DARPA-PS-26-10.pdf

Program Solicitation

Biological Technologies Office (BTO)

Generative Optogenetics (GO)

DARPA-PS-26-10

December 19, 2025

Table of Contents

1. Program Information

1.1. Background

1.2. Acquisition strategy

1.2.1. Abstracts

1.2.2. Oral Presentations

1.2.3. Phase 1 (12 months)

1.2.4. Phase 2 (30 months)

1.3. Program Description/Scope

1.3.1. Overall Scope of the GO Program

1.3.2. Research Objective 1 (RO1): De Novo Synthesis

1.3.3. Research Objective 2 (RO2): Error Mitigation

1.3.4. Program Constraints / Out of Scope

1.3.5. Program Structure

1.3.6. Program Security

1.3.7. Program Metrics

1.3.8. Program Milestones & Deliverables

1.4. Technical Guidelines for Proposals

1.5. Advisory and Working Groups

1.5.1. Biosecurity Working Group (BSWG)

1.5.2. Regulatory Policy Working Group (RPWG)

1.5.3. Independent Commercialization and Consulting Group (ICCG)

2. PS AUTHORITY

2.1. PS Procedure

3. ELIGIBILITY INFORMATION

3.1. Eligible Applicants

3.1.1. Federally Funded Research and Development Centers (FFRDCs) and Government Entities .22

3.1.2. Other Applicants

3.2. Organizational Conflicts of Interest (OCI)

4. GUIDELINES FOR ABSTRACTS, ORAL PRESENTATIONS, AND PROPOSALS

4.1. General Guidelines

4.2. Abstract Content

4.3. Abstracts – Process and Basis of Evaluation

4.4. Oral Presentations Content

4.5. Oral Presentations – Process and Basis of Evaluation

5. AWARDS

5.1. General Guidelines

5.2. Controlled Unclassified Information (CUI) and (CTI) on Non-DoD Information Systems

5.3. Representations and Certifications

5.4. Competition Sensitive Information

5.5. Intellectual Property / Data Rights

5.6. Procurement Integrity Act (PIA)

5.7. Fundamental Research Risk-Based Security Review Program (FRRBS)

6. PS DEFINITIONS

7. Appendix A: Checklist ................................................................................................................................i

PROGRAM SOLICITATION OVERVIEW INFORMATION

Federal Agency Name – Defense Advanced Research Projects Agency (DARPA), Biological Technologies Office (BTO)

Funding Opportunity Title – Generative Optogenetics Announcement Type – Initial Announcement Funding Opportunity Number – DARPA-PS-26-10 Dates o Posting Date: December 19, 2025 o Proposer Workshop: January 7, 2026 o Questions Due Date: January 12, 2026 by 5:00 PM, Eastern Time (ET) o Abstracts Due Date and Time: January 16, 2026 by 5:00 PM (ET) o Oral Presentations Due Date and Time: By Government request, estimated 29 days after Abstract submission The Defense Advanced Research Projects Agency (DARPA) is soliciting innovative concepts to design a protein complex that can be expressed in vivo (i.e., in a living cell) and responds to optical signals to synthesize a corresponding DNA or RNA sequence. This technology should enable massless transfer of genetic information to cells expressing the protein complex via a template-free mechanism that incorporates nucleotide bases in response to specific optical signals (e.g., different wavelengths of light). The GO program is focused on two main Research Objectives (ROs): (1) achieving the core capability of synthesizing DNA/RNA directly in living cells using optical signals, and (2) developing error mitigation methods that ensure high-fidelity nucleic acid synthesis. The acquisition process for GO will proceed through a two-stage process, starting with (1) written abstracts which will inform invitations to brief (2) an Oral Proposal Package (OPP) at DARPA.

Multiple awards are anticipated.

Total Funding - Phase 1 fixed price awards of $1.7M or $1.99M depending on RO selected.

DARPA anticipates multiple awards for Phase 2.

Types of instruments that may be awarded – Other Transaction (OT) for Prototype agreements Agency Contact

The Solicitation Coordinator for this effort can be reached at:

GO@darpa.mil

DARPA/BTO

ATTN: DARPA-PS-26-10

675 North Randolph Street Arlington, VA 22203-2114

Attachments A: Abstract Summary Slide Template B: Abstract Template C: Model Other Transaction (OT) Streamlined, Fixed D: Cost Spreadsheet Template E. Representations and Certifications F: GO CUI Guide mailto:GO@darpa.mil

PROGRAM SOLICITATION

Defense Advanced Research Projects Agency (DARPA) Generative Optogenetics (GO)

1. Program Information

1.1. Background

Synthetic DNA and RNA are essential molecules for technologies that address critical global and national security challenges related to resilient supply chains, advanced materials manufacturing, agriculture, and human health. However, traditional methods for the de novo synthesis of DNA and RNA sequences are constrained by the size and complexity of the desired oligonucleotides, limited scalability, and environmental concerns. Current methods for nucleic acid synthesis fall into two categories: catalyzed by proteins (i.e., enzymatic) or synthetic chemistry. Naturally, cells replicate copies of their genomes and produce RNA via enzymatic processes, achieving remarkable speed and accuracy with error rates as low as 1 in 1010. This fidelity is driven by mechanisms such as template-directed polymerization, proofreading freshly synthesized nucleic acids, and DNA mismatch repair. However, none of these processes are capable of de novo synthesis because they all require a nucleic acid template.

