De Novo DNA Synthesis Using Polymerase

Home , De novo synthesis, DNA synthesis

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. plctos uh s N nanotechnology DNA as such applications (NGS). ing and colleagues over 35 years ago years 35 over colleagues and phosphoramidite nucleoside pioneered method by Caruthers Marvin in practice in nt, 200–300 about now is synthesis oligo limit phosphoramidite-based of upper the handling, liquid in improvements and fine-tuning Center Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. Quantitative Quantitative Biosciences, UC Berkeley, Berkeley, California, USA. Rathin Rathin Bector data archiving data Received Received 3 July 2017; accepted 22 May 2018; published online 18 June 2018; addressed to D.H.A. ( 3 1 even and genes synthetic oligonucleotides as chromosomes entire such including constructs bioen longer DNA, and and (oligos) synthetic research requires biological of gineering majority overwhelming The basis of an enzymatic synthesizer.oligonucleotide to write a defined sequence. This approach may form the nucleotide in 10–20 s, and that the scheme can be iterated conjugates can extend quantitatively a primer by a single subsequent extension. We demonstrate that TdT–dNTP the incorporated nucleotide releases the primer and allows TdT–dNTP molecules. Cleaving the linkage between TdT and remains covalently bound to TdT and is inaccessible to other of incorporation the tethered dNTP, the 3 (dNTP) molecule that it can incorporate into a primer. After conjugated to a single triphosphate deoxyribonucleoside transferase (TdT). deoxynucleotidyl Each TdT molecule is that uses the polymerase terminal template-independent waste. Here, we describe an synthesis oligonucleotide strategy to the direct synthesis of ~200 mers and produces hazardous nucleoside method, phosphoramidite even though it is limited are Oligonucleotides almost exclusively synthesized using the Palluk Sebastian De novo nature biotechnology nature matically reduced the cost of high-throughput and genome-wide genome-wide and screens functional high-throughput of cost the reduced matically Technologies, Sandia National Livermore, Laboratories, California, USA. Biomolecular Biomolecular Engineering, UC Berkeley, Berkeley, California, USA. inflection point in biological research biological in point inflection Nathan J Hillson nucleotide nucleotide conjugates Department Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany. Joint BioEnergy Institute, Emeryville, California, USA. Today, essentially all synthetic DNA is manufactured using the the using manufactured is DNA synthetic all essentially Today, 9 . As a result, longer molecules must be assembled from from assembled be must molecules longer result, a As . De novo De 6 . 1 [email protected] , 4 2 and target capture for next-generation sequenc next-generation for capture target and , 1 DNA synthesis using polymerase- 1 7 1 DNA synthesis also enables other emerging emerging other enables also synthesis DNA – , , , Hratch M Baghdassarian , 2 2 3 . Massively parallel oligo synthesis oligo parallel Massively . , , 10 12

, , Daniel H Arlow advance online publication online advance

& Jay D Keasling 7 — a development that marked an an marked that development a — ) ) or J.D.K. ( 8 . However, after decades of of decades after However, . [email protected] ′ end of the primer 2 Biological Biological Systems and Engineering Division, Lawrence Berkeley National Lab, Berkeley, California, USA. 1 5 , and DNA-based DNA-based and 2 1 , 4 , 2 , 5 , 5 , 1 12 , , 6 7 2 8 Department Department of Chemistry, UC Berkeley, Berkeley, California, USA. , , , Tristan de Rond , Department Department of UC Bioengineering Berkeley, Berkeley, California, USA. 8 8 , 3 , , Alisa N Truong 11 has dra has

DOE DOE Joint Genome Institute, Walnut Creek, California, USA. ).

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Biophysics Biophysics Graduate Group, UC Berkeley, Berkeley, California, USA. o 12 i : 1 yet sufficiently terminate further elongation. further terminate sufficiently yet exncetd tihshts dTs t te 3 the stranded DNA to (dNTPs) triphosphates deoxynucleotide add indiscriminately to is activity predominant whose polymerase alone. chemistry organic using possible not are that ways in system be the optimize to can employed selections and screens high-throughput as such techniques engineering enzyme 4) and bases); the of positions nucleophilic the on groups protecting DNADNA without (i.e., natural from initiated be could synthesis 3) waste; hazardous generate not need and tions condi aqueous in place take reactions 2) oligos; longer of synthesis direct the enabling thereby depurination, as such DNAdamage and products side of formation the reduce may function they which in of and conditions mild enzymes 1) the specificity exquisite synthesis: chemical over advantages potential several promises synthesis oligo the the base 3 using to develop 3 develop to difficult be may it that suggesting approach, this on based synthesis a defined sequence date to back at (refs. 1962 least sequence a defined can be rationalized in light of the co-crystal structure of TdT with with TdT of dCTP structure co-crystal the of light in rationalized be can by synthesis by dNTPs (RTdNTPs)terminator’ to sequencing in analogous a scheme ‘reversible using synthesis DNA stepwise TdTfor employ pro to TdT.Detailed posals on based method synthesis oligo practical a of oligo However,synthesis. thus far there have no been demonstrations sequences in oligos a that and is process not failure-prone to amenable target all elongation 3 the on groups removable RTdNTPswith approach: this to obstacle These These authors contributed equally to this work. should Correspondence be 0 ′ OH that, once incorporated into a primer, directly block further further block directly primer, a into incorporated once that, OH . Terminal deoxynucleotidyl transferase (TdT) is the only known known only the is (TdT) transferase deoxynucleotidyl Terminal Proposals for Proposals enzymatic 1 0 3 2 8 1 7 ′ 1 / , ( 1 O-unblocked RTdNTPs with inhibitory groups attached to to attached groups inhibitory RTdNTPswith O-unblocked n 2 , 7 b 2 , Supplementary Fig. 1 Fig. Supplementary , , but thus far there are no reports of successful 6 1 t , , Peter W Kim 22– . , Sebastian , Barthel Sebastian 0 4 , rendering some DNA sequences inaccessible to study.to inaccessible DNA sequences some rendering , 1 ′ 1 O-unblocked RTdNTPs that are rapidly incorporated, incorporated, rapidly are RTdNTPs that O-unblocked 4 2 7 date back to 1986 (refs. (refs. 1986 to back date 6 1 3 are not incorporated efficiently by TdT 3 , , making it the natural candidate for use in enzymatic de de novo 1 , 9 , , Anup K Singh ). An alternative scheme proposes proposes scheme alternative An ). 1 synthesis of oligonucleotides with of synthesis oligonucleotides – 3

, , Justine S Kang 15– 7 Department Department of Chemical and 11 2 Novo Novo Nordisk Foundation 1 9 ). There is at least one one least at is There ). CBRN CBRN Defense and Energy 1 11 , 9 ′ s r e t t e l , n o single- of end , 1

20 2 5 de de novo , ). Enzymatic ). Enzymatic 2 Institute Institute for 1 6 , , a fact that , 2 , 7 , DNA - - © 2018 Nature America, Inc., part of Springer Nature. All rights reserved. structures of two types of TdT–linker-dNTP conjugates used in this study, based on the amine-to-thiol crosslinkers PEG crosslinkers amine-to-thiol the on study, based this in used conjugates TdT–linker-dNTP of types two of structures ( sequence. a defined by a primer extend to iterated be can cycle The extension. subsequent for primer the releasing thereby peptidase), DTT, light, nm (e.g., 365 reagent cleavage the of addition by cleaved is TdT and nucleotide incorporated the between linkage the and removed) (or inactivated are conjugates TdT–dNTP remaining the step, deprotection the In molecules. TdT–dNTP other by extensions further prevents and attached 3 the into nucleotide tethered the of incorporation Upon conjugate. TdT–dNTP of excess an to exposed is primer a DNA step, extension the In linker. a cleavable via a dNTP with labeled site-specifically molecule a TdT of consisting conjugates with a black dotted line. dotted a black with indicated is bond cleavable a scar. as The to referred are and nucleobase the to attached remain red in indicated atoms the linker, the of cleavage Upon respectively. (pa-dUTP), dUTP 5-propargylamino and (aa-dUTP) dUTP 5-aminoallyl analogs dTTP the and “TdT–dTTP”) (lower, BP-23354 and dTTP”) nucleoside triphosphate ( triphosphate nucleoside a tethered with labeled is site-specifically molecule polymerase each 1 Figure s r e t t e l crosslinker amine-to-thiol PEG disulfide-forming the using residues 4I2 ID: PDB structure crystal the on (based residues cysteine accessible surface- five TdT, has murine which of domain polymerase the fied a write sequence. to defined iterated be can deprotection and extension of cycle tion reac two-step simple This extension. subsequent for 3 primer the the of deprotect to cleaved be then can linker The conjugates. to 3 attached the covalently remains it primer, a into dNTP tethered its serine (TdTserine ID: PDB structure on crystal (based the residues cysteine surface-accessible all which in TdT mutant a purified and expressed Wefirst sequences. defined of thesis primer by precisely one nucleotide, the key property that enables syn surface. its on points some at least to ered teth nucleotides TdT that incorporate can concluded we result, this on Based nucleotides. four to up by extended been had that primers releasing complexes, the TdT, dissociated and nucleotides tethered ( mercaptoethanol incorporated into the primer ( SDS-PAGE,by indicating that detectable one or more complexes tethered nucleotides had fluorescent been formed conjugates nucleotide 5 a to tions 180, 188, 253, and 302; 302; and 253, 188, 180, tions (posi site catalytic the near enzyme the of surface the on positions at various residues cysteine single Weactivity. ase reintroduced then

a We conceived of an approach for reversible termination wherein wherein termination reversible for approach an of Weconceived To test the feasibility of this approach, we first expressed Toand puri of we expressed approach, first this test feasibility the Next, we attempted to generate conjugates that would extend a extend would that conjugates generate to attempted we Next, 7 ), and tethered the dTTP analog 5-aminoallyl-dUTP to those those to 5-aminoallyl-dUTP analog dTTP the tethered and ), 4 ′ Cleavage reagent -SPDP ( -SPDP end, blocking further elongation by other polymerase-dNTP polymerase-dNTP other by elongation further blocking end, ′ TdT fluorescein (FAM)-labeled DNA primer, these polymerase- these primer, DNA (FAM)-labeled fluorescein (e.g., DTT/βME,

