DARPA-SN-25-46 FINAL.pdf

Request for Information (RFI)

DARPA-SN-25-46

Physics-based control over de novo synthesis of DNA or RNA

Responses due March 20th, 2025, 4:00 PM ET

POC: Dr. Matthew Pava, DARPA/BTO

E-mail: DARPA-SN-25-46@darpa.mil

URL: https://www.darpa.mil/research#research-opportunities

The Defense Advanced Research Projects Agency (DARPA) Biological Technologies Office (BTO) seeks to gain a better understanding of biotechnological advancements and gaps that could contribute to the ability to synthesize de novo DNA and RNA sequences in vivo. The goal of this RFI is to gather information on the possibility and challenges in developing a platform for in vivo (i.e., in a living cell) synthesis of DNA/RNA, where the nucleic acid sequence is precisely defined by patterns of physical stimulation (i.e., optical, mechanical, sound, electrical, thermal, etc.) rather than using a DNA/RNA template strand. The ultimate aim is for this template-free, de novo synthesis (i.e., capable of producing arbitrary sequences that may not be based on natural sequences) mechanism to produce DNA/RNA that can be translated into functional proteins (Fig 1). DARPA may elect to host a workshop predicated on subject of this RFI in Arlington, VA on May 1st, 2025, and a subset of respondents to this RFI may be invited to attend this workshop, in which case, their travel costs will be reimbursed.

Background

Synthetic DNA and RNA hold promise for providing solutions to global challenges involving rapid prototyping, genetic engineering, and biopharmaceuticals. However, traditional chemical and enzymatic methods for the de novo synthesis of DNA and RNA sequences for novel protein production are constrained by the size and complexity of the desired oligonucleotides, limited scalability, and environmental concerns1,2. Consequently, alternative biocatalytic approaches are needed.

DARPA is looking to challenge the current state of the art technology and ask if it is possible to produce de novo DNA/RNA sequences in vivo

Figure 1. Schematic of de novo DNA or RNA synthesis in vivo via patterns of physical stimulation. Created in BioRender. DARPA BTO. (2024) https://BioRender.com/u13q901 mailto:DARPA-SN-25-46@darpa.mil https://www.darpa.mil/research#research-opportunities via physics-based (i.e., light, mechanical, sound, electromagnetic, thermal, etc) control of cellular processes (Fig 1).

Strategic Approach

The process of synthesizing DNA or RNA requires the coordination of multiple processes and enzymes; this RFI specifically seeks to understand possible approaches to controlling the following processes in vivo with physics-based stimulations. For simplicity, we refer to the agglomeration of all processes and enzymes necessary to execute physics-based, template-free DNA/RNA synthesis merely as a ‘synthase unit.’

1. Initiation:

• What would be a possible strategy for activating and synchronizing the initiation process of DNA/ RNA synthesis in vivo across multiple de novo synthase units within cells and across populations of cells with a physical stimulus?

2. Elongation:

• Describe a possible approach(s) to using temporal sequences of light or other patterns of physical stimuli to enable accurate control over nucleotide incorporation with single base-level precision during the elongation process in cells (Fig 2).

• Terminal deoxynucleotidyl transferase (TdT) is commonly used to synthesize de novo DNA due to its ability to non-selectively add nucleotides to DNA3,4. However, where precise control over the addition of specific bases is necessary, particularly in response to physical stimuli, a more suitable approach may involve designing chimeric enzymes that can selectively bind nucleotides for elongation. For example, could this be achieved by engineering domains from dNTP-binding proteins5,6, nucleoside diphosphate kinases7,8, or RNA-specific ribonucleotidyl transferases9,10? Alternatively, would it be necessary to develop entirely new, non-natural enzymes that can facilitate the precise elongation of DNA or RNA strands?

What would be the potential benefits or consequence of using a “non-traditional” enzyme for the elongation process?

• To make a meaningful impact in fields such as synthetic biology, bio-manufacturing, environmental monitoring, and medicine, especially in the area of RNA therapeutics, the potential system must be capable of synthesizing nucleotide

Figure 2. Potential strategies for light-controlled elongation of de novo DNA or RNA in vivo. Created in https://BioRender.com sequences with real-time functionality. DARPA is interested in the possibilities and limitations of such a system, concerning the following:

Kinetics: What would be the fastest elongation rate (base/min) possible to achieve with precision?