For template-free production of nucleic acids, chemical phosphoramidite synthesis is the gold standard.

Considering the entirety of the phosphoramidite process, DNA and RNA synthesis rates can take 20–45 min per base with an error rate of 0.01 per base. Phosphoramidite synthesis is constrained by sequence length (200 bp for DNA, 40 b for RNA) and requires extensive downstream processing to make nucleic acids biologically compatible. Recent advancements in cell-free enzymatic synthesis have improved de novo synthesis rates to 1.3–10 minutes per base, with error rates ranging from 0.01 to 0.2 per base. However, enzymatic methods remain limited by fragment size and centralized production, requiring additional steps for assembly and delivery into cells. Furthermore, while approximately 70% of human protein-coding RNAs are ≤3 kb in length, current state-of-the-art methods cannot achieve single-shot synthesis of such sequences. Instead, substantial downstream efforts are required to assemble shorter fragments into longer protein-coding sequences, a process that must occur outside the cell. This process can take anywhere between 10 days to over a month. No existing technology enables massless information transfer to relay genetic instructions to living cells. All current approaches require some mechanism predicated on moving matter that encodes the genetic information, typically DNA or RNA nucleic acids, across biological barriers like a cell wall/membrane.

Generative Optogenetics (GO) program aims to create a molecular machine that can be expressed in living cells and provide a mechanism for transducing genetic information transmitted masslessly via optical signals into the nucleic acid sequences (DNA and/or RNA), which are the native information storage for all known life. Such a capability will create a direct interface between computers used to design genetic sequences and living cells that operate on those sequences. Technology developed on GO will enable precise influence over cellular behavior by facilitating genetic programing with single-cell spatial resolution, temporal precision to deliver different messages to a cell sequentially, and remote, scalable dissemination of genetic instructions.

At its core, GO addresses the high-risk challenge of developing a novel, open-ended genetic control platform that functions in vivo (i.e., within a living cell) to accelerate the transmittal of genes to living systems. If successful, this technology is anticipated to unlock a foundational capability with ramifications for medicine, agriculture, and manufacturing, while diminishing reliance on brittle supply networks that become untenable for long distance operations, like extended human spaceflight. GO is solely focused on building a proof-of-concept to address this high-risk challenge, but performers that meet the program’s ambitious goals will pave the way for broadly transformative applications.

1.2. Acquisition strategy

Abstract submission deadline is anticipated to be January 16, 2026, 5:00PM (Eastern Time). Abstracts will be reviewed by the Government; if selected, the proposer will be asked to brief an Oral Presentation Package

(OPP). OPPs will be reviewed by the Government, and if selected, they may result in a Phase 1 award of an Other Transaction (OT) for Prototype (P) and eligibility to participate in future Phases of the program. As the GO acquisition strategy moves forward from abstracts to OPPs and finally to the Concept Design Review (CoDR) in Phase 1, DARPA expects that each team’s approach to developing an optically controlled, template-free system for synthesizing nucleic acid sequences within living cells will solidify, and consequently, the performer teams’ composition will become more finalized.

This PS encourages solutions from all responsible sources capable of satisfying the Government’s needs, including large and small businesses, nontraditional defense contractors as defined in 10 U.S.C. § 3014, and research institutions as defined in 15 U.S.C. § 638.

To facilitate this objective, the Government will use the following acquisition process for GO:

1.2.1. Abstracts

Through the forthcoming program solicitation, the Government will require proposers to submit 5-page Abstracts as the first step in the acquisition process. The Government will review all submitted Abstracts to gain a high-level understanding of each proposing team’s strategy to develop a prototype molecular machine capable of functioning in a living cell to transduce genetic information encoded in optical signals into protein-coding nucleic acid sequences. Predicated on the review of submitted abstracts for their technical comprehension and ability, the Government will decide to invite a subset of proposing teams to submit and brief OPPs in an Oral Presentation.

1.2.2. Oral Presentations

Upon the Government’s request, proposers will have the opportunity to submit and present their OPP to the DARPA program team. The Government will evaluate all OPPs from teams whose abstracts were selected in the first stage of the acquisition process. Oral presentations of the OPP are required, and these will afford the Government an opportunity to ask clarifying questions of the briefing teams. The OPP, including the briefing, will provide the detailed information needed for Phase 1 selection decisions, requiring significantly more detail than the Abstracts. The Government anticipates that teams selected on the basis of the OPP will be given fixed-price OT-P awards to address Phase 1 goals/metrics over a 12-month period of performance.

While awards made following the OPP will be for Phase 1 only, the content of the OPP should describe each team’s overall plan to develop their molecular machine, including planned Phase 2 effort, with particular emphasis placed on how the proposed Phase 1 effort will de-risk and refine the strategy for Phase 2. It is not expected that Phase 2 plans will be finalized at this time, but proposing teams should articulate a reasonably detailed draft, including a design and test plan for their proposed mechanism of transducing genetic information transmitted masslessly via optical signals into nucleic acid sequences. The draft Phase 2 plan should align to the goals of the program for demonstrating this transduction mechanism in a living cell. Based on this draft plan for Phase 2, the OPP must describe a set of clear, finalized Phase 1 tasks, and it must justify how these tasks will de-risk and inform the finalization of the Phase 2 plan by month 9 after award.

