Breaking the Ice: A Breakthrough Method for Ambient Temperature DNA and RNA Preservation
Currently, labs often rely on freezers to store their nucleic acids, but this is costly and dependent on a stable electrical grid. Encapsulation is an alternative storage method that can be cheaper, far more disaster-proof, scalable, and sustainable.
In 2001, sequencing a human genome cost over $100 million USD. Twenty years later, this process costs less than $1,000 [NIH]. The rise of sequencing has led to the proliferation of biospecimen collections, where the preservation of nucleic acids (mostly DNA, but RNA, too) is essential. Sometimes, nucleic acids are stored in their original tissues and fluids of origin, often frozen at ultra-low temperatures. Other times, the nucleic acids are extracted from those origins, but they too are almost always kept frozen to slow their degradation.
As biobanks have grown, it has become difficult to store so many samples in this frozen state. Freezers are large, expensive, energy-intensive, and — if you ask the people who depend on freezers to protect their samples — prone to breaking at the most inconvenient times. Since the early 2010s, a move towards ambient temperature storage has been predicted and called for [Yong et al. 2014]. Here, we provide a comprehensive review of the current alternatives to freezers and discuss why the field is eager to move toward ambient temperature storage. We also discuss the challenges that face most current ambient preservation methods, and discuss Caching: a new ambient preservation method that avoids the shortfalls of current ambient storage techniques.
Freezers are large, expensive, energy-intensive, and — if you ask the people who depend on freezers to protect their samples — prone to breaking at the most inconvenient times.
Before discussing how to preserve nucleic acids, we first need to understand how DNA and RNA degrade over time. We can think of DNA as a twisted ladder (a double stranded helix), and RNA is a ladder that’s been cut from top to bottom down the middle (a single stranded helix). The more breaks there are, the shorter the fragments become. Eventually, the fragments are so small they cease to have any meaning, like a book’s pages after being put through a paper shredder. For DNA, once extracted from its original source, the largest sources of degradation are water, oxygen from the air, and heat [Colotte et al. 2011]. While RNA is even more susceptible to these factors due to its single, rather than double, backbone, it has an even greater threat: RNAases, enzymes that catalyze the degradation of RNA and are commonly found throughout the environment, capable of degrading entire samples within hours [Breaker et al. 2003].
To preserve nucleic acids, there are six main strategies. We will dive deeper into each of them below.
Freezing Samples
Currently, freezing samples is by far the most widely adopted preservation method. Freezers for home use were first introduced in 1947, and lab freezers have only been in existence since 1968, with the first ultralow freezer in 1987. Although freezer technology continues to evolve, it remains a pain point in many laboratories and biobanks around the world. When natural disasters strike, freezers are at significant risk of failure due to the complete reliance on electricity. Even when there is no disaster, freezer failure still occurs with alarming regularity due to either human or manufacturer error. Perhaps most famously, in a headlining incident from 2023 a janitor turned off a freezer leading to the loss of samples worth $1 million. Freezers also take up an enormous physical and electrical footprint, sometimes limiting the number of samples that can be stored because there is no longer space or electrical capacity, even in brand-new buildings. To make matters worse, the act of thawing a sample causes degradation [Shao et al. 2012], so biobanks often implement a system much like the mythical hydra that grows two new heads with each one that is cut off: each time a frozen sample is accessed for the first time, it is split into 10 to minimize the number of freeze-thaw cycles. While an understandable tradeoff, this hydra-like explosion of samples exacerbates the problems groups were already facing.
Thankfully, there are alternatives to freezing samples.
Formalin-Fixed Paraffin-Embedded
Arguably the second most common method is fixing samples in formalin. This method preserves tissues in formalin-fixed and paraffin-embedded tissue blocks (FFPE), chemically slowing the degradation of the tissue and almost everything within it, including DNA and RNA to some degree. It’s common to walk into a hospital basement and find regular shelves at ambient temperatures full of irregularly shaped FFPE samples. While these samples are invaluable to researchers who analyze the structure or other spatial components of a three-dimensional sample, it is a poor way to store nucleic acids, with significant degradation of even DNA after 4 to 6 years of storage [Guyard et al. 2017].
