerase variants with enhanced transcriptional activity . t7 rna polymerase variants are known which have the ability to incorporate modified ribonucleotides into growing rna molecules . however , these variants have relatively low levels of transcriptional activity . presented herein are mutations that increase the transcriptional activity of the variants with broad substrate range .

Description:
various features and advantageous details are explained more fully with reference to the non - limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description . it should be understood , however , that the detailed description and the specific examples , while indicating embodiments of the invention , are given by way of illustration only , and not by way of limitation . various substitutions , modifications , additions , and / or rearrangements will become apparent to those of ordinary skill in the art from this disclosure . in the following description , numerous specific details are provided to provide a thorough understanding of the disclosed embodiments . one of ordinary skill in the relevant art will recognize , however , that the invention may be practiced without one or more of the specific details , or with other methods , components , materials , and so forth . in other instances , well - known structures , materials , or operations are not shown or described in detail to avoid obscuring aspects of the invention . the wild type t7 rna polymerase has the following sequence ( seq id no : 1 ): the present invention will be described in greater detail by way of specific examples . the following examples are offered for illustrative purposes only , and are not intended to limit the invention in any manner . those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results . the t7 rna polymerase orf was cloned into pqe - 80l ( qiagen ). all t7 rna polymerase variants were derived from this plasmid either by mega - primer pcr ( bryksin & amp ; matsumura , 2010 ) or isothermal assembly ( gibson , 2011 ). plasmids were transformed into bl21 - gold ( agilent ) e . coli cells . cells were grown in 2 × yt media at 37 ° c . overnight . subcultures were grown at 37 ° c . until reaching od600 ˜ 0 . 7 - 0 . 8 at which point 1 mm iptg was added . cells were grown four hours at 37 ° c ., pelleted , and frozen at − 80 ′ c . pellets were resuspended in binding buffer ( 50 mm tris - hcl , ph8 . 0 , 0 . 5 m nacl , 5 mm imidazole ). resuspended cells were lysed via sonication on ice using 50 % probe amplitude for 3 minutes ( 1s on , 1s off ). cell debris was pelleted by centrifugation ( 30 min : 10 , 000 g ). his - tagged t7 rna polymerase was purified by immobilized metal affinity chromatography ( imac ). the lysate was run over 1 ml ( bead volume ) ni - nta ( fisher ) gravity column pre - equilibrated with binding buffer . the column was washed with 10 column volumes of wash buffer ( 50 mm tris - hcl , ph 8 . 0 , 0 . 5 m nacl , 20 mm imidazole ). t7 rna polymerase was eluted off the column by the addition of 3 column volumes of elution buffer ( 50 mm tris - hcl , ph 8 . 0 , 0 . 5 m nacl , 250 mm imidazole ). dialysis was performed in final storage buffer ( 50 mm tris - hcl , ph 8 . 0 , 100 mm nacl , 1 mm ddt , 1 mm edta ). dialates were adjusted to 1 mg / ml and added to an equal volume of glycerol ( final concentration 0 . 5 mg / ml ). real - time transcription reactions ( fig1 , fig3 a - 3b ) contained 40 mm tris - hcl ph 8 . 0 , 30 mm mgcl2 , 6 mm spermadine , 6 mm each ntp ( or modified ntp ), 10 mm dtt , 500 mm t7 rna polymerase , 500 mm dna template , and 0 . 17 mg / ml dfhbi ( in dmso ). reactions were incubated for up to 4 hours at 37 ° c . with spinach fluorescence ( excitation / emission 469 / 501 ) reading taken one to four minutes in a safire monochromator ( tecan ). spinach templates were made by thermal cycling overlapping primers ( 5 ′- aatataatacgactcactatagaggagactgaaatggtgaaggacgggtccagt gcttcg ( seq id no : 2 ) and 5 ′- gaaaagactagttacggagctcacactctactcaacagtgccgaagcactggac ccg ( seq id no : 3 )) with accuprime pfx in its standard buffer ( 94 ° c . : 2 min , 12 cycles [ 94 ° c . : 15 s , 50 ° c . : 30 s , 68 ° c . : 30 s ], 68 ° c . : 1 min ). templates were purified by qiaquick gel extraction kit ( qiagen ). end point transcription reactions contained 40 mm tris - hcl ph 8 . 0 , 30 mm mgcl2 , 6 mm spermadine , 6 mm each ntp ( or modified ntp ), 10 mm dtt , 500 mm t7 rna polymerase , 500 mm dna template . reactions were incubated for up to 4 or 20 hours at 37 ° c . dna templates were made as above . rvmu reactions ( fig3 c , fig4 a , and fig7 ) and rrmy reaction ( fig4 b and fig8 ) were run four hours , labelled by inclusion of 0 . 17 μm ( α32p ) atp ( 3000 ci / mmol ,) and analyzed by denaturing page . rgmh reactions ( fig4 c and fig9 ) were run twenty hours , labelled by inclusion of 0 . 