1.2.3. Phase 1 (12 months)

Performers will refine and de-risk their work plan for Phase 2. The revised work plan will be provided to DARPA in a written form (i.e. a Task Description Document, TDD, that may be included in the OT agreement) as well as in an oral presentation. All Phase 1 performers will present their final revised Phase 2 plans during the CoDR, scheduled approximately ~9 months after the Phase 1 agreement award.

1.2.4. Phase 2 (30 months)

This phase DARPA may be negotiated Phase 2 using the existing OT agreement from Phase 1. Performers advancing to this phase will execute their technical plan for developing a prototype molecular machine capable of functioning in a living cell to transduce genetic information encoded in optical signals into protein coding nucleic acid sequences.

1.3. Program Description/Scope

1.3.1. Overall Scope of the GO Program

The DARPA GO program aims to develop a protein complex, referred to here as a nucleic acid compiler (NAC), that can be expressed within living cells to allow an end user to program genetic instructions into those cells, template-free, using nothing but light to transfer the genetic information to the cells (Figure 1). The central challenge of developing the NAC involves integrating protein domains / subunits for precise optical responsiveness (i.e., optogenetic domains), substrate binding, and enzymatic activity into a functional complex of proteins (i.e., a holoenzyme). While many of these domains have precedence as either engineered or naturally occurring proteins, the challenge lies in developing the interoperability and seamless integration of these domains into a functional holoenzyme, the NAC. Advances in computational design, which allow for accurate prediction of protein structures and binding interactions, are essential for optimizing substrate binding sites, allosteric interactions, and domain integration. These computational tools are crucial for designing the NAC to respond rapidly and predictably to optical signals, enabling the synthesis of long, accurate nucleic acid sequences that can precisely alter cellular function as intended. Moreover, expression of the NAC itself must not be deleterious to host cell function or viability.

To develop the NAC, the GO program consists of two Research Objectives (ROs):

1.3.2. Research Objective 1 (RO1): De Novo Synthesis

All GO performers MUST address RO1, which focuses on developing the core capability of the NAC for template-free DNA or RNA synthesis, where optical inputs precisely dictate the sequence of the nucleic acid produced by the NAC in a living cell. A NAC can be designed using a variety of architectures, ranging from extremes of a single, monolithic protein comprised of multiple domains to multi-unit complex (Figure 2). To accomplish this, performers will need to solve three critical challenges: achieving multiplexed optogenetic control, ensuring stability and the precise polymerization of the NAC-nucleic acid sequences, and successfully integrating the molecular components into the NAC.

Figure 1. Overview of the GO program. The goal of the program aims to create a protein complex that is expressed within living cells, which converts optical signals (e.g., light pulses) into nucleic acid sequences that the cell can process using its natural transcription and translation mechanisms.

1.3.2.1. Multiplexed Optogenetics

Achieving distinct multiplexed optical programming of the NAC presents a significant challenge, as it requires precise engineering of multiple protein domains capable of responding to distinct wavelengths of light.

Currently optogenetic systems have been demonstrated to support up to three distinguishable wavelengths (red, green, and blue) within a cell, but expanding this capability is essential for enabling the NAC to incorporate nucleotides with high precision. This expansion may involve optimizing existing optogenetic domains or developing new ones with improved photophysical properties, such as enhanced spectral separation, faster on/off kinetics, and reduced phototoxicity. By leveraging photons as massless information carriers, these optogenetic domains must facilitate precise molecular motion and interaction, ensuring accurate nucleotide incorporation and enzymatic activity. Computational protein design tools and directed evolution approaches offer potential strategies to overcome these limitations, enabling the multiplexed optical control required for the NAC to function effectively.

1.3.2.2. Stable and Precise Polymerization

The NAC must achieve precise polymerization, including initiating synthesis, maintaining processivity to stabilize elongating nucleic acid sequences, and efficiently releasing the synthesized strand to meet program metrics for length and accuracy. The NAC design may need to include strategies to address the challenge of selectively binding the correct nucleotide substrate at the correct time from the mixture of these substrates that exists within the cellular environment. Overcoming this challenge will be necessary for the NAC to achieve desired sequence accuracy metrics for the GO program. Additionally, the stability of the complex formed between the NAC and the nucleic acid sequence it is synthesizing must be sufficient to avoid unwanted dissociations that will result in truncated sequences. Similarly, NACs that synthesize single-stranded nucleic acids will need to overcome issues associated with secondary structures (e.g., hairpin loops) in the DNA/RNA molecule that could interfere with continued synthesis. Achieving stable NAC-based nucleic acid synthesis may necessitate designs that incorporate accessory subunits/domains to improve processivity by holding on to the newly synthesized strand and/or single-stranded binding proteins/domains that hinder the formation of problematic secondary structure in DNA/RNA molecules. Finally, the performers will need to resolve the challenge of releasing synthesized sequences, which may involve strategies such as natural termination signals or engineering inducible cleavage mechanisms.

1.3.2.3. Integration of Molecular Components

A fully functional NAC must integrate optogenetic, substrate binding, catalytic, and other domains into a cohesive holoenzyme capable of precise and predictable operation. This integration presents significant

Figure 2. A non-exhaustive subset of possible high-level designs for a NAC.

challenges, as the domains must interact seamlessly to ensure accurate nucleotide incorporation and overall system functionality. For example, optogenetic domains may need to regulate substrate binding to ensure that nucleotide incorporation into the DNA or RNA sequence aligns precisely with the optical illumination pattern.