TdT–dNTP conjugates for reversible termination of primer elongation. ( elongation. primer of termination reversible for conjugates TdT–dNTP 365-405 nm, peptidase) ∆ 5cys) and that 5cys) this confirmed mutant polymer retained Fig. 1 Fig. β b ME), which cleaves the linkage between the the between linkage the cleaves which ME), 2. Deprotection and and Fig. 1 Fig. Supplementary Fig. 2 Fig. Supplementary 5′ Supplementary Fig. 4 Fig. Supplementary Supplementary Supplementary Fig. 3a 5′ 5′ TdT-DNA complex DNA primer a 4I2 ). When a polymerase incorporates incorporates a polymerase When ). 7 TdT +1 nt ) were mutated into alanine or or alanine into mutated were ) OH 3′ 3′ OH 3′ OH cycle To next 1. Extension ). When exposed exposed When ). ) for site-specific site-specific ) for ). ). Addition of Tethered dNTP TdT-dNTP PP i TdT 5′ ′ end end OH 3′ β------

based on a photocleavable amine-to-thiol crosslinker amine-to-thiol photocleavable a on based conjugates other developed we sequences, defined of synthesis thus and extension primer of To primer. iteration the of enable extension on the upon nucleobase cleavage that could interfere with subsequent to used be can nucleotide. single by a that DNAa primer extend conjugates polymerase–nucleotide TdT in of surface results the to nucleotide single a tethering that concluded control reaction wherein TdT–dCTP was irradiated before addition addition before irradiated TdT–dCTP was wherein reaction control a in formation primer–TdT complex observe not did we contrast, In by two nucleotides. that was extended a primer nm releasing irradiation, 365 by dissociated was which complex, primer–TdT a formed When we this extension product exposed to fresh TdT–dCTP, it again protein complex nor extended primer ( DNA-primer of extended nor amount complex protein substantial a form not did conditions assay and 3b Fig. a primer extended by predominantly one nucleotide ( by dissociated be then could that complex covalent a formed enzymes mutant four of the of each DNA primer, conjugates exposure of a 5 on impact stantial TdT ( activity purification and expression ( easier enable to protein binding tose mal to fused 302, position at TdTcysteine surface the single a with mutant using conjugates prepared and 5-propargylamino-dCTP ( cleavage upon 5a Fig. scar Supplementary minimal a leaves which length, tethering of aminoallyl-dUTP by PEG of aminoallyl-dUTP tethering ing both ing the both DNA and primer the protein ( on SDS-PAGE visible in complex a resulted covalent dCTP) contain C uloie a rvae b cplay lcrpoei ( electrophoresis capillary by revealed as nucleotide, dC plex, releasing a primer that was extended by a single propargylamino- complex with 365 nm light cleaved the linker and dissociated the com Supplementary Fig. 5c Fig. Supplementary a The The attachment above described chemistry leaves a large PEG ‘scar’ ) Scheme for two-step oligonucleotide extension using TdT–dNTP TdT–dNTP using extension oligonucleotide two-step for ) Scheme b ). In contrast, TdT ). In contrast, advance online publication online advance TdT TdT ′ FAM-labeled primer to the conjugate (henceforth, TdT– ′ end of the primer, the conjugate becomes covalently covalently becomes conjugate the primer, the of end S S TdT-(PEG S TdT-(BP-23354)-pa-dUTP “TdT-PEG O O “TdT-dTTP” ). We coupled this linker to the dCTP analog analog dCTP the to Welinker ). this coupled ). The cysteine mutations do not have mutations a sub ). cysteine The H N N 4 ∆ -SPDP)-aa-dUTP O 5cys protein exposed to identical labeling labeling to identical exposed protein 5cys 4 -dTTP” O H N Supplementary Fig. 6 Supplementary O O 4 4 -SPDP (upper, “TdT–PEG (upper, -SPDP -SPDP. to the Upon exposure

O O Supplementary Fig. 3 Fig. Supplementary O nature biotechnology nature Fig. Fig. 2 O HO NO HO a O ). Irradiation of ). Irradiation the 2 OH O P O OH O P HO O O HO O O O O Supplementary Supplementary H N O β b P P ). As expected, ). As expected, H N P P O ) Chemical ) Chemical ME to release release to ME O OH O 2 OH O i. 1 Fig. 8 of similar similar of OH OH O i. 2 Fig. O O N O 4 N b - ). We ). NH NH and and O O b ). ). - - - -

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. containing 146 sequential sequential 146 containing sequence defined Wea with DNA. molecule DNA single-stranded complementary a prepared of synthesis accurate for template a as serve could DNA bases only whether scarred containing investigated ( base each on scar propargylamino a contain ultimately min 5 at ( measured a formation with product +2 in conjugates, reduction original substantial the to relative termination improved ( specificity labeling improve to reaction labeling the in (maleimide) duced another set of conjugates with reduced of loading pro linker-dNTP we dNTP,so second a with resulted off-target labeled extensions conjugates from double of majority the that hypothesized We ( process. intramolecular an suggesting incubation, subsequent of min 14 during product +2 the of mation for the on effect little had product +1 form quantitatively to ficient suf incubation an of initial a TdTafter Dilution reaction molecule). polymerase same the to tethered dNTPs multiple of incorporation (i.e., process intramolecular an suggest would which concentration- independent, or molecule), polymerase another by molecule ase polymer one to tethered dNTP the of incorporation intermolecular (i.e., an process, suggest would the on which dependent concentration, was conjugate ‘non-termination’) (i.e., extension double ( min 2 after visible primer doubly-extended of (~4%) amount small a of formation the ( tions condi these under ddNTPs three ddATP to other free the compared by TdT–dATP rate of incorporation is the with reduced in agreement and s, TdT–dATP 8 of in did the primer so in extension slower 15 s. The slightly primer nM 25 extended completely almost TdT–dTTP ( product extended gly sin respective its into primer a convert to able was conjugate Each n 7poaglmn--ez-GP ( 7-propargylamino-7-deaza-dGTP and and 5-propargylamino-dCTP as well as 7-propargylamino-7-deaza-dATP 5-propargylamino-dUTP analogs dNTP the using conjugates linker–nucleotide. the of efficiency incorporation decreased the for compensates partially site catalytic the to respect with otide of nucle tethered concentration ing effective high that the extremely ( ( ( dTTP freely incorporated When solution, from kinetics. incorporation its tested then cloned. Sequencing of cloned. Sequencing 69 an clones revealed error rate of ~6 × 10 and PCR-amplified then we which DNA, complementary (natural) ( solution from than faster much with moiety the to maleimide the and quenched dUTP linker 5-propargylamino analog dTTP photocleavable the coupled we nucleoside, the of ics nucleotides. free of ration extension products ( primer of variety a produced reactions those instead, primer; the of nature biotechnology nature do not prevent normal base-pairing normal prevent not do 12 Fig. ( errors many without attachment amplified presented be can the chemistry with conjugates using produced DNA (95% CI: 0.2 – 1.4 × 10 × 1.4 – 0.2 CI: (95% Supplementary Fig. 10b Fig. Supplementary upeetr Nt 1 Note Supplementary 8 Fig. Supplementary Supplementary Fig. 11 Fig. Supplementary Since Since DNA using synthesized the photocleavable conjugates would observed we product, singly-extended predominant the Besides Next, we prepared a complete set of photocleavable TdT–dNTP photocleavable of set complete a prepared we Next, o et h ifune f h lne o te noprto kinet incorporation the on linker the of influence the test To Supplementary Fig. 9 Fig. Supplementary ), in accordance with reports that comparable modifications modifications comparable that reports with accordance in ), upeetr Fg 7b Fig. Supplementary β ME ( ME β ME-linker-dTTP was a less efficient substrate than than substrate efficient less a was ME-linker-dTTP β Fig. Fig. 2 Fig. 2 Fig. ME-linker-dTTP, ME-linker-dTTP, Fig. 2 Fig. −3 ), the linker–nucleotide was incorporated incorporated was linker–nucleotide the ), ) and used Taq polymerase to synthesize synthesize to polymerase Taq used and ) ). These new conjugates indeed displayed displayed indeed conjugates new These ). b ). /nt), suggesting that the propargylamino propargylamino the that suggesting /nt), c