Length: What would be an appropriate length threshold for a de novo DNA/RNA sequence with such a system?

Accuracy: How would the system be optimized for maximum accuracy of nucleotide sequence?

3. Termination:

• Describe methods for controlling transcription termination of nascent, de novo DNA and RNA sequences with a physical stimulus across a population of synthase units within and between cells.

• What post-synthesis modification would need to be considered to ensure translation of the de novo sequence?

• Ideally approaches will produce a functional protein and leave the cell intact with the ability to repeat this process with a different DNA or RNA sequence.

4. State of Art:

• The current state of the art (SOA) methods (Table 1) for de novo DNA and RNA writing focus on combining chemical and enzymatic syntheses to attain sequence selection and high accuracy1. Additionally, de novo DNA/RNA synthesis-to-protein production involves several time-consuming steps, beyond just chemical or enzymatic DNA/RNA synthesis, such as plasmid construction and cell transformation (Table 2). What possible improvements over the current state of the art could be realized by an enzymatic platform that could convert patterns of physical stimuli into de novo DNA or RNA sequences in vivo?

• From the perspective of biomanufacturing workflows (industrial and/or biopharmaceutical), agriculture, and therapeutic delivery to patients, would the ability to directly write DNA or RNA in a living cell using only patterns of physical stimuli to define the sequence create new efficiencies relative to current SOA?

• Are there examples of how the improvements in efficiency over current SOA can be quantified in terms time, money, or safety? Examples ranging from cellular engineering/strain development to the supply chain and logistics of product deliver are of interest.

Table 1. State of the art DNA and RNA synthesis.

Nucleotide Sequence Method Template Rate Length (bp) Accuracy/Efficiency Ref

DNA Phosphoramidite Template-free

20-45 min/base 10 - 200

95.1% 60.6% 36.7%

DNA Taq PCR Template 15-60 sec/kb 5 kb Error rate 1 in

4.3*105 12,13

DNA High fidelity polymerase Template 15-30 sec/kb

Up to 40 kb with optimization

Error rate 1 in 2- 3*106

12,13

DNA DNA Printer Template-free

10-20 min/base 120 ~78% yield for 50 nt oligo 14,15

RNA Phosphoramidite Template-free

20-30 min/base 40 ~37% 16,17

RNA Polymerase (T7) Template ~5 sec/kb Up to 27 kb Error rate 1 in 2 ×

18–20

Table 2. Delivery of DNA/RNA into cells.

Method Size Limit (kb) Efficiency Stability Time to validate success

Cell Types Ref

Electroporation

• 10 -15 kb

• Bacterial artificial chromosome with loss of viability and efficiency

• ~109 cfu/ug pUC19

• 20% in hard to transfect cells

• 106 cfu/ug

Transient or stable

• Transient:

hours to days

• Stable:

weeks

Bacterial, yeast, plant, eukaryotic

21–24

Lipid transfection

• 15-20 kb plasmid • 60-90% Transient or stable

• Transient:

1-3 days

• Stable: 2- 3 weeks

Eukaryotic 25–27

Viral transduction • 10 kb

• Wide variation depending on vector & cell type

Transient or stable

• Transient:

1-3 days

Mammalian and insect

28,29

Bacterial transformation (chemically competent cells)

• 10 kb

• 104 to 108 cfu/ug

DNA

Stable with antibiotic pressure

• 1 day Bacteria 30,31

5. Biosecurity:

• Advances in genomic technologies allow for a deeper understanding of DNA and RNA, but they also catalyze the need for standardized specification and procedures to ensure results are consistent, reliable, and safe. With these rapid advancements, new security vulnerabilities emerge that require novel enabling technologies for the identification and prevention of potential misuse. What should biomonitoring programs aimed at developing guidelines and policy consider when trying to reduce and monitor the risk of a capability that would enable synthetic DNA and RNA production inside a cell line capable of translating those genetic instructions?

• Current frameworks, such as the 2024 Office of Science and Technology Policy (OSTP) Framework for Nucleic Acid Synthesis Screening and the 2023 Health32 and Human Services (HHS) Guidance33, aim to develop guardrails for safeguarding nucleic acid synthesis prior to procurement and use in biological systems. What are some of the biosecurity vulnerabilities arising from engineered in vivo nucleic acid synthesis, that are not addressed in the current frameworks?