Similarly, designs involving protein subunit binding must coordinate these interactions with substrate binding domains to maintain synchronization and fidelity. Addressing these challenges may involve strategies such as identifying domains that interact effectively to control the NAC, ensuring synchronous activation and deactivation of multiple NACs within a living cell, and optimizing domain interfaces for efficient communication. Potential approaches include leveraging computational tools to map allosteric pathways, modeling molecular motion to predict domain interactions, and employing high-throughput empirical methods to refine and validate integration strategies.

1.3.3. Research Objective 2 (RO2): Error Mitigation

OPTIONAL, GO performers may elect to address RO2 in addition to RO1. Note that GO performers shall not pursue RO2 without addressing RO1. RO2 addresses the challenge of achieving high-fidelity synthesis in NACs by incorporating mechanisms to detect and filter out sequence errors. Some applications of GO technology will necessitate NACs capable of synthesizing longer sequences, and it is anticipated that increasing the length of the sequence will increase the likelihood it contains errors. To this end, RO2 aims to investigate the tradeoffs involved in designing a NAC with enhanced error detection capabilities to meet stricter error tolerance requirements, including how these design choices impact overall NAC performance.

There are several potential approaches to address RO2 (Figure 3), an example includes developing double-stranded synthesis methods that incorporate components such as mismatch-binding proteins (e.g., MutS homologs). These proteins can either flag errors for downstream correction or be engineered to degrade faulty sequences, ensuring that only high-fidelity nucleic acid strands are retained. Other strategies may include utilizing base editors to identify nucleotide incorporation errors or synthesizing palindromic sequences that fold onto themselves to increase error detection. RO2 provides an opportunity to explore innovative solutions to error mitigation while considering the tradeoffs in performance, complexity, and scalability inherent to these approaches.

Figure 3. Notional non-exhaustive and non-mutually exclusive set of mechanisms for introducing error detection and mitigation to a NAC design. A, NACs synthesizing double-stranded molecules could include mechanisms based on base-pair mismatch detection;

B, palindromic nucleotide sequences could be interleaved with mismatch detection inspired mechanisms; C, iterative toggling between a ‘write’ and ‘error recognition’ mode. Proposals should clearly explain their approach along with its relative strengths and weaknesses.

1.3.4. Program Constraints / Out of Scope

To ensure alignment with program objectives, certain approaches are deemed out of scope. Specifically, the program will not support bioprospecting to identify or characterize wholly new domains or proteins from natural systems, nor exploratory work focused on phenomenological or mechanistic characterization of novel proteins that respond to light or other physical signals. Additionally, approaches requiring in vitro synthesis steps, such as lysis and ligation/annealing of smaller oligonucleotides, are excluded, as are designs seeking to create entire systems that operate in-parallel to the central dogma. While synthesis of nucleic acid sequences built from non-canonical bases is permissible, these sequences must be converted into canonical DNA/RNA for transcription or translation by the host cell’s existing enzymes. While GO performers may work in most common cell chassis including yeast, bacteria, plants, immortalized mammalian cell lines (e.g., HEK293, CHO, etc), or human induced pluripotent stem cells (iPSCs), the development of the NAC in embryonic stem cells (ESCs) is explicitly prohibited. No genes that are export-controlled or restricted for biosafety reasons will be within scope. Furthermore, substantial hardware development, including novel optical systems or sequencing platforms, is unnecessary for success on GO and is therefore excluded. Tasks to develop or discover completely novel mechanisms for error mitigation or physical signal modalities are also out of scope, though additional physical signal modalities beyond optical signals may be considered if scientifically justified and plausible within a transitional use case. All human subjects research and animal subjects research is explicitly out of scope for GO.

1.3.5. Program Structure

The GO Program will proceed across two phases spanning a total period of performance of 42 months. Phase 1 (12 months) focuses on refining molecular components and de-risking integration strategies, while Phase 2 (30 months) focuses on integration and demonstration of the NAC platform in vivo.

1.3.5.1. Phase 1 (12 months)

During Phase 1, performers will focus on refining and demonstrating the critical molecular components necessary for a functional NAC, including optogenetic domains, enzymatic polymerization, and error mitigation mechanisms. This phase is structured to culminate in a month 9 technical milestone (section 1.3.7), Figure 4. The GO program structure.

providing teams with an opportunity to de-risk their technical approach before advancing to the more complex integration work required in Phase 2. Phase 1 also allows teams to address any capability gaps identified through their progress and make adjustments to their approach. For example, experimental results from Phase 1 may reveal the need for additional expertise or resources, which can be addressed by reorganizing the team, such as adding a Co-PI or sub-awardee for Phase 2. This expertise could come from other Phase 1 participants or external collaborators. The final three months of Phase 1 will focus on the initial integration of technologies into a prototype NAC.