), ), consistent with TdT-catalyzed incorpo ). We sought to investigate whether the the whether investigate to Wesought ). advance online publication online advance ). N c ). 16 16 ). -acetyl-propargylamino nucleotides nucleotides -acetyl-propargylamino . oee, ne tahd o TdT to attached once However, ). Supplementary Fig. 7b Fig. Supplementary µ 29 M TdT–dCTP, M TdT–dGTP, and , 3 Supplementary Fig. 7a Fig. Supplementary 0 . Supplementary Fig. 10a Fig. Supplementary upeetr Fg 5b Fig. Supplementary Supplementary Supplementary Fig. 1 Fig. ), suggest ),

b ), and and ), −4 ), we ), /nt ). ). ). ). ------

performed twice with similar results. similar with twice performed was experiment This above. as described was generated standards product of The ladder the with +2 of peaks the ladders. as a co-eluting peak at 120 s quenched in the reactions is detectable non-termination) (i.e., extension of A double amount small on the of type conjugate. depending in s 10–20 product extended the into singly converted is completely The primer TdT–dGTP,by photolysis. followed conjugates, and TdT–dTTP of a 25 the by extension nM 16 DNA primer ( peak. tallest of to the the height is axis normalized and the intensity standards internal of times the elution using is axis time normalized The electropherogram to 0-4 give extensions. selected conditions TdT under using analogs dNTP free by incorporating was generated standards of product The ladder (iv). products of extension in a results distribution conjugates pre-photolyzed to of the primer Exposure iii). (ii, (i) by the a nucleotide primer single panel from products reaction imaging. Oligonucleotides are visualized using 5 using are visualized Oligonucleotides imaging. by fluorescence SDS-PAGE two-color with intermediates cycle of reaction four nucleotides with similar results. ( results. similar with nucleotides four of all conjugates with was repeated experiment This (iv). the with primer the incubation before are irradiated conjugates if the TdT–dCTP observed is formation no complex primer-TdT In contrast, (iii). 365 nm irradiation by is again dissociated which 1 a form cycle again complex, primer-TdT of products extension conjugate, to TdT–dCTP fresh exposed When (ii). of the complex primer-TdT dissociation indicating and DNA bands, protein of the migration the distinct 365 with nm recovers of light the complex Irradiation of a complex. primer-TdT the formation indicating composite), in and DNA (yellow protein both in a results new containing band conjugate (i) to of the an primer the TdT–dCTP excess Exposing channel). (green Lumitein stain the fluorescent using is visualized and protein channel) nucleotide in 10–20 s, enabling stepwise DNA synthesis. ( DNA synthesis. stepwise in s, 10–20 enabling nucleotide 2 Figure b c a (Lumitein+FAM) Ladder Protein+DNA iv. Control(unlinked) iii. Cycle2product ii. Cycle1product i. Primer Ladder 120 s 15 s

(Lumitein) 8 s 0 s TdT–dNTP conjugates can extend a DNA molecule by a can extend DNA single molecule conjugates TdT–dNTP (FAM) Protein DNA P =Primer T = TdT-dCTP c ) Capillary electropherograms of reaction time courses for courses time of reaction electropherograms ) Capillary dA PT i Primer

a +TdT-dCTP dC Retention time . Each reaction cycle quantitatively extends extends quantitatively cycle . reaction Each Cycle 1 Retention time +365 nm ii +1 nt +1 nt b ) Capillary electropherograms of electropherograms ) Capillary +TdT-dCTP yl Ctl Cycle 2 2n 3n +4nt +3nt +2 nt µ +2 nt dG +365 nm M M TdT–dATP, TdT–dCTP, ′ i iv iii FAM (red fluorescence s r e t t e l a dT ) Monitoring ) Monitoring

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. step. This ten-step synthesis was performed once. Synthesis of another sequence is described in in described is sequence another of Synthesis once. performed was synthesis ten-step This step. per <0.1% of a rate at occurred Substitutions deletions. indicate bars empty and (non-termination), insertions indicate bars hatched bases, correct indicate bars Solid sequence. target the against singletons) (excluding reads 4,861 of alignment an on based synthesis the of yields stepwise the of create a (reverse) primer binding site, PCR-amplified the product product the ( PCR-amplified site, to binding dATP primer free (reverse) with a TdT create using product the tailed poly-A then We ( laser nm a 405 using of irradiation 1 min 5 sequence the to of using conjugates and corresponding cycles extension deprotection chromatogram indicates that the starter (ending with 5 with (ending starter the that indicates chromatogram stranded DNA molecule with a 3 with molecule DNA stranded conjugates, termination will be achieved. be will termination conjugates, that the nucleotide is hindered from the accessing active sites of other enough short is tether the as long as and a primer; into incorporated be will it conformation, productive a in polymerase the of site active the reach can nucleotide tethered the as long As general. quite be to of a polymerase using two different linkers, so we expect the approach surface the on points by various to triphosphate prepared nucleoside a be tethering can conjugates polymerase–nucleotide active that We by a polymerase. have shown of extension chain termination ible ( 5 sequence the synthesized also we possible, is of repeats synthesis the the To (1.0%). errors remaining from arising insertions and that demonstrate (1.3%) errors of source predominant the as deletions with 97.7%, was steps all of yield average The 10). (step 93.4% to 6) (step ( synthesis the of steps individual ( as cleotide intended. By the aligning reads against the target sequence oligonu complete the contained (80.5%) 3,913 that found we reads, 3 a with molecule DNA for stepwise 3 Figure s r e t t e l products are PCR-amplified for subsequent analysis. ( analysis. subsequent for PCR-amplified are products Tailingprimer. a poly-T for site a binding dATP free generate to with TdT using A-tailed then are products The purification. DNA and deprotection, wise yield of (0.882) of yield wise step average an implying sequence, intended the contained (88.2%) Supplementary Fig. 15b Fig. Supplementary Fig. 3 Fig. Supplementary Fig. 15a Fig. Supplementary ′

-CCC-3 Here, we developed polymerase–nucleotide conjugates for revers conjugates polymerase–nucleotide Here, we developed Finally, to demonstrate the feasibility of TdT–dNTP conjugates conjugates TdT–dNTP of feasibility the demonstrate to Finally, a a HO-

), and analyzed the amplicon by ( by amplicon the analyzed and ), Sequence confirmation of 10-mer synthesis. ( synthesis. 10-mer of confirmation Sequence 5′ 5′ 5′ 5′ 3′ ′ and analyzed the products by NGS. Of 474 reads, 418 418 reads, 474 Of NGS. by products the analyzed and de de novo 3. PCRforNGSlibrarypreparation 2. A-tailingwithTdT+freedATP 1. Tenreactioncycles ′ -CTAGTCAGCT-3 DNA synthesis, we subjected a double-stranded a double-stranded we subjected DNA synthesis, -OH 3′ 1/3 GATC CTAG CTAG CTAG = 96.1%, assuming three independent steps steps independent three assuming 96.1%, = ′ overhang ( overhang ). ), we were able to estimate the yields of of yields the estimate to able were we ), ′ overhang is extended by the the by extended is overhang Fig. 3 Fig. ( =–CCCH Supplementary Fig. 13 Fig. Supplementary ′ . (For deprotection, we used used we deprotection, (For . c TTTTTTT AAAAAAA AAAAAAA -OH 3′ ), which ranged from 99.5% 99.5% from ranged which ), Supplementary Fig. 14 Fig. Supplementary Fig. Fig. 2 NHAc) 3 b b ′ ) Sanger sequencing chromatogram of the PCR-amplified synthesis products. The sequencing sequencing The products. synthesis PCR-amplified the of chromatogram sequencing ) Sanger a -TTT-3 ) NGS. Of 4,861 4,861 Of NGS. ) -OH -OH 3′ 3′ 5′ ) Ten-step synthesis and procedure for amplification of the products for sequencing. A double- sequencing. for products the of amplification for procedure and ) Ten-step synthesis ′ N ) had been extended by the intended sequence, followed by a poly-A tail. ( tail. a poly-A by followed sequence, intended the by extended been ) had -acetyl-propargylamino DNA sequence 5 sequence DNA -acetyl-propargylamino ) to ten ten to ) c b Frequency 1,944 2,917 3,889 4,861 972 ).) ).) - - - 0