While responses focused on cyberbiosecurity mitigations to secure software and hardware systems that would be necessary to deliver physical stimuli are welcome, approaches to securing Generative Optogenetic systems (i.e., cells expressing synthase units) at the level of biology are highly encouraged. Are there examples of additional technologies that could be developed to increase the inherent security of cells expressing Generative Optogenetic synthase units?

Programmatic Approach

Additionally, we hope to receive concise input that would help us structure a feasible and successful program that triggers development of innovative technologies with strategic capabilities.

1. Schedule: Appropriate milestones within the program and reasonable timeframes for the completion of these milestone.

• Other suggestions for how a program could be structured to best allow for solutions to be successful

2. Cost: Minimal dollar amount for the award, for solutions at different stages of maturity (e.g., ideas for promising, novel approaches, preliminary in vitro and in vivo data).

NO CLASSIFIED INFORMATION SHOULD BE INCLUDED IN THE RFI RESPONSE.

It is the submitter's responsibility to clearly label proprietary information contained in the RFI response. DARPA will not disclose information labeled as proprietary.

Responses can address any one or multiple of these considerations. RFI responses should also provide a rough estimate of achievable performance and indicate specific issues to be addressed, such as those listed earlier.

DARPA invites responses from all capable and qualified sources including, but not limited to, universities, University-Affiliated Research Centers, U.S. Government laboratories, Federally Funded Research and Development Centers, and private or public companies.

Workshop

DARPA is considering hosting an invitational workshop on May 1st, 2025 in Arlington, VA with the purpose of reviewing and discussing current and future research relevant to this RFI. DARPA may provide travel reimbursement to a subset of invited responders to RFI for participation in the workshop. Information discussed at this workshop may assist in the formulation of possible future areas of research with the objective of template-free, de novo synthesis mechanism to produce DNA/RNA that can be translated into functional proteins. If DARPA determines to proceed with workshop invitations to RFI respondents, invitations will be sent out by April 1st, 2025.

Format Each submission should be typed in 12-point, single-spaced font on 8.5- by 11-inch pages, with 1-inch margins. All submissions must be electronic, adhere to the content formatting described below, and use one of the following file formats: Adobe PDF or Microsoft Word.

Responses should include:

1- Cover Page (one page)

a. Title

b. Organization

c. Responder’s technical and administrative points of contact (names, addresses, phone numbers, fax numbers, and email addresses)

2- Technical Response to this RFI should be written narratives that provide details and insight to the questions posed, as this will enable an understanding of the current state of the art, identify key limitations and challenges, and explore the possibilities and potential applications of the technology. The information gathered through this RFI will be used to inform the development of a future program.

H1. What would be the objective of the research? Articulate with no jargon.

H2. What is the state-of-the-art and its limits?

H3. What is new about the listed approaches and why would they succeed?

H4. Who cares? If successful, what difference will it make?

H5. What are the risks?

H6. How much will it cost?

H7. How long will it take?

H8. What are the mid-term and final metrics to determine success?

3- References (no limit) 4- Appendix: Responder’s relevant experience and expertise including citations of their relevant peer-reviewed literature. Relevant published papers (limit 3).

Submission All technical and administrative correspondence, questions regarding this announcement, how to respond to this RFI, and submissions themselves should be sent to DARPA-SN-25-46@darpa.mil.

Please refer to “Generative Optogenetics RFI” in all correspondence. Emails sent directly to the Program Manager(s) may result in a delayed response or no response.

Disclaimers and Important Notes This is an RFI issued solely for information and new program planning purposes; it does not constitute a formal solicitation for proposals. This RFI is not tied to any funding for research and submission of proposals is discouraged as they will not be reviewed. In accordance with FAR 15.201(e), responses to this notice are not offers and cannot be accepted by the Government to form a binding contract. Submission is voluntary and is not required to propose to a subsequent Broad Agency Announcement (BAA) (if any) or other research solicitation (if any) on this topic.

DARPA will NOT provide reimbursement for costs incurred in responding to this RFI. NO CLASSIFIED INFORMATION SHOULD BE INCLUDED IN THE RFI RESPONSE. It is the submitter's responsibility to clearly define to the Government what is considered proprietary data.