17 μm ( α32p ) gtp ( 3000 ci / mmol ,) and analyzed by denaturing page . rgvg - m6 reactions ( fig5 ) were run twenty hours , incubated for 1 hour 37 ° c . with 0 . 03 u / ul baseline - zero dnase in its supplied buffer , analyzed by denaturing page and imaged after staining in sybr - gold . the buffer comparison ( fig1 ) used the buffers listed in the figure , was run twenty hours , incubated for 1 hour 37 ° c . with 0 . 03 u / ul baseline - zero dnase in its supplied buffer , analyzed by denaturing page and imaged after staining in sybr - gold . mn in the permissive buffer ( fig6 ) contained 200 mm hepes ph 7 . 5 , 5 . 5 mm mgcl2 , 2 mm spermidine , 0 . 5 mm each 2 ′- o - methyl - ntp , 40 mm dtt , 0 . 01 % triton , 10 % peg8000 , 1 . 5 mm mncl2 , 10 u / ml yipp , 200 nm rna polymerase , and 200 nm dna . reactions were run twenty hours , incubated for 1 hour 37 ° c . with 0 . 03 u / ul baseline - zero dnase in its supplied buffer , analyzed by denaturing page and imaged after staining in sybr - gold . 32p gels were exposed to a storage phosphor screen ( molecular dynamics ) before imaging on a storm 840 phospoimager ( ge healthcare ). autoradiographs were analyzed using imagequant ( ge healthcare ). the relative thermal stability of each t7 rna polymerase was assessed by incubating 0 . 5 mg / ml enzyme in pbs buffer with texasred dye ( invitrogen ). enzyme / dye mixtures were equilibrated at 37 ° c . for 10 minutes and heated at a rate of 0 . 07 ° c ./ s to 97 ° c . using a lightcycler 96 thermocycler , while fluorescence was monitored ( excitation 577 nm / emission 620 nm ). the first derivatives of the change in fluorescence as a function of time were used to approximate the relative t m . data were analysed using roche thermocycler software . stabilizing mutations increase the activity of the t7 rna polymerase mutant g542v h784s previous experiments selecting for rna polymerases with altered substrate specificity ( chelliserrykattil & amp ; ellington , 2004 ) focused on the four amino acids that are proximal to the incoming nucleotide ( cheetham , 1999 ; temiakov , et al ., 2004 ), and thus likely played a role in substrate recognition . one of the resulting mutants , called “ vrs ,” could incorporate 2 ′ f - modified pyrimidines . vrs had mutations at two of the randomized residues ( ie g542v and h784s ). interestingly , an h772r mutation also arose during the selection , despite h772 not being randomized . h772r is not near the substrate recognition domain , but has been seen in other selections for t7 rna polymerase activity ( ellefson , et al ., 2013 ; dickinson , et al ., 2013 ). to test whether h772r is a general stabilizing mutation , a derivative of vrs without h772r , termed “ vs ,” was constructed . purified enzymes were tested for their ability to polymerase rna composed either of natural ntps ( rn ) or of ribo - purines and 2 ′- f - pyrimidines ( rrfy ; fig1 ). real - time polymerase activity was assayed using the fluorescent aptamer spinach in the presence of dfhbi ( van nies et al ., 2013 ). spinach will bind dfhbi and fluoresce irrespective of whether it is transcribed as a purely ribo - aptamer or when substituted with 2 ′- f - pyrimidines , although the 2 ′- f - pyrimidine version is only about 30 % as fluorescent as the purely ribonucleotide version . 2 ′- o - methyl substituted spinach is not detectably fluorescent . notably , vs showed a decrease in activity for each substrate composition . this suggests that h772r contributes to the overall activity of vrs , apart from any substrate preference considerations . several more derivatives of vrs with additional mutations were created and tested for their ability to increase the activity of vrs . the so - called “ m5 ” ( s430p , n433t , s633p , f849i , and f880y ; ( u . s . pat . no . 7 , 507 , 567 ) and “ m6 ” ( m5 with the additional p266l mutation , associated with promoter clearance ( guillerez , et al ., 2005 ) sets of mutations increased activity of the vrs mutant , both for rn and rrfy incorporation . the “ m5 ” mutations increase the activity of several t7 rna polymerase substrate specificity mutants the “ m5 ” mutations arose in a t7 rna polymerase selection for transcriptional activity at higher temperatures . in a wild type background , these mutations increase the half - life of enzyme at 50 ′ c and allow for transcription at that temperature . the m5 protein was crystalized , and few gross morphological differences to the wild - type t7 rna polymerase crystal ( cheetham , 1999 ) are apparent ( fig2 a ). there is , however , an added hydrogen bond made by f880y , which may stabilize the two halves of the palm domain ( fig2 b ). it should be noted that the f880y mutation is not sufficient to increase vrs activity ( see vrsy in fig1 c - 1d ). it was then tested whether the m5 and m6 mutations could increase the activity of other t7 rna polymerase mutants . several known