De novo DNA synthesis using polymerase-nucleotide conjugates


Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of 200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3′ end of the primer remains covalently bound to TdT and is inaccessible to other TdT–dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT–dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10–20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

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Figure 1: TdT–dNTP conjugates for reversible termination of primer elongation.
Figure 2: TdT–dNTP conjugates can extend a DNA molecule by a single nucleotide in 10–20 s, enabling stepwise DNA synthesis.
Figure 3: Sequence confirmation of 10-mer synthesis.

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We thank S. Sehgal, A.K. Sreekumar, E. Baidoo, C.J. Petzold, L. Chan, and V. Teixeira Benites for assistance with experiments, G. Goyal, J. Chiniquy, and N. Kaplan for NGS of the synthesis products, C. Hoover for optimizing the NGS procedure, P.D. Adams, C.J. Joshua, J.F. Barajas, M.E. Brown, C.B. Eiben, A. Flamholz, A. Tambe, B. Wagner, S. Weißgraeber, P. Weißgraeber, S. Jager, and R. Palluk for helpful discussions, and E. de Ugarte for assistance with artwork. This work has been supported by the DOE Joint BioEnergy Institute ( by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. D.H.A. was also supported by the Synthetic Biology Engineering Research Center (SynBERC) through National Science Foundation grant NSF EEC 0540879 and by NIH training grant GM-08295 through NIGMS. T.d.R. was supported by ERASynBio (81861: “SynPath”). N.J.H. was also supported by the DOE Joint Genome Institute ( by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy. Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia a wholly owned subsidiary of Honeywell International Inc. for the US Department of Energy's National Nuclear Security Administration under contract DE-NA0003525. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains, a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (

Author information




S.P. and D.H.A. conceived the method, designed and performed experiments, analyzed data, and wrote the manuscript with input from all other authors. T.d.R., S.B., J.S.K., R.B., H.M.B., A.N.T., and P.W.K. performed experiments and analyzed data, and all authors discussed and interpreted results. A.K.S., N.J.H. and J.D.K. supervised the research. All authors read and corrected the manuscript.

Corresponding authors

Correspondence to Daniel H Arlow or Jay D Keasling.

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Competing interests

S.P. and D.H.A. have filed international patent application WO2017223517A1 on polymerase-nucleotide conjugates and are cofounders of Ansa Biotechnologies, Inc.

Integrated supplementary information

Supplementary Figure 1 The nucleotide binding pocket of TdT has little room for 3' OH modifications

(a) Rendering of the co-crystal structure of TdT with dCTP (PDB ID 4I2J) focused on the dNTP binding site. The molecular surface of the protein is rendered as a yellow mesh, the nucleotide is rendered as sticks, and two Zn2+ ions are shown as grey spheres. (b) Rendering of a model of TdT in complex with dTTP based on the co-crystal structure of TdT with ddTTP and a DNA primer (PDB ID 4I27, 3'-OH inserted using Maestro) depicting the same molecular features as panel a and a Mg2+ ion shown as a pink sphere. The 3' OH of the dCTP and dTTP molecules (magenta) make close contacts with the surface of the protein, leaving little room for 3' O-substituents.

Supplementary Figure 2 Chemical detail of extension of a primer by TdT-PEG4-dTTP conjugates

Chemical steps to prepare TdT-PEG4-dTTP and use it to extend a DNA primer. Left column: 5-aminoallyl dUTP is reacted with the amine-to-thiol crosslinker PEG4-SPDP to yield a disulfide forming linker-dNTP “OPSS-PEG4-dTTP”. OPSS-PEG4-dTTP is then used to label a TdT mutant containing a single surface-accessible cysteine residue to form the conjugate “TdT-PEG4-dTTP”. Right column: A DNA primer exposed to TdT-PEG4-dTTP forms the covalent complex TdT-PEG4-dT-DNA upon incorporation of the tethered nucleotide into the 3' end of the primer. Treatment of the complex with the reducing agent βME cleaves the disulfide linkage between the enzyme and the incorporated nucleotide, releasing a primer that has been extended by a HS-PEG4-dT nucleotide.