Any proprietary information should be clearly labeled as “proprietary.” Respondents are advised that DARPA is under no obligation to acknowledge receipt of the information received or provide feedback to respondents with respect to any information submitted under this RFI.

Reference

1. Hoose, A., Vellacott, R., Storch, M., Freemont, P. S. & Ryadnov, M. G. DNA synthesis technologies to close the gene writing gap. Nat. Rev. Chem. 7, 144–161 (2023).

2. Flemmich, L., Bereiter, R. & Micura, R. Chemical Synthesis of Modified RNA. Angew.

Chem. Int. Ed. 63, e202403063 (2024).

3. Barthel, S., Palluk, S., Hillson, N. J., Keasling, J. D. & Arlow, D. H. Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3’ Terminal Structures for Enzymatic De Novo DNA Synthesis. Genes 11, 102 (2020).

4. Kuznetsova, A. A. et al. Insight into the mechanism of DNA synthesis by human terminal deoxynucleotidyltransferase. Life Sci. Alliance 5, e202201428 (2022).

5. Ji, X. et al. Mechanism of Allosteric Activation of SAMHD1 by dGTP. Nat. Struct. Mol. Biol.

20, 1304–1309 (2013).

6. Barnes, C. O. et al. The crystal structure of dGTPase reveals the molecular basis of dGTP selectivity. Proc. Natl. Acad. Sci. U. S. A. 116, 9333–9339 (2019).

7. Postel, E. H., Berberich, S. J., Rooney, J. W. & Kaetzel, D. M. Human NM23/Nucleoside Diphosphate Kinase Regulates Gene Expression through DNA Binding to Nuclease- Hypersensitive Transcriptional Elements. J. Bioenerg. Biomembr. 32, 277–284 (2000).

8. Ernst, O. et al. A genome-wide screen uncovers multiple roles for mitochondrial nucleoside diphosphate kinase D in inflammasome activation. Sci. Signal. 14, eabe0387 (2021).

9. Martin, G. & Keller, W. RNA-specific ribonucleotidyl transferases. RNA 13, 1834–1849 (2007).

10. Martin, G., Doublié, S. & Keller, W. Determinants of substrate specificity in RNA-dependent nucleotidyl transferases. Biochim. Biophys. Acta BBA - Gene Regul. Mech. 1779, 206– 216 (2008).

11. DNA Oligonucleotide Synthesis. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis.

mailto:DARPA-SN-25-46@darpa.mil https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/genomics/pcr/dna-oligonucleotide-synthesis

12. Platinum II Taq Hot-Start DNA Polymerase - US.

https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-enzymes-master-mixes/platinum-hot-start-pcr-enzyme.html.

13. McInerney, P., Adams, P. & Hadi, M. Z. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase. Mol. Biol. Int. 2014, 287430 (2014).

14. Our Synthesizer Devices | Kilobaser - Personal DNA/RNA Synthesizer.

https://kilobaser.com/dna-and-rna-synthesizer.

15. SYNTAX System. DNA Script https://www.dnascript.com/products/syntax/.

16. Francis, A. J. & Resendiz, M. J. E. Protocol for the Solid-phase Synthesis of Oligomers of

RNA Containing a 2’-O-thiophenylmethyl Modification and Characterization via Circular Dichroism. J. Vis. Exp. JoVE 56189 (2017) doi:10.3791/56189.

17. Ryczek, M., Pluta, M., Błaszczyk, L. & Kiliszek, A. Overview of Methods for Large-Scale RNA Synthesis. Appl. Sci. 12, 1543 (2022).

18. Huang, J., Brieba, L. G. & Sousa, R. Misincorporation by Wild-Type and Mutant T7 RNA Polymerases: Identification of Interactions That Reduce Misincorporation Rates by Stabilizing the Catalytically Incompetent Open Conformation. Biochemistry 39, 11571– 11580 (2000).

19. Schelle, B. & Thiel, V. T7 RiboMAX Express: Generation of 27kb in vitro Transcripts in Minutes. https://www.promega.com/resources/pubhub/enotes/t7-ribomax-express-generation-of-27kb-in-vitro-transcripts-in-minutes/ (2002).

20. Sastry, S. S. & Ross, B. M. Nuclease Activity of T7 RNA Polymerase and the Heterogeneity of Transcription Elongation Complexes*. J. Biol. Chem. 272, 8644–8652 (1997).