Paper Matrix
Alternatively, rather than going through the relatively complex process of fixing, liquid samples (such as blood) are sometimes spotted onto paper (such as a Guthrie card). The type of paper used is typically filter paper, but anecdotally, the author of this article has heard that on three independent occasions people have found toilet paper works just as well (and is often easier to come by). Nevertheless, regardless of the type of paper used, the underlying principle is that the matrix of fibers physically stabilizes the DNA. While this process works well and has been around since the early 1960s [Woolf & Adams 2020], it is difficult to scale, and samples cannot be aliquoted for retrieval. Instead of being able to take an aliquot of the sample and leave the remainder undisturbed, the entire sample must be rehydrated before retrieval can happen. Afterward, the remainder of the sample must be re-spotted and dehydrated again. Thus, we do not consider it possible to take aliquots of a sample stored via any dehydration methodology.
Additives
Additives such as GenTegra and DNAStable can offer protection similar to a freezer [Howlett et al. 2014, Miernyk et al. 2017]. Mixing in the additive with the extracted nucleic acids slows degradation by forming a physically stabilizing matrix around the nucleic acids, and, if dehydrated, the additives essentially shrink-wrap the nucleic acids, encasing them in a protective layer. This theoretically protects samples from degradants in the environment. However, additive’s ease of use is also their undoing. It is as easy to rehydrate the additive as it is to mix it into the original sample. This means that any moisture in the air rehydrates the additive and thus cannot protect the sample against moisture, a primary cause of degradation.
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For this reason, simply dehydrating, or even lyophilizing (freeze-drying) a nucleic acid sample is not widely adopted for long-term sample storage.
Encapsulation
There are various encapsulation techniques, but the premise is that nucleic acid samples are enclosed in a protective layer impermeable to water and oxygen.
Imagene has created one of the most protective encapsulation methods ever commercialized, with a degradation rate orders of magnitude slower than other methods, making it extremely compelling [Coudy et al., 2021]. However, this method works by dehydrating nucleic acids inside a small stainless steel minicapsule with a glass insert, and then hermetically sealing the capsule with inert gas [Washetine et al. 2019]. This makes the process costly and difficult to implement and scale.
At Cache DNA, we encapsulate nucleic acids with a “micro-scale” process (which we call Caching) that is scalable and far less costly. Caching was designed to utilize commonly found laboratory equipment, and with a standard benchtop liquid handling system, users can Cache tens of thousands of samples per year.
In the Caching encapsulation process, the negatively charged nucleic acids are mixed with an inert bead core with a positive charge, and their opposite charges attract and leave the nucleic acids weakly bound to the bead cores. An encapsulant is then mixed into the solution, where it forms a protective shell around the nucleic acids and shields the sample from degradants. This allows Cached samples to be kept at ambient temperature with degradation rates equivalent to or better than a freezer.
At the end of the Caching process when either a total or partial amount of the Cached sample is retrieved, a simple etching buffer is added. In 10 minutes, the buffer dissolves both the encapsulant and bead core, but does not react with the nucleic acids, leaving them free in solution for downstream applications.
Importantly, no part of the Caching process requires freezers. With no need for freezers, users can now reclaim valuable physical space, significantly reducing the need for electricity and monitoring. Critically — users can now take aliquots of a sample without harming the nucleic acids, unlike other preservation methods, where samples are degraded when thawed or rehydrated. With Caching, the sample is protected by the encapsulant, easily allowing for aliquoting when a user wishes to access the sample, or further disaster-proofing if a lab wishes to create a mirror site at a separate location. Currently, Caching is the only encapsulation technique that enables scalable storage and protected aliquoting.
Summary Discussion
In short, the field of nucleic acid preservation has come a long way since the field of genomics began in earnest in the mid-1900s. Yet with the relatively recent explosion of sequencing and the promises of personalized medicine on the horizon, there is an urgent need to accelerate the adoption of new technologies that are more scalable and sustainable [Coppola et al. 2019]. Coupled with the increasing popularity of initiatives like the Freezer Challenge which encourages labs to operate more sustainably (now with 31 participating countries and over 1,100 laboratories registered for the Freezer Challenge so far this year), the field is entering a new era where labs are being forced to reevaluate their storage methods more than ever before. To support this changing landscape, Caching has developed an uncompromising preservation technology that performs just as well, if not better than freezers, while offering all the benefits of ambient temperature storage at a reduced cost over the lifetime of the sample.