Supplementary Figure 3 TdT-PEG4-dNTP conjugates with one tethered nucleotide can extend a primer by a single nucleotide

SDS-PAGE showing the ability of different TdT variants to incorporate a tethered nucleotide after labeling reactions with OPSS-PEG4-dTTP. Both gels were imaged for the fluorescence of oligo P1 (5' FAM-dT35), the ladder used (L) was generated by extensions of P1 by free OPSS-PEG4-dTTP. (a) The oligo before the reaction (O), reaction products formed during incorporation with labeled TdT (P) and after cleavage of the linker by βME (B) are shown. TdTwt and TdTc302 form high-molecular weight complexes containing the primer, indicating tethered incorporation, while TdTΔ5cys does not. After treatment with βME, the complexes dissociate, and the migration of the released oligo indicates multiple extensions by TdTwt, a single extension by TdTc302, and no extensions by TdTΔ5cys, respectively. (b) Products formed during extension reactions (-BME) and after cleavage of the linker by βME (+BME) are shown for TdTwt (wt), TdTΔ5cys (Δ), and TdT-PEG4-dTTP conjugates with a single surface exposed cysteine in positions 180, 188, 253 and 302. The formation of a fluorescent high-molecular weight complex occurs for all TdT variants except for TdTΔ5cys. Upon cleavage, the oligo shows multiple extensions for TdTwt, and a single extension for TdT variants with a single surface-exposed cysteine. These experiments were each performed twice with similar results.

Supplementary Figure 4 Attachment positions used for preparation of TdT-PEG4-dNTP conjugates

Rendering of the region surrounding the active site of the co-crystal structure of TdT with ddTTP and a primer (PDB ID: 4I27, primer not shown) indicating the positions that were used as attachment points for tethering aminoallyl dUTP to TdT via the amino-to-thiol crosslinker PEG4-SPDP. To prepare the conjugates, all 5 surface-accessible cysteine residues of TdT were mutated to serine or alanine, and then one of the indicated residues (positions 180, 188, 253, and 302) was mutated to cysteine for labeling with OPSS-PEG4-dTTP (see Fig. S2). Distances shown are measured from the 5-methyl group of ddTTP to the Cα atom of the indicated residue. Molecular graphics were made using PyMol.

Supplementary Figure 5 Chemical detail of extension of a primer by TdT-dNTP conjugates

(a) Chemical steps to prepare TdT-dCTP and use it to extend a DNA primer. Left column: Propargylamino dCTP is reacted with the photocleavable amine-to-thiol crosslinker BP-23354 to form a photocleavable linker-nucleotide “linker-dCTP”. The maleimide moiety of linker-dCTP is then used to label a TdT mutant containing a single accessible cysteine residue to form “TdT-dCTP”. Right column: A DNA primer exposed to TdT-dCTP forms the covalent complex TdT-dC-DNA upon incorporation of the tethered dCTP into the 3' end of the primer. Irradiation of the complex with 365 nm light cleaves the carbamate linkage between the enzyme and the incorporated nucleotide, releasing a primer that has been extended by a single propargylamino dC nucleotide. (b) Structures of the complete set of propargylamino dNTPs used in this study. (c) Diagram of the MTdTc302 construct. The fusion protein consists of an N-terminal His-tag followed by MBP and Mus musculus TdT residues 132-510 (PDB ID: 4I27 numbering). The only cysteine residues in the construct are two buried cysteines depicted in black (Cys155, Cys404) and Cys302 (yellow) that serves as attachment point for the linker.

Supplementary Figure 6 Effect of the mutations in MTdTc302 on TdT activity

Extension of oligo P2 (FAM-dT60) with dTTP using 10 nM M(BRCT)TdTwt, MTdTwt or MTdTc302, respectively, analyzed by fragment analysis. The mutants show a similar distribution of elongation products, suggesting that the absence of the BRCT domain compared to full-length wt TdT and the mutations introduced to generate a TdT variant with a single surface-exposed cysteine do not significantly alter TdT activity. The electropherogram time axis is normalized using the elution times of internal standards (see Methods) and the intensity axis is normalized to the height of the tallest peak. This experiment was performed twice with similar results.

