RNAES

The RNAES solution team includes a multidisciplinary group of collaborators within the Emory University School of Medicine and the National Public Health Laboratory of Sudan.

We need simple and economical RNA & DNA extraction and storage. Despite major advancements in molecular testing and sequencing technology over the past 30 years, no innovation in commercial nucleic acid (RNA and DNA) extraction and storage has addressed long-standing barriers to accessible and equitable use of these methods. Molecular tests, such as PCR and RT-PCR, and next-generation sequencing provide the most accurate detection of many infectious diseases, and these are particularly important for RNA viruses that cause devastating outbreaks and pandemics. However, all available methods require nucleic acid extraction from clinical samples for optimal performance. This creates massive inequalities in access to best-in-class laboratory methods based on the resources and social vulnerability of a community. Central barriers to nucleic acid extraction at many sites lie in 1) a lack of reliable access to commercial reagents, 2) erratic and uncontrolled markups on supplies and 3) a need for extensive laboratory infrastructure, such as ‑80°C freezers, centrifuges, and consistent power to perform commercial protocols.

Pre-existing inequalities, exacerbated by conflict & climate. In Sudan, where we seek to implement our solution, the central barriers to nucleic acid extraction are further compounded by ongoing conflict that has led to 10.7 million displaced persons, the world's largest displacement crisis, and severe weather events including drought and flooding. As a result, the people of Sudan have suffered outbreaks of viral illnesses such as cholera, hepatitis E, measles, polio, dengue, and chikungunya, as well as drug resistant tuberculosis and malaria, which is endemic throughout the country and throughout the year. Vaccine derived polio viruses have been detected, and cases of HIV, hepatitis B, and hepatitis C are on the rise. Beyond what has been detected, there is a risk for outbreaks of Ebola, yellow fever, Rift Valley fever, and Crimean-Congo hemorrhagic fever viruses. Medical installations that would have been tasked with testing and managing such infections have been raided for supplies and even instruments. As such, there are currently few highly centralized facilities that can perform molecular testing in the country. Laboratories can be supplied with stable, lyophilized reagents for PCR and RT-PCR performance, but this does not address barriers created by the lack of nucleic acid extraction and storage infrastructure which was tenuous prior and now has been decimated by the conflict.

Available methods do not offer solutions. Protocols developed for nucleic acid extraction in resource-constrained settings continue to utilize some combination of hazardous chemicals, single-source reagents, and complicated workflows. These all require ultra-cold (-80°C) storage and transport of samples and nucleic acids on dry ice or in liquid nitrogen. Sample-to-answer devices are far too expensive for general use, and these cannot be scaled up or sourced to meet clinical needs, even in high-resource environments. Lateral-flow antigen and antibody tests represent one alternative to molecular methods. However, these are significantly less sensitive than molecular tests for most pathogens, and there are issues with assay quality, bait-and-switch supplier tactics, and even target loss for previously accurate tests (e.g. malaria).

The RNAES solution: What it is and what it does. RNAES is a unique nucleic acid purification and storage solution that can be performed virtually anywhere. It utilizes biosafe and shelf-stable ingredients, simple papers, and low-cost membranes to achieve rapid nucleic acid extraction and ambient temperature stabilization from a wide range of samples including serum, plasma, respiratory samples, and even capillary whole blood. RNAES has been further streamlined with reusable 3D-printed components to improve feasibility and acceptability for a variety of use cases.