RTdNTPs accept better can that synthesis by sequencing for polymerases neer polymerases 3 3 of use enables it that is RTdNTPs,modified we propose that a key advantage of our approach DNA damage tethered 3 tethered lag behind natural dNTPs in incorporation kinetics incorporation in dNTPs natural behind lag base via a hydroxymethyl linkage can be rapidly incorporated by a by incorporated rapidly be can linkage hydroxymethyl a via base However, to group attached the dNTPs photocleavable a with similar ( inhibition the for responsible is linker able of TdT speed on natural dNTPs, our that results suggest the bulky photocleav incorporation the match not do conjugates presented the not reported) not O 3 TdTusing by primer a to nucleotides 4 of addition the onstrated Mathews Recently, oligonucleotide. 10-mer a of thesis in 10 s using a more powerful 365 nm light source light nm 365 a more powerful s 10 using in can be cleaved moieties nitrobenzyl similar though for deprotection, DNA synthesis DNA 97.7%, comparable to early demonstrations of phosphoramidite-based (C, G, and T) and 3 min (A) and an achieved average stepwise yield of by changing the attachment chemistry. attachment the by changing conjugates future in eliminated or reduced be could Scars omitted. be can or yields improves this whether determined conclusively not have we but step, photolysis each by produced scar the neutralize to expect they can achieve native-like incorporation kinetics. While While kinetics. incorporation native-like achieve can they expect we so site, catalytic conserved highly the contacts that region the in ′ 47,493 ntcorrect/48,610total=97.7%stepwise 93 80 84 83 91 95 88 80 41 93.4% 94.1% 98.0% 98.8% 99.5% 99.1% 98.3% 98.4% 98.0% 99.3% -nitrobenzyl RTdNTPs-nitrobenzyl with 60-min coupling times (stepwise yields O-modified RTdNTPs are typically poor substrates for natural natural for substrates poor typically are RTdNTPs O-modified Using TdT–dNTP conjugates, we report enzymatic enzymatic report we conjugates, TdT–dNTP Using oprd o eesbe emnto srtge epoig 3 employing strategies termination reversible to Compared C 10 9 8 7 6 5 4 3 2 1 TT TT advance online publication online advance T 3 ′ 4 -unblocked dNTPs are identical to the native substrates substrates native the to identical are dNTPs -unblocked . In spite of these efforts, 3 efforts, these of spite In . C 3 3 3 2 2 , and considerable effort has been expended to engi to expended been has effort considerable and , 1 . . Our demonstration included a 3 min acetylation step 7 . Our demonstration used coupling times of 1.5 min min 1.5 of times coupling used demonstration Our . T A . We used 1 min of irradiation with a 405 nm laser laser nm 405 a with irradiation of min 1 Weused . Supplementary Figure 15b Figure Supplementary A G G ′ -CTACTGACTG-3 Synthesis step ′ T -unblocked dNTPs. It is recognized that that recognized is It dNTPs. -unblocked T C A C G

′ ′ A C O-modified RTdNTPs still still RTdNTPs O-modified via ten cycles of extension, extension, of cycles ten via nature biotechnology nature . T Supplementary Fig. 7 Fig. Supplementary G A A A A 3 1 without causing causing without C 3 5 c T G C A . By contrast, contrast, By . ) Estimates ) Estimates de novo de et al. et T TT GG CC AA dem syn ′ ). ). - - - - - ′ © 2018 Nature America, Inc., part of Springer Nature. All rights reserved. are removed after the synthesis is completed is synthesis the after are removed disrupting secondary structures include using base modifications that for Strategies ends. recessed and blunt on activity reduced TdThas since in times, order synthesis during to extension consistent achieve structures secondary DNA of formation the reduce to necessary be may it Also, times. coupling adjusted extending require so may termini sequence, different terminal its on extent certain a to depend four bases of the primer the last of the bases four contacts TdT Because identified. be to remains variability this of cause the and 93–94%, of yields had steps two remaining the 98%, exceeding had eight steps of yields synthesis our demonstration Though synthesis. demonstration our in step per ~1% of mination non-ter remaining the of origin the investigating currently We are ently exceed 99% to enable the accurate synthesis of longer molecules. to consist must be increased yields the extension be Second, must identified. DNA growing the for support solid suitable a First, gates. conju TdT–dNTP on based synthesizer oligonucleotide enzymatic specificity. high with rapidly cleaved be could that esters or peptides as such linkers able conjugates. Alternately, cleav interest are of enzymatically particular practical enzymatic DNA synthesis technology. a DNA synthesis enzymatic of practical development the for point starting promising a offers scheme method the ( of implementation the hamper will costs reagent that expect not do we enzyme, TdT of quantity stoichiometric a sumes con cycle synthesis each While levels. practical to down brought be must costs manufacturing conjugate commercialization, for Finally, to at function elevated temperatures or in the presence of co-solvents. This This work has supportedbeen by the DOE Joint InstituteBioEnergy ( R. Palluk for helpful discussions, and E. de Ugarte for assistance with artwork. A. Flamholz, A. Tambe, B. Wagner, S. Weißgraeber, P. Weißgraeber, S. Jager, and procedure, P.D. Adams, C.J. Joshua, J.F. Barajas, M.E. Brown, C.B. Eiben, N. Kaplan for NGS of the synthesis products, C. Hoover for optimizing the NGS V. Teixeira Benites for assistance with experiments, G. Goyal, J. Chiniquy, and We thank S. Sehgal, A.K. Sreekumar, E. Baidoo, C.J. Petzold, L. Chan, and o Note: Any Supplementary Information and Source Data files are available in the the in available are references, and codes accession statements of including Methods, and data availability any associated M polymerase nature biotechnology nature t j Genome Institute (h ERASynBio (81861: “SynPath”). N.J.H. was also supported by the DOE Joint by NIH training grant GM-08295 through NIGMS. T.d.R. was supported by (SynBERC) through National FoundationScience grant NSF EEC 0540879 and D.H.A. was also supported by the Synthetic Engineering Research Biology Center Lawrence NationalBerkeley Laboratory and the US Department of Energy. and Environmental Research, through contract DE-AC02-05CH11231 between Ac to do so, for United States Government The purposes. Department of Energy to publish or reproduce the published form of this manuscript, or allow others States Government retains, a nonexclusive, paid-up, irrevocable, worldwide license publisher, by accepting the article for publication, acknowledges that the United under contract DE-NA0003525. The United States Government retains and the for the US Department of Energy’s National Nuclear Administration Security Solutions of Sandia a wholly owned of subsidiary Honeywell International Inc. laboratory managed and operated by National Technology and Engineering the US Department of Energy. Sandia National Laboratories is a multimission DE-AC02-05CH11231 Lawrence between NationalBerkeley Laboratory and of of Office Science, Biological and Environmental Research, through contract b h Supplementary Note 2 Note Supplementary n e l etho e Several challenges remain in the implementation of a practical practical a of implementation the in remain challenges Several i i kn . n

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5. 4. 3. 2. 1. claims jurisdictional in published maps and affiliations. institutional R polymerase-nucleotide conjugates and are cofounders of Ansa Biotechnologies, Inc. S.P. and D.H.A. have filed international patent application WO2017223517A1 on manuscript. the corrected and read authors All research. the J.D.K. and supervised N.J.H. A.K.S., results. interpreted and discussed authors all and data, analyzed and A.N.T., P.W.K. and H.M.B., R.B., experiments J.S.K., T.d.R., S.B., performed authors. other all from input with manuscript the wrote and data, analyzed experiments, performed and designed method, the S.P. conceived D.H.A. and accordance with the DOE Public Access Plan ( providewill public access to resultsthese of federally sponsored research in 25. 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. r CO AUTHO d e o eprints and permissions information is available online at at online available is information permissions and eprints p e r

