Patent Application: US-80326110-A

Abstract:
this disclosure provides novel reversibly terminated ribonucleotides which can be used as a reagent for dna sequencing reactions . methods of sequencing nucleic acids using the disclosed nucleotides are also provided .

Description:
we have previously devised a strategy for performing direct sequencing of single dna molecules ( wo 00 / 53805 ). this method is reliant on the use of nucleotides which function as reversible terminators , most likely enabled by modification of the sugar ring reversible by light or chemical environment ( other configurations may also be possible ). furthermore it ideally requires the use of processive polymerase enzymes as release of the template under study would cause it to be released from the physical location at which the sequencing reaction is occurring . accordingly we have begun studies employing polymerases known to be processive in character , and have identified t7 rna polymerase ( and its relatives in other phages such as t3 and sp6 ) as ideal for our purposes . these polymerases combine high processivity with the possibility of greater flexibility for the use of sugar - modified nucleotides . furthermore as they function on double - stranded dna , naturally unzip and then re - zip the helix behind them , they obviate some of the difficulties encountered in assembling processive dna polymerases , and the fact that such dna polymerases are rarely strand displacing and / or difficulties may occur in the preparation of single - stranded templates . for the purposes of our studies we have employed several different assays for the utilisation of nucleotides by t7 rna polymerase and variants that we have engineered . t7 rna polymerase can incorporate ribonucleotides into a growing chain in at least two different synthetic modes . when the polymerase recognises a bona fide promoter sequence it initiates the synthesis of short runs of rna in a distributive mode , but in a template - dependent manner . the polymerase may slip back and forth releasing short rna fragments , but eventually locks onto the template in a new stable mode known as the elongation mode . additionally t7 rna polymerase has been reported to add a nucleotide to the transcript when it reaches the very end of a template in a non - template directed manner . finally , whilst t7 rnap normally works in the context of a promoter element encoded in the template as described , a model system has been devised by temiakov et al . ( temiakov ), in which the rna polymerase extends the rna chain of a rna - dna hybrid . in this experimental situation the rna oligomer acts as a primer . together with the template and polymerase it forms a structure that has been described as mimicking the elongation mode of transcription , however some of the properties of the polymerase enzyme may be slightly different under these conditions . here we report the incorporation of an ratp analog , 2 ′-( 2 - nitrobenzyl )- atp , into a growing rna chain by complexes assembled according to temiakov et al . this incorporation occurs as a non template - dependant addition of nucleotides to the 3 ′ end of the rna , and we have not observed this activity with elongation mode complexes on duplex dna substrates . we suspect that this activity resembles the so - called ‘ n + 1 effect ’, a phenomenon reported earlier for t7 rna polymerase ( milligan ). furthermore we provide evidence that the 2 ′-( 2 - nitrobenzyl )- atp acts as a terminator for further extension of the rna chain by t7 rna polymerase . we have used uv irradiation to remove the 2 - nitrobenzyl moiety leaving a canonical 2 ′- oh group on the ribose ring of the terminal nucleotide . transcription appears to be subsequently able to resume , thereby reversing the termination effect as desired for use in a sequencing methodology such as that which we have suggested . the observation that nucleotides with a 2 - nitrobenzyl moiety at the 2 ′ position can be incorporated into rna by t7 rna polymerase transcription complexes , even under unusual conditions , suggest a general limited degree of compatibility between these nucleotide analogs and the catalytic site of the enzyme not necessarily similar for other reagents . indeed we have been unable to mimic such as effect with a similar compound in which the 3 ′- ribose position is modified instead . crystal soaking studies of both bst dna polymerase ( johnson et al .) and t7 rna polymerase ( yin and steitz ) reveal the protein / nucleotide motion during catalysis and provide some insight into the possible source of this observation . most particularly they reveal that in addition to interactions favoring pre - catalytic binding of the nucleotide , the catalytic process also involves substantial motion of the protein and substrate during which the nucleotide is ‘ ratcheted ’ up until it is positioned deeper within a protein channel . during this complex motion the substrate nucleotide must move past a number of side chains , and notably the 3 ′- hydroxyl seems to move closely past a variety of residues during transit , and to sit snugly and tightly against the