polymerases with altered ribose specificity namely wt , y639f , fa , rgvg , vrs , and r425c ( table 1 ) were tested . to each of these specificity mutants was added a set of stability mutations , namely “ l ” ( p266l ), m5 , and m6 . also included was a recently described mutant , 2p16 , which is likely a stabilized version of rgvg . these 25 polymerases were purified and assayed for transcriptional activity in vitro ( fig3 ). whether transcribing natural ribotides ( rn ; fig3 a ), 2 ′- f - pyrimidines ( rrfy ; fig3 b ), or 2 ′- o - methyluridine ( rvmu ; fig3 c ) the m5 and m6 mutations increased activity of the mutants fa , rgvg , and vrs . wt and y639f activity on rn was slightly increased by the m5 mutations , but this trend did not hold up with rrfy or rvmu incorporation . it is evident that the 2p16 is indeed more active than rgvg ( as previously reported ( siegmund , et al ., 2012 )) but is not as active as either rgvg m5 or rgvg m6 . no transcription was detected from the r425c family of polymerases . a subset of the most active polymerases were assayed for the ability to incorporate 2 ′- o - methyluridine ( rvmu ), 2 ′- o - methylpyrimidines ( rrmy ), and 2 ′- o - methyladenosine and 2 ′- o - methylpyrimidines ( rgmh ) ( fig4 , fig7 - 9 ). as was case for rn and rrfy above , the m5 mutations enhanced the activity of the fa and rgvg enzymes for each set of substrates . rgvg - m6 was the most active enzyme in all conditions , yielding at least 25 - fold more rna than the fa mutant , which is the most commonly used enzyme for generating 2 ′- o - methyl rna . thermal - melt assays confirmed that , for all t7 rna polymerase variants tested , addition of the m5 mutations increased their thermal stability ( fig1 ). the weakly active rgvg mutant has a t m almost 5 ° c . lower than that of wt t7 rna polymerase , but this loss of stability and rgvg &# 39 ; s activity are rescued by the m5 mutations . contrary to expectations , however , the similarly weak vrs and fa mutants do not have low melting temperatures , and the h772r mutation did not have the expected effect on t m . it seems that the increase in activity due to the addition of these mutations cannot be solely attributed to an increase of stability . t7 rna polymerase r6 is effective for high - yield transcription of fully modified rna after demonstrating that rgvg - m6 could catalyse the formation of rna containing three 2 ′- o - methylnucleotides , its ability to generate fully - modified rna was assayed . rgvg - m6 was able to polymerase using a combination 2 ′- f - purines and 2 ′- o - methylpyrimidines ( frmy ) as well as a combination of 2 ′- f - guanosine , 2 ′- o - methyladenosine , and 2 ′- o - methylpyrimidines ( fgmh ) ( fig5 ). fully 2 ′- o - methyl rna ( mn ) was not obtained . previous reports of mn incorporation have used more permissive buffer compositions , including manganese as well as rgmp and / or rgtp . rgvg - m6 &# 39 ; s ability to synthesize mn rna in several such permissive buffers was tested ( fig1 ) and it was determined that effective mn polymerization was achieved in buffers that included rgmp or rgtp . a panel of enzymes for mn polymerization in this permissive buffer ( 200 mm hepes ph 7 . 5 , 5 . 5 mm mgcl 2 , 2 mm spermidine , 0 . 5 mm each 2 ′- o - methyl - ntp , 40 mm dtt , 0 . 01 % triton , 10 % peg8000 , 1 . 5 mm mncl 2 , 10 u / ml yeast inorganic pyrophosphatase , 200 nm rna polymerase , and 200 nm dna ) was tested . fa - m5 and fa - m6 show an increase in activity relative to the parental fa mutant . rgvg - m5 , rgvg - m6 , and 2p16 showed a marked improvement over the parental rgvg . in addition , rgvg - m5 and rgvg - m6 generate substantially more rna in this buffer than the y639l h784a mutant ( u . s . pat . no . 8 , 105 , 813 ). although certain embodiments have been described above with a certain degree of particularity , or with reference to one or more individual embodiments , those skilled in the art could make numerous alterations to the disclosed embodiments without departing from the scope of this invention . further , where appropriate , aspects of any of the examples described above may be combined with aspects of any of the other examples described to form further examples having comparable or different properties and addressing the same or different problems . similarly , it will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments . the claims are not to be interpreted as including means - plus - or step - plus - function limitations , unless such a limitation is explicitly recited in a given claim using the phrase ( s ) “ means for ” or “ step for ,” respectively . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . u . s . pat . no . 7 , 507 , 567 u . s . pat . no . 8 , 105 , 813 beaudry , et al ., chem biol . 7 : 323 - 34 , 2000 . brieba & amp ; 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