MP - hn Y-. Goe, . Msa, .. Sei, . N nntcnlg fo the from nanotechnology DNA G. Seelig, & R.A. Muscat, B., Groves, Y.-J., Chen, cells human in screens Genetic E.S. Lander, & D.M. Sabatini, J.J., T.,Wei,Wang, and technologies synthesis: DNA novo de Large-scale G.M. Church, & S. Kosuri, S.M. Richardson, Gibson, D.G. utr D. Hutter, D.R. Bentley, Wu, J. H. Ruparel, 3 C. Montemagno, & H. Yang, A.S., Mathews, F.J.Bollum, L.M.S., Chang, transferase. terminal of biology Molecular R.C. Gallo, & for primer for nucleotides Photo-cleavable C. enzymes Montemagno, & Yang,H. A.S., Mathews, template-independent Modified J.E. Sylvester, & J.W. Efcavitch, acids. nucleic synthesizing for J.W.apparatus Efcavitch, and Methods S. Siddiqi, & DNA long of synthesis template-free for model theoretical A S.M. Ud-Dean, Minhaz template-independent catalyzed enzyme for Compositions F. Rose, & A.C. Hiatt, story F. Chen, the transferase: deoxynucleotidyl Terminal A.J. Berdis, & E.A. Motea, Oligonucleotide Enzymatic Template-Independent R.W. Davis, & M.A. Jensen, thymus calf by catalyzed reactions Oligodeoxyribonucleotide-primed F.J. Bollum, demystified. synthesis Gene J. Peccoud, & J.S. Bader, J.C., Anderson, M.J., Czar, science. to gift our DNA/RNA: of synthesis chemical The M.H. Caruthers, synthesis. chemical RNA and DNA of review brief A M.H. Caruthers, class new phosphoramidites—A Deoxynucleoside M.H. Caruthers, & S.L. Beaucage, in storage information digital Next-generation S. Kosuri, & Y. Gao, G.M., Church, genome. 1040–1044 (2017). 1040–1044 Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. Chem. using the CRISPR-Cas9 system. CRISPR-Cas9 the using applications. Crit. Rev. Biochem. Rev. Crit. synthesis. (2016). DNA mediated enzyme free (2015). US20160108382A1. Patent US synthesis. polydeoxynucleotide (2014). US8808989. Patent US Patent US sequence. nucleotides. protected using bonds (1998). US5872244A. phosphodiester of creation Challenges. and Prospects, History, Its (TiEOS): Synthesis polymerase. TrendsBiotechnol. synthesis. deoxypolynucleotide for (1981). 1859–1862 intermediates key of DNA. cell. the to tube test terminator chemistry. terminator Trans. f msudd N polymerase. DNA misguided a of Acad. Sci. USA Sci. Acad. synthesis. by sequencing DNA for terminator reversible a as nucleotide 34–40 (2013). 34–40 technology. sequencing of generations (2010). groups. aminoalkoxyl triphosphates for light-mediated, enzyme-catalyzed, template-independent template-independent enzyme-catalyzed, (2017). light-mediated, synthesis. DNA for triphosphates 1821–1832 (2018). 1821–1832 (2010). i n p t u s b E / i l n TIN R R i Science c d , 575–580 (2011). 575–580 39, et al. ′-O-modified3 nucleotides as reversible terminators for pyrosequencing. , 1420–1427 (2013). 1420–1427 288, - NTRIBUTI CO e a x Science c h hsoy n avne o rvril triaos sd n new in used terminators reversible of advances and history The al. et G . c Syst. Synth. Biol. Synth. Syst. h e INT aee ncesd tihshts ih eesby terminating reversibly with triphosphates nucleoside Labeled al. et t Design and synthesis of a 3 a of synthesis and Design al. et J. Biol. Chem. Biol. J. s m et al. Creation of a bacterial cell controlled by a chemically synthesized Nat. Methods Nat. s cuae hl hmn eoe eunig sn reversible using sequencing genome human whole Accurate al. et - , 1628 (2012). 1628 337, l . . Publisher’s note:

p , 5932–5937 (2005). 5932–5937 102, E l a , 52–56 (2010). 52–56 329, ur Poo. uli Ai Chem. Acid Nucleic Protoc. Curr. R n , 63–72 (2009). 63–72 27, ein f snhtc es genome. yeast synthetic a of Design al. et

E ). , 27–52 (2008). 27–52 21, STS Nat. Nanotechnol. Nat. Nature uloie Ncetds uli Acids Nucleic Nucleotides Nucleosides O NS

, 499–507 (2014). 499–507 11, , 1945–1949 (1962). 1945–1949 237, , 16462–16467 (2007). 16462–16467 104,

, 67–73 (2008). 67–73 2, , 53–59 (2008). 53–59 456, Science S pringer Nature remains neutral with neutral Nature remains regard to pringer ici. ipy. Acta Biophys. Biochim. eois rtois Bioinformatics Proteomics Genomics , 748–760 (2015). 748–760 10, , 80–84 (2014). 80–84 343, r. iml Chem. Biomol. Org. h t t p -O-allyl photocleavable fluorescent photocleavable ′-O-allyl : / / e n ′ - e O r cgd 2 -caged g

y 71 . g h o t t , 13.17.1–13.17.38 13.17.1–13.17.38 , v p erhd. Lett. Tetrahedr. / s r e t t e l : 1151–1166 1804, d / / o w Biochemistry ′ -deoxynucleoside -deoxynucleoside 8278–8288 14, w

w n 879–895 29, w Biochem. Soc. Biochem. Science l . o n a a Proc. Natl. Proc. d t u s

/ r J. Biol. J. e

. c 355,

o 22, 11, 57, m /

otn A, ez K, idrcs K & ax A Srcua bss o te KlenTaq the for basis Structural A. Marx, & K. Diederichs, K., Betz, A., Hottin, He, J. & Seela, F. Propynyl groups in duplex DNA: stability of base pairs incorporating Photocleavable R. Weissleder, & H. Lee, V.M., Peterson, M., Liong, S.S., Agasti, of Structures M. Delarue, & P. Beguin, F., Romain, S., Rosario, J., Gouge, D.C. Knapp, terminators: synthesis and use in primer extension. extension. (2011). primer in use and synthesis terminators: N plmrs ctlsd noprto o akn- ess alkyne-modified versus alkene- of incorporation nucleotides. catalysed polymerase DNA Res. pyrimidines. 5-substituted or 8-aza-7-deazapurines 7-substituted cells. single in analysis protein multiplexed and sensitive allow conjugates barcode-antibody DNA (2013). deoxynucleotidyltransferase: terminal of mechanism. ion two-metal the of aspects dynamical cycle catalytic the along intermediates , 5485–5496 (2002). 5485–5496 30, Chemistry J. Am. Chem. Soc. Chem. Am. J. t al. et Fluoride-cleavable, fluorescently labelled reversible reversible labelled fluorescently Fluoride-cleavable, , 2109–2118 (2017). 2109–2118 23,

, 18499–18502 (2012). 18499–18502 134, J. Mol. Biol. Mol. J. Chemistry

, 4334–4352 425, 17 uli Acids Nucleic , 2903–2915 2903–2915 ,

36. 35. 34. 33. 32. 31.

Lefler, C.F. & Bollum, F.J. Deoxynucleotide-polymerizing enzymes of calfthymus of enzymes Deoxynucleotide-polymerizing F.J. &Bollum, C.F. Lefler, Gardner,A.F. Chen, C.-Y. DNA polymerases drive DNA sequencing-by-synthesis technologies: both Chen, F. Litosh, V.A. B.P.Stupi, 3 J. Biochem. poly J. of Preparation III. gland. present. and past USA detection. SNP and sequencing for terminators reversible terminators. reversible fast-cleaving for determinants key as groups 2-nitrobenzyl of modification eesbe emntr b mlclr uig f -irbny akltd HOMedU alkylated 2-nitrobenzyl of tuning triphosphates. molecular by terminators reversible ′ -OHunblocked reversible terminators. , 1948–1953 (2010). 1948–1953 107, advance online publication online advance et al. Reconstructed evolutionary adaptive paths give polymerases accepting Stereochemistry of benzylic carbon substitution coupled with ring with coupled substitution carbon benzylic of Stereochemistry al. et et al. Improved nucleotide selectivity and termination of 3 Angew. Chem. Int. Edn Engl. Edn Int. Chem. Angew. etal. , 594–601 (1969). 594–601 25, Nucleic Acids Res. Acids Nucleic Rapid incorporation kinetics and improved fidelity of a novel class of Front. Microbiol. Front. aeydoyunlt ad polydeoxyguanylate. and N-acetyldeoxyguanylate

, e39 (2011). e39 39, , 305 (2014). 305 5, NucleicAcids Res.

