[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"doc-detail-43185-en":3,"doc-seo-43185-105":30,"detail-sidebar-cat-0-en-105":91},{"code":4,"msg":5,"data":6},0,"success",{"doc_id":7,"user_id":8,"nickname":9,"user_avatar":10,"doc_module":4,"category_id":11,"category_name":12,"doc_title":13,"doc_description":14,"doc_content":15,"file_id":16,"file_url":17,"file_type":18,"file_size":19,"view_count":20,"is_deleted":4,"is_public":21,"is_downloadable":21,"audit_status":21,"page_count":22,"language":23,"language_code":24,"site_id":25,"html_lang":24,"table_of_contents":26,"faqs":27,"seo_title":13,"seo_description":14,"update_tm":28,"read_time":29},43185,1099514068035,"Ezra","https://ap-avatar.wpscdn.com/davatar_276721f389ce27ea32af1340a28f341c",8,"Research & Report","De novo DNA synthesis using polymerase-nucleotide conjugates","Oligonucleotide synthesis using the nucleoside phosphoramidite method is constrained to short ~200-mers and generates hazardous waste. The work presents a template-independent enzymatic strategy using terminal deoxynucleotidyl transferase (TdT) conjugated to single deoxyribonucleoside triphosphates (dNTPs). After TdT incorporates the tethered nucleotide, the 3′ primer end remains covalently bound to TdT, preventing re-incorporation by other conjugates. Cleavage releases the primer, enabling iterative single-nucleotide extensions in 10–20 s and writing defined sequences, suggesting an enzymatic oligonucleotide synthesizer platform.","© 2018 Nature America, Inc ., part of Springer Nature . All rights reserved .  \nl ett e rs  \nDe novo DNA synthesis using polymerasenucleotide conjugates  \nSebastian Palluk1–3, 12, Daniel H Arlow1,2,4,5, 12, Tristan de Rond 1,2,6, Sebastian Barthel 1–3, Justine S Kang 1,2,7, Rathin Bector 1,2,7, Hratch M Baghdassarian 1,2,8, Alisa N Truong 1,2, Peter W Kim 1,9, Anup K Singh 1,9, Nathan J Hillson 1,2, 10 & Jay D Keasling 1,2,5,7,8, 11  \nOligonucleotides are almost exclusively synthesized using thenucleoside 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.  \nThe overwhelming majority of biological research and bioengineering requires synthetic DNA, including oligonucleotides (oligos) and longer constructs such as synthetic genes and even entire chromosomes 1,2. Massively parallel oligo synthesis3 has dramatically reduced the cost of high-throughput and genome-wide functional screens4 and target capture for next-generation sequencing (NGS) . De novo DNA synthesis also enables other emerging applications such as DNA nanotechnology5 and DNA-based data archiving6.  \nToday, essentially all synthetic DNA is manufactured using thenucleoside phosphoramidite method pioneered by Marvin Caruthersand colleagues over 35 years ago7—a development that marked an inflection point in biological research8. However, after decades of fine-tuning and improvements in liquid handling, the upper limit of phosphoramidite-based oligo synthesis is now about 200–300 nt, in practice9. As a result, longer molecules must be assembled from  \noligos in a process that is failure-prone and not amenable to all target sequences 10, rendering some DNA sequences inaccessible to study.  \nProposals for enzymatic de novo synthesis of oligonucleotides with a defined sequence date back to at least 1962 (refs. 11, 12) . Enzymatic oligo synthesis promises several potential advantages over chemical synthesis: 1) the exquisite specificity of enzymes and mild conditions in which they function may reduce the formation of side products and DNA damage such as depurination, thereby enabling the direct synthesis of longer oligos; 2) reactions take place in aqueous conditions and need not generate hazardous waste; 3) synthesis could be initiated from natural DNA (i.e., DNA without protecting groups on the nucleophilic positions ofthe bases); and 4) enzyme engineering techniques such as high-throughput screens and selections can be employed to optimize the system in ways that are not possible using organic chemistry alone.  \nTerminal deoxynucleotidyl transferase (TdT) is the only known polymerase whose predominant activity is to indiscriminately add deoxynucleotide triphosphates (dNTPs) to the 3′ end of singlestranded DNA 13, making it the natural candidate for use in enzymatic oligo synthesis. However, thus far there have been no demonstrations of a practical oligo synthesis method based on TdT. Detailed proposals to employ TdT for stepwise DNA synthesis using ‘reversible terminator’ dNTPs (RTdNTPs) in a scheme analogous to sequencing by synthesis 14 date back to 1986 (refs. 15–21) . There is at least one obstacle to this approach: RT","cbCainG5nNjesleJ","https://ap.wps.com/l/cbCainG5nNjesleJ","pdf",556578,2,1,9,"English","en",105,"# Background and motivation\n## Limitations of phosphoramidite chemistry\n# Enzymatic synthesis concept\n## Advantages of enzymatic approaches\n# TdT-based synthesis design\n## Template-independent TdT and conjugate mechanism\n# Prior proposals and challenges\n## Reversible terminators and obstacles\n# Experimental performance and implications","[{\"question\":\"Why is phosphoramidite oligonucleotide synthesis limited in length?