How it works. RNAES involves 4 simple steps that take a total of approximately 15 minutes to perform, with only 3 minutes of hands-on time. No steps require precise timing, and all reagents can be dispensed and mixed with low-cost transfer pipettes. The 4 steps are the following:

1. Lyse. A unique lysis sphere is dropped in the sample. Spheres contain dried lysis buffer reagents sufficient for a single reaction. The buffer is reconstituted by the sample itself and allowed to incubate for 10 minutes. This step is extremely flexible; nucleic acids remain stable for a week at this stage.
2. Bind. The lysis solution is mixed with an amino acid binding buffer and added to a RNAES membrane placed in a simple 3D printed cartridge. Fluid is pulled across the membrane into a blotter pad by capillary action. The membrane is washed once with an amino acid buffer.
3. Store. RNAES membranes are transferred to a drying tube, which is closed and dropped in a zipper-locked bag. Nucleic acids, including viral RNA, are now stable for over 30 days at 37°C and ready for ambient temperature transport in a standard envelope.
4. Test. When ready for testing, the membrane is dunked in elution buffer (TE buffer, pH 8.0) for 60 seconds and then discarded. Nucleic acids are ready for molecular testing or next-generation sequencing.

What it achieves. The simplicity of our solution and the stability of both reagents and purified nucleic acids allow RNAES to be deployed to remote, austere environments with performance at the point of sample collection or any field laboratory. Pathogens are inactivated during the protocol, which ensures safe transfer of dried membranes to testing laboratories that do not require extensive biosafety controls. All steps are performed at ambient temperature and require no electricity (no centrifuges, vortex mixers, heating blocks, or ultra-cold freezers). 3D printed components have been designed to be reusable following decontamination in 10% bleach. This further reduces the cost of an already economical solution and decreases plastic waste. Finally, RNAES cartridges trap all chemical waste in the blotter pad, which reduces the biohazardous liquid waste typical of current nucleic acid extraction protocols.

In summary, RNAES connects patients with world class molecular testing and sequencing by providing simple, accessible nucleic acid extraction and storage wherever and whenever it is needed.

A highly vulnerable population with poor healthcare access. In April 2023, a brutal and deadly war between Sudan’s Armed Forces and the Rapid Support Forces broke out, with both vying for control of Sudan. The war has been ongoing for over a year with multiple cease fires being violated and no end to conflict in sight.  Both forces have been accused of committing atrocities against the Sudanese people, who remained caught in the middle of this bloody power grab. In January 2024, the International Organization for Migration estimated that 10.7 million people had been displaced from their homes in Sudan, including 9 million displaced internally. The Office of the UN High Commissioner for Refugees describes internally displaced persons as among the world’s most vulnerable populations as they face significantly higher rates of mortality than the general population. Women and children comprise the majority of this group, and they are frequently deprived of access to basic healthcare services.

The devastation wrought upon Sudan's healthcare infrastructure is a profound tragedy with far-reaching implications, particularly in infectious diseases management. According to the UN Office for the Coordination of Humanitarian Affairs, an estimated 65% of Sudanese people lack access to healthcare and upwards of 80% of hospitals in conflict-affected areas are no longer functional. Before the conflict, the focal point of healthcare provision and infectious diseases testing was in the capital (Khartoum). However, Khartoum became the central hotspot of the conflict, resulting in the destruction of many vital healthcare facilities. The National Public Health Laboratory (NPHL) was occupied and looted by renegade security forces. NPHL was home to the malaria and HIV control programs; reference laboratories for influenza, measles, polio, tuberculosis, and bacteriology; the National Epidemiology Laboratory; and the newly established Genomics Unit. This incursion halted many medical and public health services, and it hindered efforts to detect, treat, and prevent infectious diseases that affect the people of Sudan and may stigmatize vulnerable communities, such as internally displaced persons.

RNAES as a part of the solution – simple, rapid, and without electricity. There is no single solution to the medical crisis that is being faced by healthcare workers and internally displaced persons in Sudan. However, we believe that RNAES addresses an important need by 1) increasing capacity and resilience in healthcare laboratory systems and 2) addressing barriers to molecular testing caused by breaks in the supply chain and infrastructural demands of current protocols. This will support expedited testing locally at public health laboratories that lack consistent electricity for sample storage but that have intermittent electricity or even battery power to run testing instruments. RNAES will allow testing to happen rapidly and at low cost in geographic regions of common outbreaks. This in turn impacts disease control and case management and curbs the spread of disease by expediting diagnosis, especially in internally displaced and refugee camps where outbreaks may impact thousands. This is expected to increase access to and quality of healthcare services for underserved groups in resource-constrained and vulnerable communities in Sudan and worldwide.