1.3.5.2. Concept Design Review (CoDR; month 9)

Performers must present a refined Phase 2 plan during a CoDR at month 9, including any planned restructuring of their team. The CoDR serves as a critical down-selection point, requiring performers to present compelling technical plans supported by demonstrable progress in Phase 1 to de-risk those plans. At the CoDR, performers will need to communicate a project plan, including any changes in teaming, to establish confidence that the technical work is achievable within the required timeframe for the program. Key technical and project risks will be evaluated to ensure that only the most viable and impactful approaches are selected to advance, contributing to the overall success of the program. The presentation and accompanying written material on the performer’s concept at the design review will include a refined Phase 2 strategy, complete work plan and cost proposal for the revised Phase 2 work plan, a Science and Technology Protection Implementation Plan for safeguarding program components, and a data rights plan (section 1.3.6).

1.3.5.3. Phase 2 (30 months)

Phase 2 focuses on integration of the components developed in Phase 1 to create a functional NAC and ultimately demonstrate functionality within a living cell. The first major technical milestone in Phase 2 (month

19) requires performers to demonstrate their NAC design either cell-free (in vitro) or in vivo. However, by the second milestone (month 31) all demonstrations for both RO1 and RO2 must be completed within a living cell, showcasing the full functionality of the NAC platform. Components of research conducted in Phase 2 will be categorized as controlled unclassified information (CUI) since a functional NAC will have similar metrics to export controlled synthesizers.

1.3.5.4. Additional program events

Program Workshops:

Phase 1: To support the development of a research community around GO technology and to facilitate team refinement during Phase 1, the program incorporates a DARPA-sponsored workshop scheduled for month 5.

All Phase 1 performers will be required to present their research at this event, which will also be open to relevant research communities beyond GO performers. The workshop will serve as a platform for performers to identify talent to augment their teams and foster discussions on barriers to adoption and ethical development of GO technology. DARPA will determine attendees, but Phase 1 performers will provide statements of interest/disinterest regarding abstracts submitted by non-performers to inform whether their presentations align with GO program goals.

Phase 2: Workshops will continue annually in Phase 2 to accelerate GO technology adoption and expand its impact. These events will showcase performer progress and remain open to the broader community to help address challenges and identify organizations with critical capabilities. While team restructuring becomes more challenging in Phase 2, the month 19 workshop will support this process. Performers will be expected to continue providing feedback to DARPA in the form statements of interest/disinterest on research synopsis submitted by the broader community regarding their interest in proposed presentations. Workshops in months 31 and 41 will increasingly emphasize commercial and research applications of GO technology, focusing on transitioning fundamental advancements beyond the program’s scope.

Pitch Events:

Technologies emerging from GO will have an enormous range of potential applications, likely necessitating the commercial transition from the program. Beginning in Phase 1, performers will develop commercialization strategies with the guidance of the Independent Commercialization and Consulting Group (ICCG; Section 1.5.3). Performers will be expected to attend commercialization workshops that begin in Phase 1 and present their business hypotheses at “Pitch Events” starting in Phase 2. These workshops and Pitch Events will be hosted by the ICCG throughout the program and GO performers will be expected to progressively revise their commercialization strategy. Importantly, the ICCG workshops and Pitch Events are a program-wide resource to aid technical performers in developing ideas and strategies to commercialize the technology they develop on GO.

1.3.6. Program Security

Phase 1: Will be conducted as unclassified fundamental research, allowing teams the flexibility to de-risk and refine their technical approaches. However, performers must submit a draft Phase 2 CUI S&T PIP by month 6, with a finalized version due by month 9. This plan must include a timeline for acquiring CUI-compliant systems and equipment, data protection requirements, and other security measures.

Phase 2: Will require performers to implement the S&T PIP to safeguard all information, materials, and processes generated during the program, particularly the DNA sequence of the NAC. Performers are subject to controls outlined in the program-specific CUI guide, which provides a framework for identifying, protecting, and marking CUI in accordance with DoD policies and security classification guides. The scope of protection includes all aspects of NAC sequence development, design, optimization, and integration into living cells, with strict prohibitions on public disclosure or publication of the NAC sequence.

Throughout the program performers are encouraged to publish findings, however, all publications, external engagements, and investor discussions must be coordinated with DARPA and the Program Security Officer (PSO) to ensure compliance with CUI guidelines and export control regulations. Additionally, all publications must be submitted to DARPA’s Public Release Center (DISTAR) for review and approval prior to public dissemination.

All individuals accessing CUI information must complete approved training and ensure that sensitive information is safeguarded on systems compliant with NIST 800-171 standards. This security strategy balances the need for commercialization with the protection of sensitive information, ensuring the program’s success. A detailed CUI guide is provided as an attachment to this solicitation and further described in section 5.2.

1.3.7. Program Metrics

Table 1. GO program metrics and milestone by RO.

Table 1. GO program metrics by milestone and RO.

The GO program contains four major technical milestones spread across the two program phases, and each of these milestones are associated with a set of quantitative metrics (Table 1) designed to assess technical progress on the program. Ultimate success on the program involves meeting or exceeding the final metrics in Phase 2 via NAC design that functions in a living cell (i.e., in vivo) to synthesize nucleic acids template-free. Of note, the metrics listed in Table 1 represent the minimal set of metrics used to define success on the program, and these DARPA-defined metrics are system-level requirements that are generally agnostic to performer-specific approaches. Proposing teams are strongly encouraged to include additional quantitative metrics to characterize success reflective of specific aspects of their unique NAC design. Similarly, DARPA reserves the right to include additional metrics to awards on a per team basis as necessary to manage risks unique to individual teams’ different technical strategies for designing a NAC.