electron - density wall in the post - catalytic position . this suggests that placing bulky groups at the 3 ′ position is likely to generate significant steric hindrance during a variety of stages of catalysis , and that identifying catalytically active ( wild type or mutant ) enzymes that incorporate bulky 3 ′ groups is quite unlikely . conversely , our catalysis data with the 2 ′- modified atp supports the observation that this position is less obviously involved with contacting multiple amino acids in the active site during catalysis , and in particular appears to be positioned ‘ poking out ’ of the free channel after catalysis . this could provide the space for the bulky modification to sit without perturbing structure significantly , but would most likely impede binding and / or catalysis of the next incoming nucleotide , consistent with the observed chain - terminating behavior . clearly the 2 ′- modified nucleotide is not free of inhibitory characteristics , but combining the known biochemical evidence , crystal structures , and our own data paves a way to a line of investigation in which a biochemical or genetic screen of mutants is undertaken to find t7 rnap variants capable of incorporation of 2 ′-( 2 - nitrobenzyl ) ribonucleotides also in the processive mode of template dependant transcription . we can already identify the candidate amino acid residues that would be initially mutated to random variants singly , or in combination . these are lysine 631 , methionine 635 , tyrosine 639 , and phenylalanine 644 , all located on the nucleotide binding face of the 0 helix which shows the largest movement during catalysis ( tyrosine 639 is already implicated in 2 ′- selection in t7 rna polymerase ( brieba and sousa . see also fig4 ). on the ‘ back wall ’ of the binding pocket residues histidine 784 and aspartic acid 787 are obvious candidates for random mutagenesis ( histidine 784 has also already been implicated in 2 ′- selection — brieba and sousa ). additionally we would consider substitutions of glycine 542 ( the equivalent to the ‘ steric gate ’ in dna polymerases inhibiting ribose incorporation , gao et al . ), and further deletions in this region . finally , we could consider debulking the back pocket of the binding site , for example mutating residues 782 , 783 , 785 , 786 to glycine or alanine . any mutants that incorporate 2 ′-( 2 - nitrobenzyl ) ribonucleotides in the processive mode of template dependant transcription would be of great utility for the implementation of the novel single molecule sequencing technologies as proposed by our previous disclosure ( armes / stemple patent application wo 00 / 53805 ). a similar approach may be taken with dna polymerases , providing that some effort is first made to generally permit the access of ribose ands its possible derivatives by removing the steric gate ( this involves mutating the structurally equivalent region to glycine 542 in t7 rna polymerase , for example e710a in the e . coli klenow fragment . astatke et al .). a polymerase with a steric gate deletion may include a deletion of an amino acid sequence comprising any one or more of the residues listed above . similarly , a polymerase with amino acid substitutions in nucleotide binding and catalytic pocket may comprise a an amino acid substitution in any one or more of the residues listed above . the nucleotides of the invention may be used for sequencing methods which involves single nucleic acid templates , such as those described in pending pct application wo 00 / 53805 . in addition , the nucleotides of the invention may be used for sequencing methods which involves multiple nucleic acid templates , such as , for example , pyrophosphate based sequencing method . in addition , the nucleotides of the invention may be substituted in any reaction where a terminating nucleotide is employed . for example , the nucleotides of the invention may be substitute for ddntps in sequencing by the sanger method . the nucleic acid scaffolds were formed by incubating 1 nmol rna - 1 ( 5 ′- biotin - aacugcggcgau - 3 ′ ( seq id no : 1 )) and 1 nmol dna - 1 ( 5 ′- gggtcctgtctgaaatacctatcgccgc - 3 ′ ( seq id no : 2 )) in 100 μl transcription buffer ( 200 mm tris - hcl ( ph 7 . 9 at 25 ° c . ), 30 mm mgcl 2 , 50 mm dtt , 50 mm nacl , 10 mm spermidine ) for 10 minutes at 70 ° c . annealing occurred by slowly lowering the temperature to 25 ° c . the concentration of hybrid was assumed to be 10 μm . for the primer extension reactions 2 pmoles of scaffold were mixed with 10 units of t7 rna polymerase ( fermentas ) in 9 μl transcription buffer to allow the transcription complex to form . reactions were started by the addition of 1 μl of 1 mm nucleotide solution and incubated for 20 minutes at 37 ° c . reactions were stopped by addition of 2 μl 500 mm edta and cooled on ice . uv irradiation was done for 10 minutes on a uv lamp . samples were precipitated , dissolved in formamide loading buffer and resolved on a denaturing 16 % polyacrylamide gel ( 8m urea ). gels were electroblotted onto nylon - membrane ( osmonics , usa ) in blotting buffer ( 0 . 