Developed out of need. The RNAES project began with the goal of providing an RNA extraction and storage solution for collaborators operating in resource-constrained settings. While trying to detect and study viral diseases in their respective communities, they faced two fundamental challenges to molecular testing: 1) reliably sourcing RNA extraction kits and 2) maintaining ultra-cold chain storage and transport for samples and nucleic acids. Historical inequalities in access to reagents, further exacerbated by stock-outs and volatile pricing during COVID, drove our laboratory team to address not only these challenges but also the limitations of previous extraction protocols developed for resource-constrained settings. We utilized low-cost reagents that could be easily sourced and emphasized protocol speed and simplicity for the extraction of dengue and SARS-CoV-2 virus RNA from serum and nasal swabs, respectively. Beyond identifying the best performing reagents, we defined a set of alternatives to allow flexible sourcing if necessary.

Improved by community feedback. After development of RNAES version 1, we began working with global partners to determine what worked and what failed in real-world settings. Based on initial feedback, it was clear that version 1 still relied too heavily on laboratory infrastructure for widespread implementation and the use case was limited to RNA virus detection from relatively simple specimens. Building off this information, we reformulated RNAES chemistry and streamlined performance with reusable 3D-printed components. RNAES version 2, which is the cornerstone of our proposed solution, now provides RNA and DNA extraction from viruses, bacteria, and parasites (such as malaria) even when starting with complicated specimens (e.g., capillary whole blood). This version has allowed collaborators to stabilize nucleic acids and transport them across country without any cold storage and even ship them between continents in a simple envelope.

Focused on vulnerable populations. Our team has the unique blend of expertise and local experience that is necessary to now deliver the RNAES solution to internally displaced populations in Sudan. Jesse J. Waggoner, MD, is Team Lead and has over a decade of experience in developing and implementing molecular tests for diseases that affect the world’s most vulnerable populations. David R. Myers, PhD, is a biomedical engineer and expert in developing novel, low-cost, accessible devices that integrate infectious disease diagnostics with micromechanical systems design. Ahmed Babiker, MBBS, MSc, is a Sudanese Infectious Diseases clinician and Clinical Microbiologist, and a member of the Sudanese Infectious Diseases Association, on the ground. Shahinaz Bedri MBBS, is Director of the National Public Health Laboratory (NPHL) of Sudan, and Muatsim Ahmed, MBBS, is a Sudanese molecular laboratorian at NPHL. Our team of Infectious Diseases specialists, biomedical engineers, and Sudanese clinicians and laboratorians is focused on improving the access to and quality of healthcare for persons displaced by the Sudanese conflict. We have experience in the laboratory and on the ground to implement the RNAES solution and quickly iterate design improvements to meet the needs of end users and affected populations in Sudan. 

RNAES has been implemented in several collaborating laboratories in South America, Africa, and South Asia. Using valuable feedback from our in-country partners, we continue to iterate aspects of the protocol to improve extraction efficiency and the enhance key value propositions of the technology – simplicity and stability. It is our expectation that additional advancements will be made following RNAES launch in Sudan, and this will both tailor the solution to specific in-country needs and improve the technology for expansion to other locations. Though we have not incorporated, we expect to do so in the coming months, and we are investigating business models that remain consistent with our goal of providing an accessible product to serve vulnerable communities while maintaining a viable business.

Community. This community includes an esteemed group of investigators and entrepreneurs who address vast inequalities that exist in our world through the development of sustainable technological solutions. Although this idea is widely championed in theory, it can be very difficult to advance promising technologies that do not clearly fit into traditional funding streams. Moving forward requires vision, determination, and strategy, and the path to sustainability will not be identical for any two solutions. Therefore, our primary goal for applying to this challenge is to join and learn from an inspiring community that has faced, and continues to face, challenges in developing sustainable technological solutions for the greater good.