1.3.7.1. Phase 1 metrics

In Phase 1, RO1 focuses on de-risking the integration of NAC components and demonstrating critical capabilities. By month 9, all performers must demonstrate optogenetic domains capable of responding to at least four distinct wavelengths of light with minimal (≤ 1%) co-activation. Meeting this milestone will establish the minimal amount of confidence required to advance teams to Phase 2 because co-expression of multiple optogenetic domains will be required to enable precise incorporation of each nucleotide base into a nucleic acid sequence. Additionally, performers must show that these optogenetic domains can be integrated into a polymerase to control its function. This integration is essential to show that the optogenetic domains can regulate nucleotide incorporation in response to optical signals, which is a critical step in de-risking the development of the NAC for Phase 2.

Performers pursuing RO2 must meet the RO1 metrics, and they must also demonstrate a mechanism for error detection that is highly sensitive. When multiple NACs are expressed in a single cell, less specific error detection mechanisms may mistakenly label correct sequences as errors, reducing the yield of sequences per cell. The metrics for RO2 are established under the assumption that over assessing errors will limit yield of sequences per cell, if error containing sequences are filtered out of the population. However, the hindrance on yield could be overcome by increasing the expression level of NAC proteins in the cellular chassis. Thus, RO2 metrics at this milestone are established to bias development toward highly sensitive approaches. All Phase 1 must be achieved using in vitro (cell-free) preparation, with optional in vivo results for additional validation.

The final three months of Phase 1 will focus on initial integration of technologies into a prototype NAC and preparation for Phase 2.

1.3.7.2. Phase 2 metrics

Phase 2 builds on Phase 1 progress and focuses on developing a fully functional NAC capable of synthesizing nucleic acid sequences in vivo. By the month-19 milestone, RO1 performers must demonstrate a NAC capable of integrating four optogenetic domains to regulate nucleotide incorporation into a 300-mer oligonucleotide.

RO2 performers must additionally show a 10% relative reduction in error compared to designs without error mitigation mechanisms. For RO1, the month-19 milestone can be achieved through either in vitro or in vivo experiments; however, RO2 performers must demonstrate error reduction metrics using in vitro data.

Metrics for the months 31 and 41 milestones require performers to demonstrate synthesis of longer nucleic acid sequences (up to 6 kb for RO2) with significantly reduced error rates. RO2 metrics supersede RO1 metrics at these milestones, with relaxed synthesis rate requirements to accommodate error mitigation mechanisms.

All month 31 and 41 milestone data must come exclusively from in vivo experiments. A key attribute of GO systems is their ability to achieve short-latency sequential transmission of multiple genetic sequences to living cells. As part of the month 41 milestone, all GO performers must demonstrate the capability to write two distinct nucleic acid sequences to the same cell or population of cells, with less than 1 hour required to reset the system between write events. Error rate metrics for the months 31 and 41 milestones represent absolute maximum error rates but do not prescribe sequence-specific error rates. Performers will be required to characterize sequence-specific error rates, particularly for sequences known to be problematic for de novo synthesis technologies, such as those with high GC content. Additional guidance and a common set of test sequences will be provided by DARPA in Phase 1 ahead of the CoDR, allowing performers to refine their approaches to address sequence-specific challenges in vivo.

1.3.8. Program Milestones & Deliverables

A detailed breakdown of tasks, deliverables and reporting requirements are presented in Table 2. In addition to the milestones listed below, performers will be expected to provide monthly and quarterly status updates to DARPA, including technical and financial summary reports.

Table 2. GO Milestones and Deliverables.

Phase 1 (12 mo.)

Month Milestone Deliverables/Exit Criteria

1 Phase 1 Kickoff Slides detailing the Phase 1 project plan and summarizing the initial plan for Phase 2.

5 Workshop #1

Approximately 8 weeks (40 working days) prior to the workshop, provide interest/disinterest feedback on research synopses submitted by the broader community to DARPA.

At least 3 weeks (15 working days) prior to the workshop, performers will submit slides to DISTAR and provide them to DARPA: Present at a DARPA-hosted workshop slides including details of their technical plan highlighting risks and potential gaps in approach/design to help identify potential organizations not selected for Phase 1 that may possess critical technologies to support their technical plan.

There is No Milestone Associated with this Deliverable

Submit a Phase 2 CUI S&T PIP, to include a timeline for acquiring CUI compliant equipment/systems, data requirements, etc.

There is No Milestone Associated with this Deliverable

Revised Phase 2 Task Description Document (TDD) for pursuing either RO1 or RO2.

Cost Proposal for Phase 2.

Slides for refined Phase 2 plan that include details for all experimental components, timeline for development, expected costs for Phase 2, potential risks and mitigation strategies, team capabilities, and notional transition plan for commercialization.

Highlighting any changes from initial proposal.

9 Technical Milestone #1

Technical report on molecular components developed for NAC design assembly, focused on performer-specific RO metrics. Note: Performers selecting RO2 must also demonstrate RO1 metrics.

RO1 & RO2: Demonstrate in vitro 1% co-activation in 4 co-expressed optogenetic domains and construct 1 Opto-gated template-free polymerase

RO2 only: Demonstrate in vitro error mitigation mechanisms with 99% sensitivity and 80% specificity

Concept Design Review (CoDR)

Present slides for refined Phase 2 plan and review TDD and Cost Proposal with DARPA. Finalize CUI S&T PIP and get approval from DARPA.