, 1724–1727 (2012). 1724–1727 51, nature biotechnology nature

40 , 7404–7415, (2012). rc Nt. cd Sci. Acad. Natl. Proc. ′-OH unblocked

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. boring the plasmids were added to the JBEI strain archive and are available available are and archive strain JBEI the to added were plasmids the boring - experiments shown in experiments the in used protein The activity. of loss without Buffer TdTin Storage 6.5 pH °C −80 at stored and nitrogen liquid in snap-frozen be could MTdTproteins the that found we Subsequently, glycerol. 50% of addition the after °C −20 at upon upon request (Data and availability PEG crosslinker amine-to-thiol disulfide-forming on the based conjugates the ing in described are Figures Supplementary in are shown that of experiments description The METHO ONLINE doi:10.1038/nbt.4173 at 37 °C an until OD grown were cultures Expression dilution. 1/60 a using shake baffles in without flasks cultures expression inoculate to used then and grown °C were 37 at cultures overnight Starting expression. the throughout r.p.m. 200 at ing 100 with (Miller) medium LB in grown were pET19-M(BRCT)TdTwt or pET19-MTdTwt, pET19-MTdTc302, harboring TdT.MBP-fusedof purification andexpressionProtein demonstration. synthesis 5c Figure extinction coefficient of 108,750 M of 108,750 coefficient extinction estimated by absorbance spectrophotometry on a NanoDrop 2000 assuming an Vivaspin 20 columns (MWCO 10 kDa, Sartorius). Protein concentrations were homogenizer followed by centrifugation at 15,000 by centrifugation followed homogenizer 8.3) pH NaCl, M 0.5 Tris-HCl, purification steps were performed at 4 °C. protein Cells were lysed in All Buffer A (20 mM centrifugation. by harvested and °C 15 at overnight grown were cells and IPTG mM 1 at with induced was shaken expression Protein min. 45 then for °C 15 and shaking without min 45 for (RT) temperature room to initial demonstration of tethered dNTP incorporation employing the PEG the employing incorporation dNTP tethered of demonstration initial number: number: 132–510 from the short isoform (TdTS) of DNA construction. assembly for codon-optimization TdTThe gene encoding was ordered from Integrated DNA Technologies with SPDP crosslinker, TdT was expressed fused to an N-terminal His-Tag (TdTwt). residues were identified in the crystal structure of TdT (PDB ID: ID: TdT(PDB of structure crystal the in identified were residues TdT variants can be downloaded from the JBEI Public registry ( TdT registry from the Public JBEI can be variants downloaded Note 3 Supplementary generated. likewise were and the full-length TdT protein including the BRCT domain (M(BRCT)TdTwt) (MTdTwt), TdT sequence wild-type the with protein MBP-fusion a addition, into the pET19b plasmid harboring the TdT gene using isothermal assembly. In encoding MBP from the His-Tag and the TdT domain of TdTc302 between to yield MTdTc302.inserted was (MBP) protein The sequence binding maltose linker, photocleavable (Ala302Cys) TdTc302 and ( (Ser253Cys), TdTc253 (Glu180Cys), TdTc180 (Ala188Cys), TdTc188 mutants the generating mutagenesis, site-directed using site catalytic the near positions four into cysteines of re-insertion the by constructed then were attachment linker for cysteine surface-exposed gle fractions were buffer-exchanged into TdT pH 6.5 Storage Buffer (200 mM mM (200 KH Buffer Storage 6.5 pH TdT into buffer-exchanged were fractions purest The NaCl. mM 200 at eluted protein The NaCl. M 1 to 0 of gradient a tography (HiTrap Q HP 5mL, GE Healthcare) in 20 mM Tris-HCl pH 8.3 using chroma anion-exchange to subjected and 8.3, Tris-HCl pH mM 20 into 1:40 diluted were pooled, purity sufficient with Fractions A + 500 mM imidazole). A (Buffer + gradient with 5 an to Healthcare) mM Buffer imidazole imidazole GE mL, 5 FF (HisTrap chromatography affinity nickel to subjected was tant the mutations Cys188Ala (PDB ID ID (PDB Cys188Ala mutations the y38l, n Cs3Sr ee nrdcd sn st-ietd mutagen site-directed esis using introduced were Cys438Ser and Cys378Ala, Supplementary Fig. 4 Fig. Supplementary r e The amino acid sequences of all TdT mutants used can be found in in found be can used mutants TdT all of sequences acid amino The g 2 3 i PO 8 4 s . Based on this surface-cysteine free TdT variant, mutants with a sin a with mutants variant, TdT free surface-cysteine this on Based . -SPDP. t r y 4 . , 100 mM NaCl, pH 6.5) and concentrated to ~30 mg/mL using using mg/mL ~30 to concentrated and 6.5) pH NaCl, mM 100 , j N 3 b shows a diagram of the MTdTc302 construct that was used for the the for used was that MTdTc302 the of construct diagram a shows 7 P e T gnrt te TdT the generate To . i _ . o Supplementary Note 4 Note Supplementary 0 r 0 g 1 / 0 fo 3 l The TdT amino acid sequence used consists of the residues 6 d 600 E. E. coli D 6 e 9 S r Figure Figure of 0.40–0.45 was reached. The flasks were then cooled were cooled then The flasks was reached. of 0.40–0.45 3 . In addition, sequences of the plasmids coding for all for all coding plasmids of the sequences . In addition, s ). To generate the TdT variant that was used with the the with used was that TdT the variant To ). generate . / 1 E. coli E. 3 ) ) and lacks BRCT the N-terminal domain was amplified from pMAL-c5X (NEB) and inserted 5 5 ). Respective cloning and har cloning strains expression ). Respective 2 3 was stored at −20 whereas °C in 50% glycerol, and inserted into pET19b using isothermal isothermal using pET19b into inserted and 9 + 5 mM imidazole using an Emulsiflex C3 C3 Emulsiflex an using imidazole mM 5 + ∆ −1 Supplementary TableSupplementary 1 4 5cys mutant, surface-exposed cysteine cysteine surface-exposed mutant, 5cys I cm . This includes all experiments involv experiments all includes This . 2 7 numbering), Cys216Ser, Cys302Ala, Cys302Ala, Cys216Ser, numbering), −1 Mus musculus at 280 nm. The was protein stored µ g/mL carbenicillin with shak with carbenicillin g/mL g for 20 min. The superna The min. 20 for TdT (NCBI Accession

E. coli E. ). ). Supplementary Supplementary h BL21(DE3) BL21(DE3) tp 2 4 s 7 I : . . For the / 2 / 7 p ) and and ) u b l 4 i c ------

performed using 0.8 mL spin columns (Pierce) that were filled with 250 with that were filled 0.8 mL (Pierce) using spin columns performed was purification column spin A dNTPs: untethered) (i.e., free remove to phy and TdT–dNTP conjugates were using purified amylose chromatogra affinity 0.2 mM (dATP, The was dTTP). reaction labeling then incubated for 1 h at RT, (dGTP, mM of 0.1 and dCTP) concentrations nucleotide nominal with pared trituration.) during Unless quantitatively indicated otherwise, all conjugates precipitate employing linker BP-23354 (linker-)dNTPs were pre all that tion assump on the based calculated was concentration nucleotide nominal (The on experiment. from the 0.1 mM ranged to 2.5 mM, depending reactions ling TdT pH 6.5 Storage Buffer. The (nominal) nucleotide concentration in the labe Storage Buffer and added to MTdTc302 6.5 TdT pH 1× in resuspended (conc.was pellet 10–15 linker-dNTP dried a linker-dNTP, conjugates. °C. at −80 stored and lyophilization or by speed-vac dried were pellets linker-dNTPs. The supernatant was removed and the linker-dNTP-containing at 15,000 and centrifuged mL) (~2 acetate ethyl using triturated were salts) buffer (and products crude The dissolves. fully linker (unreacted) progress, product enough is linker-dNTP (soluble) formed that the remaining some makes reaction the after but limit, solubility the above is concentration last. The was reaction at incubated RT for 1 h with Initially,shaking. the linker 2 ice. Prior to binding, the amylose resin was washed twice with 500 500 with twice washed was resin amylose the binding, to Prior ice. on precooled were procedure the throughout used buffers and reagents All RCF. at 50 performed were steps centrifugation all and (NEB), resin amylose washing step involved 1) addition of 500 500 of addition 1) involved step washing with TP8 Buffer (50 mM potassium acetate, 20 mM Tris-acetate, pH 7.9). Each TdT with twice was washed pH Buffer, column Storage the 6.5 twice and then Next, binding. for min 10 for r.p.m. 800 at block shaker a in was incubated which then resin, amylose the containing column spin the onto loaded and ~200 ing pH pH 6.5 Storage Buffer. A 15 typical laal mliieNS abnt cosikr (BP-23354). crosslinker carbonate maleimide-NHS photo cleavable the and MTdTc302 using conjugates TdT–dNTP of Preparation °C. at −80 stored and nitrogen liquid using aliquots in snap-frozen was experiments other all in used protein the conjugates was performed by 1) the addition of 150 150 of addition the 1) by TdT–dNTP of performed was conjugates Elution flow-through. the of removal 4) and min, 1 for g 50 at centrifugation r.p.m., 3) 800 at shaking while min 1 for column the of tion dNTP (100 nmol), 1 dNTP (100 nmol), in a 30 (maleimide) “linker-dNTP” thiol-reactive a form to BP-23354 to coupled was pa-dNTP the First, Biotechnologies. TriLink from purchased was (pa-dNTPs) dNTPs Broadpharm (catalog ( number BP-23354). The complete set of crosslinker propargylamino- maleimide in shown is linker-dNTPs. of to ~2.5 ~2.5 to concentrated and concentration, maltose the reduce to Buffer TP8 with 1:10 combined and concentrated were using eluates a the 30 kDa and MWCO columntwice, (Corning), repeated diluted was procedure elution The trifugation. cen 3) and r.p.m., 800 at shaking while min 5 for incubation an 2) maltose, Capillary electrophoresisCapillary (CE). cobalt. of presence the in conjugates the storing when activity of loss substantial a observed we Notably, °C. −80 experimental design is experimental available in the and software analysis data on information Further authors. the from request the Peak Scanner software from Applied Biosystems. R scripts are available toupon comparable (r-project.org) functionality with R in written software custom using processed were files data Electropherogram °C. 35 buffer: °C, 68 oven: taining POP-7 Polymer, with 15 s of injection at 1.5 kV andan Applieda Biosystems 41 3730xl DNAmin Analyzer run with a at50 cm capillary array 15 con kV, illary electrophoresis (CE, also called fragment analysis). CE samples wereHi-Di run formamideon were submitted to the UC Berkeley Sequencing labeledFacility oligonucleotides for cap and ~0.3 µ TT32 aeig ih ikrdTs n prfcto o TdT–dNTP of purification and linker-dNTPs with labeling MTdTc302 L of 100 mM BP-23354 dissolved in anhydrous DMSO (200 nmol) added added nmol) (200 DMSO anhydrous in dissolved BP-23354 mM 100 of L µ g/ µ g g of MTdTc302 200 into diluted was To site-specifically label TdT at surface cysteine residues with a with residues cysteine surface at TdT label Tosite-specifically µ Supplementary Figure 5 Figure Supplementary L. The conjugates can be frozen in liquid nitrogen and stored at stored and nitrogen liquid in frozen be can conjugates The L. The scheme for the preparation of TdT–dNTP conjugates conjugates TdT–dNTP of preparation the for scheme The µ µ L reaction containing L 1 containing reaction L L of 10× TdT pH 7.4 Storage Buffer, 26 upeetr Fg 5a Fig. Supplementary 20 µ L GeneScan 600 LIZ dye Size Standard in 75% µ µ L linker-dNTP labeling reaction contain reaction L labeling linker-dNTP L L samples containing 0.5–1.5 nM 5 Life Sciences Reporting Summary Life Sciences . The photocleavable NHS carbonate- NHS photocleavable The . µ L buffer to the column, 2) incuba 2) column, the to buffer L µ L L of 100 mM the pa- respective nature biotechnology nature µ L L TdT Buffer Storage 6.5 pH µ g/ ) was purchased from from purchased was ) µ µ L TP8 Buffer + 10 mM mM 10 + Buffer TP8 L L by absorbance) in 1× µ L L of dH g to pellet the to pellet