Mentorship. As we look to incorporate this year, our team would benefit from mentorship on less traditional business models, such as B-corp and L3C. These models are not emphasized in typical technology incubators but complement both the key values of our technological solution and our mission as clinician scientists. This challenge provides an opportunity to receive guidance about selecting between these models and connecting with young leaders who have utilized a variety of business structures.

Branding & marketing. Developing and maintaining a business that provides accessible technology to improve and expand access to care among socially vulnerable populations requires a good branding and marketing plan. We hope to receive guidance about presenting RNAES technology to global stakeholders and building a web presence that highlights the utility and impact of our solution. In addition, we seek to gain a better understanding of the challenges to marketing in different regions across the globe, regulatory hurdles that may arise, and barriers to distributive RNAES manufacturing.

Awareness. Sudan has long since fallen from global headlines, yet the country has been wracked by conflict. We believe that the RNAES solution provides a unique strategy to address certain healthcare needs for persons displaced by this conflict. Acceptance into the community would not only provide increased visibility for this work but the chance to network with organization leaders focused on developing tractable solutions globally that may address other challenges faced in Sudan.

RNAES can disrupt where and how we think about using molecular testing and sequencing. Medical providers, global health experts, and laboratorians have long faced the challenge of bringing together patients (and really, their samples) and the laboratory equipment necessary for molecular testing. To date, we have had to choose from a menu of cumbersome, expensive, and often unrealistic options. Rather than providing accessible and feasible solutions, commercial products have only become more expensive and automated, which exacerbates long-standing inequalities in healthcare access. Expensive robotic extractors, or costly sample-to-answer devices, lock users into a specific set of consumable reagents and restrict performance to high complexity laboratories or high-resource sites. In addition, these require costly contracts for regular maintenance, though this is unsustainable in resource-constrained sites. As a result, we have seen numerous automated extractors collecting dust in the field and even one used to store biscuits for afternoon tea because it could not hold up under real-world conditions (but it did keep the mice out).

Imagine molecular labs without the demands of nucleic acid extraction and storage. The vast majority of the infrastructure and cost associated with molecular testing derives from the demands of sample processing, nucleic acid extraction, and ultra-cold storage. The RNAES solution allows us to reimagine molecular testing in laboratories that do not need centrifuges, mixers, -80°C freezers, or even extensive biosafety controls. Nucleic acids that are purified and stabilized in the field are simply dipped in elution buffer and tested on any existing molecular or sequencing platform. Dried and stable PCR and RT-PCR reagents are readily available, and even after reconstitution, these can be stored at 4°C or, at worst, in a standard freezer. This all becomes possible because no processing or extraction remains to be done in the laboratory – a vast difference from methods touted to stabilize samples in the field.

Distributive nucleic acid extraction and stabilization with RNAES technology offers a powerful and innovative solution that can reshape who has access to highly sensitive molecular testing and where this can be performed.

RNAES Theory of Change

The RNAES theory of change, shown in the framework, centers around the idea that simple, feasible, and economical nucleic acid extraction and storage will increase access to molecular testing and sequencing for patients in vulnerable communities as well as their close contacts. In turn, this will improve identification and characterization of pathogens that circulate in a community, which will lead to improved health through judicious use of outbreak response measures and issuance of targeted clinical guidance.  

Evidence to support Links 1 and 2 comes from research with RNAES at Emory University and international partnering sites. This technology has allowed us to detect new cases of dengue virus in returned travelers and expand testing for emerging viruses to new populations in South America and South Asia. Extracted and stabilized nucleic acids have been transported from outbreak sites to laboratory facilities in-country and even shipped between continents to address limitations in molecular testing capacity. Therefore, RNAES can expand molecular testing to new populations and provide concrete targets by which to evaluate healthcare interventions. Future work will address Link 3 by closing the loop with communities and implementing control strategies and management guidance to improve health outcomes.