\",\"answer\":\"It is practical up to roughly 200–300 nucleotides, and longer molecules require assembly from many oligos, which is failure-prone and not compatible with all target sequences.\"},{\"question\":\"How do TdT–dNTP conjugates enable stepwise extension?\",\"answer\":\"Each TdT is conjugated to a single tethered dNTP that can be incorporated into a primer. After incorporation, the 3′ end remains covalently bound to TdT, making it inaccessible to other TdT–dNTP molecules until cleavage releases the primer for the next step.\"},{\"question\":\"What performance and output does the method demonstrate?\",\"answer\":\"TdT–dNTP conjugates extend a primer by one nucleotide quantitatively in about 10–20 seconds. The scheme can be iterated to write a defined sequence, pointing toward an enzymatic oligonucleotide synthesizer.\"}]",1783378283,23,{"code":4,"msg":31,"data":32},"ok",{"site_id":25,"language":24,"slug":33,"title":13,"keywords":34,"description":14,"schema_data":35,"social_meta":86,"head_meta":88,"extra_data":90,"updated_unix":28},"de-novo-dna-synthesis-using-polymerase-nucleotide-conjugates","",{"@graph":36,"@context":85},[37,53,68],{"@type":38,"itemListElement":39},"BreadcrumbList",[40,44,47,50],{"item":41,"name":42,"@type":43,"position":21},"https://docshare.wps.com","Home","ListItem",{"item":45,"name":46,"@type":43,"position":20},"https://docshare.wps.com/document/","Document",{"item":48,"name":12,"@type":43,"position":49},"https://docshare.wps.com/document/research-report/",3,{"item":51,"name":13,"@type":43,"position":52},"https://docshare.wps.com/document/de-novo-dna-synthesis-using-polymerase-nucleotide-conjugates/43185/",4,{"url":51,"name":13,"@type":54,"author":55,"headline":13,"publisher":57,"fileFormat":60,"inLanguage":24,"description":14,"dateModified":61,"datePublished":62,"encodingFormat":60,"isAccessibleForFree":63,"interactionStatistic":64},"DigitalDocument",{"name":9,"@type":56},"Person",{"url":41,"name":58,"@type":59},"DocShare","Organization","application/pdf","2026-07-17","2026-07-06",true,{"@type":65,"interactionType":66,"userInteractionCount":20},"InteractionCounter",{"@type":67},"ViewAction",{"@type":69,"mainEntity":70},"FAQPage",[71,77,81],{"name":72,"@type":73,"acceptedAnswer":74},"Why is phosphoramidite oligonucleotide synthesis limited in length?","Question",{"text":75,"@type":76},"It is practical up to roughly 200–300 nucleotides, and longer molecules require assembly from many oligos, which is failure-prone and not compatible with all target sequences.","Answer",{"name":78,"@type":73,"acceptedAnswer":79},"How do TdT–dNTP conjugates enable stepwise extension?",{"text":80,"@type":76},"Each TdT is conjugated to a single tethered dNTP that can be incorporated into a primer. After incorporation, the 3′ end remains covalently bound to TdT, making it inaccessible to other TdT–dNTP molecules until cleavage releases the primer for the next step.",{"name":82,"@type":73,"acceptedAnswer":83},"What performance and output does the method demonstrate?",{"text":84,"@type":76},"TdT–dNTP conjugates extend a primer by one nucleotide quantitatively in about 10–20 seconds. The scheme can be iterated to write a defined sequence, pointing toward an enzymatic oligonucleotide synthesizer.","https://schema.org",{"og:url":51,"og:type":87,"og:title":13,"og:site_name":58,"og:description":14},"article",{"robots":89,"canonical":51},"index,follow",{"doc_id":7,"site_id":25},{"code":4,"msg":5,"data":92},[93,97,101,105,110,115,120,123,127,130,134],{"id":21,"doc_module":4,"doc_module_name":46,"category_name":94,"show_sort_weight":95,"slug":96},"Story & Novel",90,"story-novel",{"id":20,"doc_module":4,"doc_module_name":46,"category_name":98,"show_sort_weight":99,"slug":100},"Literature",80,"literature",{"id":52,"doc_module":4,"doc_module_name":46,"category_name":102,"show_sort_weight":103,"slug":104},"Exam",70,"exam",{"id":106,"doc_module":4,"doc_module_name":46,"category_name":107,"show_sort_weight":108,"slug":109},5,"Comic",60,"comic",{"id":111,"doc_module":4,"doc_module_name":46,"category_name":112,"show_sort_weight":113,"slug":114},6,"Technology",50,"technology",{"id":116,"doc_module":4,"doc_module_name":46,"category_name":117,"show_sort_weight":118,"slug":119},7,"Healthcare",40,"healthcare",{"id":11,"doc_module":4,"doc_module_name":46,"category_name":12,"show_sort_weight":121,"slug":122},30,"research-report",{"id":22,"doc_module":4,"doc_module_name":46,"category_name":124,"show_sort_weight":125,"slug":126},"Religion & Spirituality",20,"religion-spirituality",{"id":125,"doc_module":4,"doc_module_name":46,"category_name":128,"show_sort_weight":125,"slug":129},"World Cup","world-cup",{"id":131,"doc_module":4,"doc_module_name":46,"category_name":132,"show_sort_weight":131,"slug":133},10,"Lifestyle","lifestyle",{"id":135,"doc_module":4,"doc_module_name":46,"category_name":136,"show_sort_weight":106,"slug":137},19,"General","general"]