21. Søndergaard, J. N. et al. Successful delivery of large-size CRISPR/Cas9 vectors in hard-to-transfect human cells using small plasmids. Commun. Biol. 3, 1–6 (2020).

22. Sheng, Y., Mancino, V. & Birren, B. Transformation of Escherichia coli with large DNA molecules by electroporation. Nucleic Acids Res. 23, 1990–1996 (1995).

23. Thermo Scientific Competent Cells - US.

https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-molecular-cloning/competent-cells.html.

24. An Introduction to Electroporation – A Tool for Transfection and Competent Cell Generation.

Cell Science from Technology Networks http://www.technologynetworks.com/cell-science/articles/an-introduction-to-electroporation-a-tool-for-transfection-and-competent-cell-generation-363195.

25. Maeki, M. et al. Development of Polymer–Lipid Hybrid Nanoparticles for Large-Sized Plasmid DNA Transfection. ACS Appl. Mater. Interfaces 16, 2110–2119 (2024).

26. TransFectinTM Lipid Reagent | Bio-Rad. https://www.bio-rad.com/en-us/product/transfectin-lipid-reagent?ID=680dace0-c2a1-451c-ba7f-d15a2294aa52.

27. Transfection Guide | Overview of Transfection Methods | Promega.

https://www.promega.com/resources/guides/cell-biology/transfection/.

28. Viral Transfection - US. https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/methods/viral-transfection.html.

29. Selecting a Viral DNA Delivery System - US.

https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/methods/viral-transfection/selecting-a-viral-dna-delivery-system.html.

https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-enzymes-master-mixes/platinum-hot-start-pcr-enzyme.html https://www.thermofisher.com/us/en/home/life-science/pcr/pcr-enzymes-master-mixes/platinum-hot-start-pcr-enzyme.html https://www.dnascript.com/products/syntax/ https://www.promega.com/resources/pubhub/enotes/t7-ribomax-express-generation-of-27kb-in-vitro-transcripts-in-minutes/ https://www.promega.com/resources/pubhub/enotes/t7-ribomax-express-generation-of-27kb-in-vitro-transcripts-in-minutes/ https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-molecular-cloning/competent-cells.html https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-molecular-cloning/competent-cells.html http://www.technologynetworks.com/cell-science/articles/an-introduction-to-electroporation-a-tool-for-transfection-and-competent-cell-generation-363195 http://www.technologynetworks.com/cell-science/articles/an-introduction-to-electroporation-a-tool-for-transfection-and-competent-cell-generation-363195 http://www.technologynetworks.com/cell-science/articles/an-introduction-to-electroporation-a-tool-for-transfection-and-competent-cell-generation-363195 https://www.bio-rad.com/en-us/product/transfectin-lipid-reagent?ID=680dace0-c2a1-451c-ba7f-d15a2294aa52 https://www.bio-rad.com/en-us/product/transfectin-lipid-reagent?ID=680dace0-c2a1-451c-ba7f-d15a2294aa52 https://www.promega.com/resources/guides/cell-biology/transfection/ https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/methods/viral-transfection.html https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/methods/viral-transfection.html https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/methods/viral-transfection/selecting-a-viral-dna-delivery-system.html https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/methods/viral-transfection/selecting-a-viral-dna-delivery-system.html https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/transfection-basics/methods/viral-transfection/selecting-a-viral-dna-delivery-system.html

30. drsnowflack. What is transformation efficiency and why is it important? The Official Blog of Edvotek® https://blog.edvotek.com/2021/11/11/what-is-transformation-efficiency-and-why-is-it-important/ (2021).

31. Addgene: Protocol - Bacterial Transformation.

https://www.addgene.org/protocols/bacterial-transformation/.

32. Fast Track Action Committee Synthetic Nucleic Acid Procurement Screening. Framework For Nucleic Acid Synthesis Screening. (2024).

33. U.S. Department of Health & Human Services. Screening Framework Guidance for Providers and Users of Synthetic Nucleic Acids. (2023).

https://blog.edvotek.com/2021/11/11/what-is-transformation-efficiency-and-why-is-it-important/ https://blog.edvotek.com/2021/11/11/what-is-transformation-efficiency-and-why-is-it-important/ https://www.addgene.org/protocols/bacterial-transformation/