We have the following three impact goals for the RNAES solution:

Goal 1. Ensure capacity for nucleic acid extraction and storage. Successful extraction and storage using RNAES technology will be objectively measured by testing for a human specimen control that is detected in whole blood samples. Extraction success is defined as detection of the control in >95% of samples extracted at an implementing site. Storage success is defined as >95% detection of the control in nucleic acids stored for at least 30 days at local ambient temperature. A secondary outcome measure of impact will be the number of laboratories actively contributing to testing and monitoring efforts compared with historical numbers.

Goal 2. Expand molecular testing and sequencing. Outcome measures for this Goal are based on the number of patients who receive molecular testing and the number of cases for which a pathogen (virus, bacteria, or parasite) is identified. These measures will be recorded as a running tally and as a percentage of RNAES extractions performed. A secondary outcome measure will be the number of pathogen sequences generated, and all sequence data will be uploaded to public databases for use by the scientific community.

Goal 3. Improve community health through outbreak management and clinical guidance. Impact on community health will be measured by the number of cases of an identified disease (dengue, leptospirosis, malaria) before and after public health and clinical interventions that are informed by RNAES use. Secondary outcome measures will be the number of interventions themselves, including public health measures (e.g., vector control programs, water treatment, vaccination campaigns) and issuance of clinical management guidance that emphasize disease-specific treatments.

RNAES represents the coming together of methods from various disciplines to yield affordable, sustainable, and scalable technology for nucleic acid extraction and storage. We opted to combine a simple, capillary-driven approach with a unique sugar-and-salt lysis buffer inspired by protocols used in botany. From this starting point, we systematically evaluated various non-toxic buffers, additives, and membranes to create an economical, flexible, and widely accessible process. With the addition of reusable 3D printed components, RNAES now provides a streamlined and safe technology to prepare total nucleic acids from any liquid sample and stabilize these on dried membranes for ambient temperature storage and transport. 

We have a staff of 8 individuals on the RNAES solution team. This includes a single full-time individual and others who devote part of the effort to the project. With additional support, we hope to shift this balance to include more full-time members on the team.

We have worked to develop this solution over the past 3 years

RNAES, for anyone with DNA (and RNA). For Team RNAES, diversity, equity, and inclusivity are more than just ideals to which we are committed; these form the very core of our approach every day and move us forward on a journey to improve testing access for everyone. The RNAES solution developed out of our need to look at a common problem in a new way. To address long-standing inequalities in test access, we embraced concepts that came from across the sciences and listened to partners from all corners of the globe, at all stages of their careers. We strive to foster an open and safe environment for all members of our team to present and test new ideas and concepts, and we support equitable representation of all contributors on the end products of our work. We encourage team members to pursue their passions and to use our work as an avenue for lasting positive change in the lives of anyone with DNA (and RNA).  

RNAES provides a unique nucleic acid extraction and storage solution for laboratories and research groups in academia, public health/government, the NGO/non-profit sectors, and even industry. As such, we provide products and services to individual consumers/stakeholders (B2C), organizations (B2B), and governments (B2G). We plan to sell kits directly to these institutions and the end users who will be working with the technology. In addition to providing goods, our group can provide molecular testing of extracted nucleic acids and consultation services regarding technology implementation. These goods and services benefit the primary institutions and end users as well as the individuals tested with our technology. We will reach customers through web presence that we hope to establish as part of Solve, professional channels (annual meetings, publications, presentations), international networks, and word of mouth. In working with our collaborating partners around the world, we have identified two key value propositions of RNAES – simplicity and stability. As we continue to advance this technology, we will ensure that these two key value propositions are retained and offered at a reasonable and accessible price point.

Our current plan leading up to and immediately following startup launch is to obtain non-dilutive funds from governmental and non-governmental sources. We have identified a low-cost space in which to start our company that is conveniently located near our academic laboratories and provides access to regional partners. In later stages, we believe that RNAES can be sustainable through direct-to-consumer sales as the technology requires only modest capital investments for kit production and QC.