Supplementary Figure 7 Tethering increases the incorporation speed of the linker-nucleotide

(a) Structures of nucleoside triphosphates used in this experiment. (b) Capillary electropherograms of elongation products of oligo P2 (FAM-dT60) by 1.4 μM TdT for 30 seconds with: 1.4 μM dTTP, 1.4 μM βME-linker-dTTP; or by 1.4 μM TdT-dTTP conjugate in the absence of free nucleotides. In the same amount of time, the primer is elongated by many dTTP molecules, but the rate of incorporation of βME-linker-dTTP is much slower, indicating that it is a worse substrate than the natural nucleotide. However, attaching linker-dTTP to TdT results in quantitative extension of the primer, suggesting that tethering can partially compensate for a poor substrate by increasing its effective concentration with respect to the catalytic site of the polymerase. The ladder of product standards for oligo P2 extension by βME-linker-dTTP and TdT-dTTP (after photolysis) was generated by elongating the primer with free pa-dUTP using TdT (see Methods). (dTTP extension products have different retention times than pa-dUTP extension products.) This experiment was performed twice with similar results.

Supplementary Figure 8 Rendering of a model of TdT-dTTP in complex with a primer

A model of TdTc302 was generated by inserting the specific mutations (Cys188Ala, Cys216Ser, Cys378Ala, and Cys438Ser) into the crystal structure of the TdT ternary complex (PDB ID: 4I27) using PyMOL. The linker structure was modeled off of the 5 position of the ddTTP using Avogadro, and the maleimide moiety of the linker was attached to Cys302 (as a succinimide). For the geometry optimization of the linker, all atoms in TdT except the adjacent three amino acids on each side of Cys 302 (Leu299 to Arg 305) were fixed. The 3'-OH of the nucleotide was inserted using Maestro. The rendering of the model shows the linker extending through a large groove connecting the nucleotide binding site and Cys302.

Supplementary Figure 9 Incorporation rate comparison of all four bases using free nucleotides

Primer elongation time courses of oligo P2 (FAM-dT60) by MTdTc302 using all four different dideoxynucleoside triphosphates (ddATP, ddCTP, ddGTP, ddTTP) analyzed by capillary electrophoresis. MTdTc302 incorporates ddATP slower than the other ddNTPs, suggesting a base-dependence of the incorporation speed under these conditions, in accordance with the slower extension of the primer by TdT-dATP compared to the other conjugates. The electropherogram time axis is normalized using the elution times of internal standards (see Methods) and the intensity axis is normalized to the height of the tallest peak. This experiment was performed twice with similar results.

Supplementary Figure 10 Non-termination (2nd extension) products arise predominantly from a concentration-independent process

The electropherogram time axes are normalized using the elution times of internal standards and the intensity axes are normalized to the peak area of the depicted region. The ladder of product standards was generated by elongating the primer with pa-dNTPs using TdT (see Methods). (a) Capillary electropherograms of extension reactions of oligo P2 (FAM-dT60) (grey) by TdT-dCTP prepared with 1 mM nucleotide in the labeling reaction after 1 minute of incubation (black), 15 minutes of incubation (cyan), and 1 minute of incubation followed by ten-fold dilution with reaction buffer and an additional 14 minutes of incubation (red). The predominant product is singly-extended primer in all reactions, and the amount of doubly-extended product formed (indicated by the arrow) is similar in the diluted and not diluted 15-minute reactions. (The shoulder on the left side of the peaks is a -1 nt impurity from the original phosphoramidite-based synthesis of the dT60 primer.) This experiment was performed twice with similar results. (b) Capillary electropherograms of 5 minute extension reactions of oligo P2 by TdT-dTTP conjugates prepared with different concentrations of nucleotide in the labeling reaction. The most abundant product is singly-extended primer in all reactions, and the amount of doubly-extended product formed (indicated by the arrow) decreases with the concentration of linker-dTTP in the labeling reaction. This experiment was repeated independently three times with similar results.

Supplementary Figure 11 Linked incorporation time courses at low and intermediate concentrations of TdT-dNTP

Capillary electropherograms of reaction time courses for the extension of 100 nM oligo P2 (FAM-dT60) by TdT-dATP, -dCTP, -dGTP, and -dTTP conjugates, followed by photolysis. Reactions were performed with 11 μM (a) or 2.7 μM (b) TdT-dNTP. The ladder of product standards was generated by elongating the primer by pa-dNTPs using TdT. The electropherogram time axis is normalized using the elution times of internal standards (see Methods) and the intensity axis is normalized to the height of the tallest peak. These experiments were repeated on three independent preparations of conjugates with similar results.