µ Synthesis Synthesis L L of TdT 2 O, and ′ -FAM µ . L L of ------

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. pa-dNTPs for use as size standards (ladder) in CE assays. CE FAM-dT in (ladder) standards size as use for pa-dNTPs eeain f xeso pout o oio 2 (5 P2 oligo of products extension of Generation bicarbonate. sodium mM 200 Unless CE. and 20 mM contained NHS-acetate reactions acetylation before otherwise, specified NHS-acetate using derivitized prop were containing groups samples argylamino DNA all Therefore, charges. positive added to the due likely CE, in migration inconsistent and yield injection reduced had CE. before desalted or formamide 75% with 50-fold diluted either were reactions extension from DNA samples so peaks, nature biotechnology nature using TdT. Reactions contained 100 nM oligo P2, 100 100 P2, oligo nM 100 TdT.contained using Reactions size standards were(ladders) generated by the incorporation of free pa-dNTPs (Bio-Rad). The gel was imaged on a MultiImager III (Alpha Innotech) for for Innotech) (5 (Alpha fluorescence green III MultiImager a on imaged was gel The (Bio-Rad). 1% + (Novex) buffer loading SDS 2× with combined PAGEwere for Samples CE. for OCC by desalted and acetylated were samples photolyzed all of Aliquots by CE. analysis for formamide 75% with eluted then and buffer, B&W 0.01× buffer, 1× B&W buffer (5 mM Tris HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl), 0.1× B&W conjugates. using TdT–dCTP experiment and Two pre-photolysis demonstration cycle ladders. as use for +2 selected were and products +1 extension the pa-dNTP as well as P2 oligo for peaks detectable with Samples CE. by analyzed and Research), Zymo (OCC; Kit Concentrator and Clean Oligo the mM using after 2, desalted 5, and samples were 33 10 acetylated, then min. Quenched of concentration final a to EDTA with quenched were aliquots and U/ 0.05 either tris-acetate, 10 mM magnesium acetate, 0.25 mM cobalt chloride, mM pH 7.9), 20 and acetate, potassium mM 50 (RBC: cobalt with buffer dNTP,reaction were performed in a 37 °C room by adding 4.5 in the maleimide-labeling reaction. Oligo P2 extension yield by 1.5 mg/mL mg/mL (~16 1.5 by yield extension P2 Oligo reaction. maleimide-labeling the in mM at 1 nucleotides wereusing generated experiment this conjugatesin used TdT–dNTPfour conjugates. all of courses time extension primer Fast PAGE CE. and for taken were aliquots and P2, oligo with reaction extension an in used then ture MTdTc302(linker) of unlinked + pa-dCTP. were The products photolysis mix stoichiometric a generate to ice on h 1 for light nm 365 with irradiated PAGE CE. and for reactions PAGEextension both for reactions and photocleavage after both reac extension after another taken were to TdT–dCTP, Aliquots with tion by photolysis. followed again subjected and OCC by purified then were ucts P2 was performed and the reaction products were photolyzed. The DNA prod Photoshop. Adobe using composited and aligned were images Gel protein). (total fluorescence red for imaged (Biotium), UV the 365 nm setting for 1 h on ice. The measured irradiance was ~5 mW/cm ~5 was irradiance measured The ice. on h 1 for setting nm 365 the using linker was a performed Benchtop 2UV Transilluminator (UVP, LLC) on of of volume mM 200 EDTA. of an 2 the by equal min addition the Photolysis after quenched and °C at 37 performed were Reactions RBC. and below), see contained 50 nM oligo P2, 0.25 mg/mL TdT–dCTP (or photolyzed TdT–dCTP, reactions extension All MTdTc302-labeling the reaction. in 1 nucleotide mM 4.5 4.5 gate to 1.5 Scientific) that were saturated with 5 buffer and then captured onto M-280 DynaBeads Streptavidin (Thermo Fisher bicarbonate mM 400 in NHS-acetate mM 100 using acetylated were products with 365 nm light on a Benchtop 2UV Transilluminator for 30 min. Photolysis quenched with 6 Formamide with 10 mM EDTA) after 8 or 15 s. The remaining reaction volume was High ionic strength in a CE sample causes poor injection and distorted distorted and injection poor causes sample CE a in strength ionic High It was observed that DNA containing multiple propargylamino groups groups propargylamino multiple containing DNA that observed was It Control Control (“pre-photolyzed conjugate”) experiment: Two cycle experiment: µ L L of the reaction were quenched in 18 µ β M) TdT–dNTP conjugates was measured at 8, 15, and 120 s. Reactions Reactions s. 120 and TdT–dNTP 15, M) 8, at measured conjugates was ME, and run on an 8–16% PAA-gradient Mini-PROTEAN TGX gel gel TGX Mini-PROTEAN PAA-gradient 8–16% an on run and ME, 60 µ ; ; L of 100 nM oligo P2 (final: 25 nM), both in RBC. After rapid mixing, The conjugates used in this experiment were generated using using generated were experiment this in used conjugates The Supplementary Table 2 Table Supplementary µ L or 0.03 U/ 0.03 or L µ L Quenching Solution after 2 min. The samples were irradiated ′ FAM-labeled primer) and, with FAM-labeled Lumitein primer) after staining a containing reaction TdT–dCTP conjugate and oligo µ L NEB TdT. Reactions were performed at 37 °C, °C, at 37 TdT. NEB L performed were Reactions ′ -biotin-dA ) extension products that were used as as used were that products extension ) µ L L Quenching Solution (94% Hi-Di µ L of 2 mg/mL TdT–dNTP conju 60 oligo. Beads were washed with TdT–dCTP conjugate was µ M of one type of pa- of type one of M ′ -FAM-dT Oligo P2 (5 P2 Oligo 60 ) with with ) The The ′ 2 ------.