5 × tbe ). blots were incubated in blocking buffer ( 1 × tbstw / 1 % blocking reagent ( roche )) for 1 hour at room temperature to avoid non - specific noise , incubated with 0 . 5 μg / ml streptavidin - hrp ( sigma ) in blocking buffer for 1 hour and washed extensively in tris buffered saline containing 0 . 1 % tween - 20 . detection was performed using a chemiluminescent substrate according to the instructions of the manufacturer ( pierce ). for the reversible termination experiment 5 pmoles of scaffold were mixed with 6 . 5 pmoles of t7 rna polymerase ( possessing an n - terminal histidine tag used in purification ) in 180 μl transcription buffer to form active transcription complexes . the reaction was started by the addition of 20 μl of 10 mm 2 ′-( 2 - nitrobenzyl )- atp and incubated for 2 hours at 37 ° c . subsequently the reaction was split into 4 samples and subjected to deprotection : two samples were exposed to uv irradiation as described above , two untreated samples were incubated for the same period of time at ambient temperature . deprotection was followed by addition of 1 volume of either extension mix containing 250 μm rntps in transcription buffer , or addition of transcription buffer only . samples were then incubated for another 1 hour at 37 ° c . processing , separation and detection of products was done as described above . use of an rna oligomer of an rna - dna hybrid acts as primer in the scaffold assay temiakov et al . ( temiakov ) have shown that a short rna oligomer can serve as a primer for t7 rna polymerase catalyzed transcription when hybridised to single - stranded dna acting as a template . the polymerase can then be ‘ walked ’ along the coding template by the stepwise addition of cognate nucleotides . we confirmed these results , but observe that under the conditions used here additional nucleotides are incorporated in a non template dependent manner . an rna oligomer and a single - stranded dna template , such as those shown in fig2 a , can form a nucleic acid scaffold that interacts with t7 rnap in a transcriptional complex . when presented with a cognate nucleotide ( here ratp ) the rna primer is extended by one or two bases ( fig2 b , lane 3 ). a scaffold will be extended by 3 or more bases ( 1b lane 4 ) when offered the next two nucleotides encoded by the dna ( here ratp and rgtp ). if , on the other hand , the sole incoming nucleotide is non - cognate ( here rctp ), the extension is limited to one base only ( 1b lane 5 ). the incorporation of one or more nucleotides in addition to the encoded base is evidently not a template dependant event . this effect is reminiscent of the so - called n + 1 effect ( milligan ). the formation of a stable transcription complex by a rna / dna scaffold and t7 rna polymerase offers a simple system for testing the potential of nucleotide analogs to be incorporated into a growing rna chain . 2 ′-( 2 nitrobenzyl - atp is incorporated into an rna chain by t7 rna polymerase in this study we used 2 ′-( 2 - nitrobenzyl )- atp as a substrate for t7 rna polymerase . the 2 - nitrobenzyl modification increases the molecular weight of the nucleotide analog and any nucleic acid into which the latter is incorporated . consequently the electrophoretic mobility of an rna containing a nb - moiety is altered correspondingly , allowing modified transcripts to be identified . due to the nature of its preparation , the 2 ′-( 2 - nitrobenzyl )- atp used in this experiment is contaminated with ratp by an estimated 5 % ( see materials and methods ). as shown in fig2 b , lane 6 , the mobility of the main product of a reaction containing predominantly 2 ′-( 2 - nitrobenzyl )- atp appears to migrate at a position between 10 nucleotides and 11 nucleotides of unmodified rna ( compare to lanes 3 and 4 ). this product of intermediate molecular weight suggests the incorporation of at least one 2 - nitrobenzyl - modified nucleotide . how many 2 ′-( 2 - nitrobenzyl )- atp nucleotides per molecule have been incorporated ? a faint band at the 9 nucleotide position ( compare lane 6 and 7 ) signifies that contaminating unmodified ratp has also been utilized ( note that the concentration of ratp will be around 5 μm ; even at this low concentration it can be readily used by the t7 rnap ( song )). the absence of any product of 10 nucleotides in length rules out the possibility that any unmodified ratp has been incorporated in an n + 1 type activity ( as was the case at high ratp concentrations ; see lane 6 ). the main product could be the result of the incorporation of a single 2 ′-( 2 - nitrobenzyl )- atp , of two 2 ′-( 2 - nitrobenzyl )- atp ( the second of which would be non - template dependant , i . e . n + 1 type ), or of one 2 ′-( 2 - nitrobenzyl )- atp added to a canonically incorporated ratp . further evidence for the identity of the main product of transcription in the presence of high concentrations of 2 ′-( 2 - nitrobenzyl )- atp comes from a deprotection experiment . the 2 - nitrobenzyl moiety is a photolabile group used in many application for its ability to absorb light energy and cleave the covalent bond between itself and a ‘ protected ’ molecule ( givens ). in the case of 2 ′-( 2 - nitrobenzyl )- modified nucleotides , cleavage of this bond generates a free 2 ′- oh group on the ribose ring . the product of this photo - deprotection should consequently run at the same position as an rna oligomer extended by the incorporation of unmodified ratp . as shown in fig2 b , lane 7 , uv treatment of the main product of 2 ′-( 2 - nitrobenzyl )- atp incorporation leads to an increase of its electrophoretic mobility . the main band now runs at the same position as an rna of 10 nt in length ( compare lane 3 and 7 ). this observation confirms the notion of 2 ′-( 2 - nitrobenzyl )- atp incorporation into the nascent rna chain by t7 rnap ( unmodified rna oligomers do not alter their electrophoretic mobility in response to uv treatment ; data not shown ). it also allows us to rule out one of the above - mentioned alternatives for the composition of the main product of 2 ′-( 2 - nitrobenzyl )- atp incorporation : the incorporation of a single 2 ′-( 2 - nitrobenzyl )- atp should lead to a loss of any band greater than 9 nucleotides and an increase in intensity of the 9 nucleotide band as a response to uv treatment . since neither is the case , this possibility can be discarded . does the main product contain one 2 ′-( 2 - nitrobenzyl )- atp incorporated in the n + 1 mode of t7 rnap activity ( succeeding a cognate ramp ), or two 2 ′-( 2 - nitrobenzyl )- atps ? although the experimental results presented so far cannot formally distinguish between these two possibilities , the latter alternative requires two assumptions to be made which render it much less likely . firstly , the incorporation of 2 ′-( 2 - nitrobenzyl )- atp would have to be far more efficient than that of ratp , i . e . the modified nucleotide would have to be the better substrate . offering 2 ′-( 2 - nitrobenzyl )- atp does not result in the formation of any oligomers with only a single incorporated modified atp ( which would have to run at a position between 9 and 10 nucleotides in length ; 1b , lane 6 ) suggesting that any incorporation of one 2 ′-( 2 - nitrobenzyl )- atp is necessarily followed by the addition of a second ( n + 1 type activity ) modified nucleotide . when , on the other hand , the transcription complex is presented with unmodified ratp only , the product of the reaction is a mixture of rna oligomers extended by one or two nucleotides ( fig2 b , lane 4 ). secondly , the 3 ′- oh group of a terminal 2 ′-( 2 - nitrobenzyl )- atp in a nascent rna chain would still have to be a target for 17 rna polymerase dependent extension , i . e . 2 ′-( 2 - nitrobenzyl )- atp would have to be non - terminator . the latter requirement has been addressed in an additional experiment . a potential application for the incorporation of nb - modified nucleotides by a polymerase is their use as reversible terminators in a novel sequencing technology ( wo 00 / 53805 ). we therefore examined the ability of 2 ′-( 2 - nitrobenzyl )- atp to act as a terminator of transcription . in the experiment shown in fig3 scaffolds where incubated with t7 rnap and 2 ′-( 2 - nitrobenzyl )- atp . as in the experiment described earlier the product of such a reaction has an apparent electrophoretic mobility that lies between those of oligomers of 10 and 11 nucleotides ( fig3 , lane 1 ). when subjected to uv irradiation , the nb moiety incorporated into the rna is removed and the mobility of the product increases to that of a 10 nucleotide oligomer ( fig3 , lane 2 ). transcription can resume upon the addition of unmodified rntps . when the 2 - nitrobenzyl moiety of the terminal base nucleotide has been removed by photodeprotection , this leads to a decrease in signal intensity of the main band , presumably because the product is being extended ( fig3 , lane 4 ). in contrast , rna chains still bearing the nitrobenzyl group can not be extended by the transcription complex , and the amount of product remains effectively unchanged ( fig3 , lane 3 ). we note that complete utilisation of the deprotected material has not occurred . this may be the case for a variety of reasons including the fact that the polymerase will necessarily have to extend from a mismatch in these ‘ chase ’ experiments . nevertheless the data is best explained we believe by the suggestion that the 2 ′-( 2 - nitrobenzyl )- atp has functioned as a terminator , which can be reversed on deprotection . in conclusion , we have shown that 2 ′-( 2 - nitrobenzyl )- atp acts as a bona fide terminator of transcription . elongation of the transcript can resume upon removal of the protecting 2 - nitrobenzyl group by uv irradiation . this quality of the 2 ′-( 2 - nitrobenzyl )- atp also rules out the possibility mentioned above that the main product of transcription in presence of high concentrations of 2 ′-( 2 - nitrobenzyl )- atp could be an rna primer extended by two rather than one modified nucleotide . all patents , patent applications , and references cited in this specification is hereby incorporated by reference . astatke m , ng k , grindley n d f , joyce c m ; proc natl acad sci u . s . a . 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