Supplementary Figure 12 Propargylamino DNA can serve as a template for accurate complementary DNA synthesis

(a) Scheme for synthesizing a DNA molecule containing 146 sequential N-acetyl-propargylamino nucleotides for use as a PCR template. First, a ~600 nt DNA molecule is produced by PCR from a plasmid template, completely substituting dTTP with dUTP. One PCR primer is 5' phosphorylated, enabling the selective digestion of one strand by lambda exonuclease (step 1). Next, a 5' FAM-labeled primer is annealed to the template and then extended by 146 nt by Klenow(exo-) using N-acetyl-propargylamino dNTPs to produce propargylamino DNA (paDNA) (step 2). A control reaction is also performed in which no dNTPs are added. After one hour of incubation, ddNTPs are added in excess to both reactions to block the products from elongation in subsequent steps. Next, the dU-containing template is removed by digestion with USER enzyme, enabling isolation of the paDNA elongation product (step 3). Finally, the paDNA products are used as templates for complementary DNA synthesis by Taq to produce a natural DNA product containing a 124 bp region derived from “reading” paDNA (step 4). The natural DNA products are then PCR-amplified and cloned for sequencing (step 5). (b) Capillary electrophoresis of the paDNA primer extension products of step 2. Template-dependent elongation of the FAM-labeled primer by Klenow(exo-) was performed using N-acetyl-propargylamino dNTPs (ii), and a control reaction was also performed without dNTPs (i). (This particular fill-in reaction was only performed once in the context of this experiment.) (c) qPCR amplification curves using the Taq-synthesized complementary DNA produced in step 4 as templates, in duplicate. Amplification is only observed in the product that is derived from propargylamino DNA. 69 clones of the amplicon were sequenced and only 5 mutations were identified in the roughly 8.6 kb of paDNA-derived bases sequenced, implying an error rate of approximately 6 x 10-4/nt.

Supplementary Figure 13 Generation of DNA starter used for multi-step synthesis

A 359 bp double stranded PCR amplicon was obtained from a pET19b template. To prevent extensions of the complementary strand during the synthesis procedure, the amplicon was digested with PstI to generate a 3' overhang that was subsequently blocked with ddTTP by TdT, (eliminating the 3'OH group necessary for further dNTP additions). Finally, the amplicon was digested with BstXI to generate a 3' overhang (5'-ATTT-3') on the other strand that serves as the initial substrate for the multi-step synthesis.

Supplementary Figure 14 The photocleavable linker can be rapidly cleaved using 405 nm light

SDS-PAGE of a photolysis time course of a TdT-oligo extension complex using a 405 nm laser. TdT-dATP was incubated with oligo P2 (5'-FAM-dT60) to form a TdT-primer complex. Subsequently, the complex was irradiated for varying durations using a 405 nm laser. The oligo (P), the reaction product before (0 min), and after multiple irradiation periods up to 4 min are displayed. This experiment was performed twice with similar results.

Supplementary Figure 15 Multiple sequence alignment of reads of the synthesis products

(a) Multiple sequence alignment of all NGS reads of the 10-mer synthesis products with a frequency of at least 0.25%. Single deletions are the most common errors, followed by single insertions caused by non-termination (i.e. double-extension during the same reaction; see Fig. S11). A different type of insertion, which is an artifact caused by nucleotide carryover through the AMPure XP DNA purification between synthesis steps, can be found in the last sequence and is indicated by a grey “G”. Such nucleotide carryover artifacts were identified in 0.7% of all reads and were manually removed before estimation of stepwise yields (see Methods, “Amplification and next-generation sequencing analysis of synthesis products”). (b) Frequencies of all non-singleton reads of the 5'-CCC-3' synthesis products.

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Palluk, S., Arlow, D., de Rond, T. et al. De novo DNA synthesis using polymerase-nucleotide conjugates. Nat Biotechnol 36, 645–650 (2018).

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