quenched by the addition of 16 TP8. The resulting 16 product of the previous cycle were mixed with 2 2 with mixed were cycle previous the of product 10 the cycles, following For the 8.5). pH TWEEN-20, DNA 10 the with and eluted was s, 90 for dried were beads the Subsequently, ethanol. 70% of 400 with first were washed beads the incubated for 2 min for sedimentation of the beads. The liquid was removed and and rack plate into on of a well a a magnetic 96-well transferred then was tion solu The performed. was min 5 of step binding a and reaction, phosphatase beads (Beckman Coulter): 115.2 analysis of synthesis products “ to (see leading cycle, insertions reaction next the in incorporated be could dNTPs the that and cleanup, XP AMPure the during carry-over dNTP of amount substantial a was there that foratwe found 1 min RT. because performed was treatment phosphatase The incubated was reaction the and dNTPs, released digest to products photolysis atScientific) 0.32 U/ 32 to proceed (NHS-acetate is a component of the Quenching Buffer).light”. Subsequently, A 3 min incubation at RT was performed to allow the acetylation reaction in tion was photolyzed for 1 min using a 405 nm laser as described in “as described at ~30 nM was mixed with 2 2 with mixed was nM ~30 at 5 synthesize 6) TdT–dCTP, 7) TdT–dATP, 8) TdT–dGTP, 9) TdT–dCTP, 10) TdT–dTTP. steps: To 1) TdT–dCTP, 2) TdT–dTTP, 3) TdT–dATP, conjugates were in used the the extension 4) following For TdT–dGTP,synthesis, the 10-mer step. 5) TdT–dTTP,extension next the to subjected was DNA recovered the and beads, XP light.” After each cleavage step, the DNA products were purified using AMPure 40 mM NHS-acetate; NHS-acetate was added immediately before use). use). “ before immediately added was Photolysis was forperformed 1 min using a 405 nm diode laser NHS-acetate as in described NHS-acetate; Azide, mM Sodium mM 40 20 EDTA, mM 50 (Sigma-Aldrich), 20 TWEEN 0.1% NaCl, mM 300 NaHCO3, mM (100 Buffer Quenching of volume equal an of addition the by performed was reactions the of Quenching s. 180 for dATP TdT– with extensions s, 90 for performed were TdT–dTTP and TdT–dGTP TdT–dCTP,with reactions at at in Extension 37 RBC 1 conjugates °C. mg/mL a 3 generate to BstXI with digested and (DCC) purified DNA was the tailing, After min). 30 for °C 37 at RBC in starter can be found in in found be can starter of for A synthesis the preparation the scheme Kit (Zymo Research). Recovery Gel the using gel-extracted and electrophoresis gel TAE-agarose DNA 2% by TdT–dNTP conjugates. The from product was digestion separated undigested 12 12 pH pH 7.9). The 12 chloride, cobalt mM 2 and acetate magnesium mM 80 Tris-acetate, mM 120 the the 3 with strand the block to ddTTP with tailed and (DCC) purified was 3 product a generate to C1, by inserted site restriction the cutting PstI, with digested and (“DCC”, Research) kit Zymo Concentrator & Clean DNA the using purified was product PCR The min). 1 for °C 72 s, 10 98 °C for protocol: of 98 two-step °C for 35 cycles 1 program: then (PCR min, (see C2 and C1 primers using and er’sinstructions manufactur the following Scientific) Fisher (Thermo per Phusion using was formed PCR The plasmid. pET19b the from derived product PCR bp 359 a from prepared was (starter) synthesis the for substrate initial as DNAused sequence was synthesized. sequence way as for the first synthesis step. The procedure was repeated until the complete acetylated, phosphatase-treated, and purified using AMPure beads in the same on depending the type of TdT–dNTP. The was reaction quenched, photolyzed, 16 resulting The TP8. Extension of a DNA strand by the sequences 5 5 sequences the by strand DNA a of Extension DNA molecule with a 3 Supplementary Note 4 Supplementary ′ -CCC-3 Supplementary Supplementary Note 4 Detailed protocol of the extension cycles. extension the of protocol Detailed Synthesis Synthesis overview. µ µ L mixture was added to 4 4 to added was mixture L ′ L of FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Fisher (Thermo Phosphatase Alkaline Thermosensitive FastAP of L overhang from further incorporations (0.5 mM ddTTP, 1U/ ′ using TdT–dNTPconjugates. using ′ -CCC-3 µ L L mixture was then added to 4 µ µ ′ L of Buffer EBT (1 mM Tris-HCl, 10 10 Tris-HCl, mM (1 EBT Buffer of L Nucleotide additions were performed using TdT–dNTPwere additions Nucleotide performed L L in 40 mM Tris-HCl and 60 mM MgCl was added to the , three cycles with TdT–dCTP with performed. were cycles three , µ ′ µ overhang used as synthesis starter. L reaction L in was for reaction RBC incubated 90 s, before it was Supplementary Figure 13 Figure Supplementary L reaction was then incubated for either 90 or 180 s, s, 180 or 90 either for incubated then was reaction L , section Linker photolysis time course using 405 nm using course time photolysis Linker , section , , section Linker photolysis time course using 405 nm ”). ”). Next, the DNA was using purified AMPure XP µ µ µ L Quenching Buffer. After quenching, the reac L Cofactor Mix (300 mM potassium acetate, acetate, potassium mM (300 Mix Cofactor L µL AMPure XP beads were added to the 64 Amplification and next-generation sequencing L of the respective TdT–dNTP 4 at respective the of L ′ overhang (5 overhang µ L of 70% ethanol and then with 200 200 with and L then of ethanol 70%

Generation of the double stranded stranded double the of Generation For the first step, 10 10 step, first the For µ ′ L L of TdT–dCTP at 4 -ATTT-3 µ . L of Cofactor Mix, and the the and Mix, Cofactor of L ′ ′ -CTAGTCAGCT-3 Supplementary Table 2 Supplementary overhang. The digested digested The overhang. µ L of purified synthesis synthesis of L purified The double-stranded doi:10.1038/nbt.4173 ′ ) for extensions by ) for extensions µ M EDTA, 0.04% 0.04% EDTA, M µ L L NEB TdT µ µ L starter starter L µ g/ g/ µ ′ µ and and L in in L L L in µ µL L - - - - )

© 2018 Nature America, Inc., part of Springer Nature. All rights reserved. taining the “target region”con Reads sequence 5 (Illumina). MiSeq a on performed was NGS service. the of users following the photolysis (see “Detailed protocol of the extension cycles”), but cycles”), of extension the protocol “Detailed (see photolysis the following 15a Fig. in “CTAGTCAGC insertion extension next the error, G the (e.g., insertion into of type (non-double) step, a causing over characteristic carried are reaction extension quenched a of As to washing. a some of result, the dNTPs photolysis during that are released dNTPs can in retain a XP that that is beads AMPure manner shown) resistant switching. to index due misassignment read as such errors artifactual avoid to analysis from and for of were set excluded data 1.5% the accounted regions target Singleton analysis. for retained were region target the in bases all for nt) ~1/2,500 rate: identified using a BioPython script, and reads with a Q-score of at least 34 (error doi:10.1038/nbt.4173 described previously as described preparation library (Illumina) Nextera barcoded for Service Sequencing DiVADNA JBEI the to submitted and DCC by purified were Amplicons s. 30 for °C 68 s, 20 for °C 49 s, 30 for °C 98 of: cycles 35 then min, 12 for °C 68 the manufacturer’s PCR program: 98 °C for instructions. 2 min, 49 °C for 20 s, ( C4 and C3 primers with Taq(NEB) The tailing products were by purified and DCC PCR-amplified using HotStart °C. 37 at min 30 for (NEB) Buffer TdT Reaction in dATP U/ mM 1 with 0.4 (NEB) using A-tailed were products synthesis 3-cycle and 10-cycle As mentioned above, it in (data was experiments not independent observed Amplification and sequencing analysis next-generation of synthesis products. ). The effect was mitigated by a brief alkaline phosphatase treatment treatment phosphatase alkaline brief a by mitigated was effect The ). 4 0 , multiplexed with other samples submitted by other other by submitted samples other with multiplexed , G T” observed in 0.27% of reads, of in reads, 0.27% observed T” ′ -TCCAGATTT(N Supplementary Table 2 Table Supplementary 0–20 )AAAAAA-3 Supplementary Supplementary ) according to to according ) µ L TdT L ′ were -

corresponding authors upon request. upon authors corresponding of from study are the this available the findings that support software analysis 40. 39. 38. 37. l strain JBEI archive ( the in deposited been have demonstration this in used variants availability. Data to linked Summary Reporting article. this Research Nature the in available is design Summary. Reporting Sciences Life yields. stepwise of tion in 0.7% identified of all reads total and were manually removed before estima definitively were insertions carryover-type dNTP eliminated. completely not i c - r kilobases. hmsn M.G. Thompson, of expression High-level C. Papanicolaou, F.& Rougeon, E., Johnson, J.B., Boulé, Site-directed L.R. Pease, & J.K. Pullen, R.M., Horton, H.D., Hunt, S.N., Ho, D.G. Gibson, pSC101 origin that increase copy number. copy increase that origin pSC101 eprtr ad vrxrsig rU tRNA. argU overexpressing and in temperature transferase deoxynucleotidyl terminal murine 51–59 (1989). 51–59 reaction. chain polymerase the using extension overlap by mutagenesis (1998). e g i s t Supplementary TableSupplementary 1 r y . j b Nat. Methods Nat. e i . Enzymatic assembly of DNA molecules up to several hundred several to up molecules DNA of assembly Enzymatic al. et o r g Cloning and expression strains with plasmids of all TdT all of plasmids with strains expression and Cloning / slto ad hrceiain f oe mttos n the in mutations novel of characterization and Isolation al. et f o l d e r , 343–345 (2009). 343–345 6, s / 3 5 5 ). The raw electropherogram data and custom ). The raw electropherogram ) ) and are available upon request ( Further information on experimental experimental on information Further Sci. Rep. Sci. nature biotechnology nature o. Biotechnol. Mol. rw a low at grown coli Escherichia , 1590 (2018). 1590 8, 199–208 10, h t t p Gene s : / / p u 77, b - -