Phase 2 (30 mo.)

Month Milestone Deliverable/Exit Criteria

Phase 2 Kickoff and Pitch Event #1

Kickoff presentation of detailed technical plan, including preliminary data generated in Phase 1.

Pitch deck slides; Present a business hypothesis for in vivo de novo nucleic acid synthesis capabilities that could be commercialized, either by venture creation or integration into an existing business; identify a value proposition for a NAC that addresses a market need.

There is No Milestone Associated with this Deliverable

Fully execute CUI S&T PIP.

Technical Milestone #2

Technical report on NAC assembly and initial performance, focused on performer-specific RO metrics. Note: Performers selecting RO2 must also demonstrate RO1 metrics. Performers addressing RO1 should include justification of assay choice (e.g.

in vitro vs in vivo).

RO1: Demonstrate in vitro or in vivo synthesis of >300 bp-long nucleic acid sequence of arbitrarily defined sequences containing all four bases.

RO2: Demonstrate in vitro functional integration of error mitigation with the NAC to reduce error by 10%19

Workshop #2

Approximately 8 weeks (40 working days) prior to the workshop, provide interest/disinterest feedback on research synopsis submitted by the broader community.

At least 3 weeks (15 working days) prior to the workshop, performers will submit slides to DISTAR and provide them to DARPA: Present at a DARPA-hosted workshop that communicates technical progress and current challenges.

Program Meeting Slides; Technical presentation on performer specific ROs.

Pitch Event #2

Pitch deck slides; Present a refined business hypothesis and value proposition for a NAC platform, including target market sizing and segmentation, possibilities for a minimum viable product, analysis of alternative approaches, and evaluation of partnership needs for advanced technology development.

No Later Than

Phase 1 Final report

All performers: Phase 1 final report.

For performers selected to advance to Phase 2, all agreements and sub-agreements required for Phase 2 work are fully executed.

For performers selected to advance to Phase 2, briefing slides for Phase 2 Kickoff (at least 1 week in advance of meeting) and a draft pitch deck for the Pitch Event.

Technical Milestone #3

Technical report on in vivo functionality of the NAC, focused on performer-specific ROs.

RO1: Demonstrate synthesis of 1.5 kb-long nucleic acid sequence with an error rate less than 1x10-3 / bp and a synthesis rate less than or equal to 10 sec / bp.

RO2: Demonstrate synthesis of 1.5 kb-long nucleic acid sequence with an error rate less than 1x10-5 / bp and a synthesis rate less than or equal to 20 sec / bp. 31

Workshop #3

Approximately 8 weeks (40 working days) prior to the workshop, provide interest/disinterest feedback on research synopses submitted by the broader community to DARPA.

At least 3 weeks (15 working days) prior to the workshop, performers will submit slides to DISTAR and provide them to DARPA: Present at a DARPA-hosted workshop that communicates technical progress and current challenges.

Program Meeting Slides; Technical presentation on performer-specific ROs.

Pitch Event #3

Pitch deck slides; Present a polished pitch showcasing developed NAC technology to investors that includes a specific ask for follow-on investment (e.g. raise estimate needed) or identifies a transition partner that would enable commercialization.

Technical Milestone #4

Technical report on in vivo functionality of the NAC, focused on performer-specific ROs. less an o RO1: Demonstrate synthesis of 3 kb-long nucleic acid sequence with an error rate less than 1x10-6 / bp and a synthesis rate less than or equal to 1 sec / bp. Synthesize two distinct sequences sequentially with < 1 hr between the 1st and 2nd sequence.

RO2: Demonstrate synthesis of 6 kb-long nucleic acid sequence with an error rate less than 1x10-7 / bp and a synthesis rate less than or equal to 2 sec / bp. Synthesize two distinct sequences sequentially with < 1 hr between the 1st and 2nd sequence. equal to 5 sec / bp.

Workshop #4

Approximately 8 weeks (40 working days) prior to the workshop, provide interest/disinterest feedback on research synopses submitted by the broader community to DARPA.

At least 3 weeks (15 working days) prior to the workshop, performers will submit slides to DISTAR and provide them to DARPA; Present developed GO technology at a DARPA-hosted workshop aimed at aligning commercialization strategies with regulatory and national security considerations.

42 Final Report Final report: content will include (but is not limited to) a detailed and comprehensive summary of approaches, data from all relevant milestones and metrics, limitations of technical solutions, and potential future directions.

1.4. Technical Guidelines for Proposals

As discussed in section 1.2 above, DARPA's staged acquisition approach for the GO program enables agile management and control of technical risks. With each stage (i.e. abstracts, OPPs, Phase 1), DARPA expects technical approaches and team composition to evolve and solidify. 2.1 belowThis section discusses specific content required for preparing abstracts and/or OPPs in the context of the GO program’s technical goals and milestones, while section 2.1 below provides procedural guidance. In addition to these sections, Appendix A provides a preparation checklist addressing all solicitation requirements.

Abstracts are required to provide high-level descriptions of proposed NAC designs and Phase 1 technical plans that will enable DARPA to assess whether the innovation and feasibility of the proposed ideas merit additional discussion in the OPPs. By contrast, OPPs must include detailed Phase 1 plans and justify these plans in the context of Phase 2 goals to make it clear how Phase 1 work will aid in the refinement and finalization of the Phase 2 by the CoDR at month 9. The Oral Presentation itself affords the Government with an opportunity for deeper discussion with a proposing team about their proposed approach to develop a NAC. Both abstracts and OPPs must clearly describe how their proposed approach addresses each of the challenges associated with RO1 and RO2, if applicable, detailed in sections 1.3.1 – 1.3.3 above. In their OPP, proposing teams must discuss any approach-specific challenges requiring resolution on the Phase 1 as part of their justification for the proposed Phase 1 work. Proposing teams may pursue multiple approaches for risk mitigation, but they must clearly describe their primary NAC development strategy and prioritize any additional approaches.

Approach-specific challenges identified should be accompanied by a detailed risk mitigation strategy provided in the OPP that is in alignment with meeting program metrics on schedule.

As described in sections 1.3.1 – 1.3.2 and Figure 2 above, any NAC design addressing program metrics is acceptable, provided:

1. The resulting NAC can function inside a living cell to synthesize nucleic acid sequences with light (i.e., an optical signal) as the sole source of information encoding the nucleic acid sequence. Abstracts and OPPs must discuss how this could be achieved with their proposed NAC design.

2. The resulting system (i.e., the cell engineered to express the NAC) does not require any exogenous substrates (e.g., engineered, non-canonical nucleotides) beyond H, C, N, O, S and P containing molecules that are typically included in standard broth or media. However, cell lines may be engineered to synthesize substrates for the NAC, if these are not produced by the cell’s native metabolism. Abstracts must state any metabolic engineering requirements, and OPPs must detail these strategies.

3. The NAC and/or resulting cellular system incorporates plausible, synchronous activation/deactivation strategies for population-level control of optical signal transduction by NACs expressed inside a cell.

Abstracts should provide a high-level overview of the team’s approach and discuss how it will support both synchronous initiation of NAC-based transduction and deactivate to eliminate unwanted transduction. OPPs must discuss notional mechanisms and any Phase 1 work necessary to de-risk or revise the approach. Proposers are strongly encouraged to consider mechanisms that will facilitate overall security of the resulting system by pursuing approaches that minimize inadvertent (i.e. non-specific) activation of the NAC.

4. The NAC is designed for in vivo single-shot synthesis of long, coding nucleic acid sequences capable of modulating cellular function via translation into functional proteins that results in quantifiable effect on the cell (e.g., expression of a reporter, secretion of a molecule, cell division, differentiation, etc).

Approaches requiring in vitro steps to obtain the desired sequence (e.g., oligonucleotide synthesis within cells followed by lysis and ligation/annealing in vitro) are out of scope for GO. Abstracts and OPPs must be clear that their NAC design is capable of in vivo single-shot synthesis, and OPPs must discuss a notional Phase 2 plan for how they intend to test the ability of NAC-derived sequences to affect cellular function.

5. Genetic messages produced by the NAC must intersect with the cell’s natural machinery for producing RNA and/or protein. Thus, approaches seeking to design entire systems that operate in parallel to the central dogma are out of scope. However, approaches using non-canonical bases may be permissible if abstracts and OPPs provide: 1) plausible, known means of engineering host cells to synthesize all required non-canonical bases and 2) a known mechanism for transliterating these bases into a canonical DNA or RNA sequence. Proposers taking this approach must provide clear evidence as part of their OPP for the feasibility of the metabolic pathways and transliteration mechanism, and this evidence must include proof that the enzymatic machinery to perform these functions is known.

Both abstracts and OPPs must provide a clear conceptual mechanism for the final NAC they aim to develop in Phase 2 and articulate necessary Phase 1 de-risking plans. All submissions must specify whether they intend to develop a NAC that will synthesize RNA or DNA, single-stranded or double-stranded molecules, utilize canonical nucleotides (ATP, GTP, etc) or non-canonical molecules, etc. Both abstracts and OPPs should clearly describe the approach to synchronize initiation and termination of synthesis across a population of NACs expressed within a living cell, and they should discuss the primary cell line that the team intends to use for the NAC. The choice of cell line should be justified both scientifically and in terms of a notional use case (i.e., commercial transition or clinical translation that could follow successful development of the NAC on the GO program). The OPP should discuss any additional cell lines required for development or risk mitigation strategies. If different cell lines are proposed for Phase 1 development compared to the final Phase 2 design, the OPP should explain its relevance and how it will yield insights that de-risk development for Phase 2 work.

Proposals submitted for RO2 must also address RO1. Proposals responding solely to RO2 will be deemed non-compliant with this PS, and they will be excluded from review. RO2 proposals must be explicit in their intent to respond to both ROs in their abstract and OPP. Additionally, RO2 proposals must clearly describe their primary approach to error mitigation along with any supporting strategies. If RO2 teams propose a combinatorial strategy for error mitigation that combines several mechanisms to reduce error rates, their abstracts must clearly indicate this intent, and their OPP should include an experimental plan to evaluate both the combined mechanism and individual components in isolation. Ideally, these proposals will also include Phase 1 work plans to simplify (i.e., down-select) error mitigation mechanisms for integration in Phase

2. Proposals are not required to make their technical…