The stability of aptamers impacts performance in therapeutics and diagnostic assays. One of the key benefits of using aptamers instead of antibodies is how you store and use your reagent. Over the past 60 years the stability of oligonucleotides has been rigorously studied and well defined.

Most providers of synthetic oligonucleotides recommend storing the suspended DNA in a neutral buffer, free from divalent metal ions (e.g Tris EDTA) at -20°C. Stored like this, most manufacturers guarantee a shelf life of 2 years. However, even at 37°C, it is estimated that the oligonucleotide would be stable for in excess of 6 weeks. Other studies have shown that aptamer beacons with fluorescent tags have retained their functionality for 5.5 years when stored in lyophilised formats.

Reagent stability is critical for convenient reagent use and simple global logistics. We are working with many partners to help them overcome this exact problem with antibody based assays.

As it is becoming more commonplace, to see oligonucleotide-based therapeutics and diagnostics it is crucial to understand the stability of oligonucleotides in vivo and how we can manipulate their chemistry to suit your needs.

Stability of aptamer diagnostics

For diagnostic use, aptamers can be highly stable making shipping around the globe simple and low cost. Often the need for cold-chain is removed – we ship aptamers at room temperature without any issues. Not only is this easier and more cost efficient for our customers, it’s also better for the environment to remove shipping at -20°C. Taking away the need for cold-chain logistics makes it easier and more affordable for aptamers to be used in diagnostic assays in low-income regions.

In biosensor diagnostic platforms our aptamers have shown excellent stability. Application in plasma matrices has allowed biosensor chips to be reused a number of times without loss of the aptamer’s stability.

Another option for use of aptamers in blood matrices for diagnostics includes adding phosphorothioate groups to the backbone.

When we modified our libraries with phosphorothioate, incubation in 90% blood matrix for up to 24 hours showed complete stability of the aptamers, ideal for diagnostics and compatible with a range sample processing times.

Beyond blood we have tested the functionality of aptamers in blood, sweat and tears… well not quite. But in common diagnostic sample matrices from river water to urine. When tested in an ELISA the aptamer showed high sensitivity and concentration-dependent responses in all matrices. Indeed, our COVID binders were developed into a monitoring system for SARS-CoV-2 in wastewater and installed at six separate sites across the UK water system for continuous pathogen monitoring.

More than this, if you have a specific matrix that you know your diagnostic assay will be used with, whether this is saliva, nasal mucous or bacterial broth, we routinely build this into our discovery process to ensure that the stability of the aptamer in the required matrix, the functionality of the aptamer in the matrix and minimal matrix effect for the best assay performance.

Stability of aptamer therapeutics

DNA was thought to have a very short half-life in vivo (30-60 minutes) due to the abundance of endogenous nucleases. However certain indications that require short half life for acute conditions, such as stroke and inflammation, will benefit from this ability of the aptamer to offer a ‘hit-and-run’ approach. Current unmodified DNA aptamers in the clinic certainly show sufficient half life over a few days to allow the desired treatment. For Aptoll, an unmodified DNA aptamer in Phase 2 trials, the half life is 9.5 hours.

If a longer half life is needed this is often done by modifying the oligonucleotide backbone; removing or replacing functional groups involved in enzymatic degradation. One of the most widely known examples of the application of stabilising modifications is the use of 2’Fluoro and 2’O-Methyl nucleotides in the drug Macugen. Using these modifications has been shown to increase the half-life of the aptamers from minutes (for unmodified RNA) to days. The half life for Macugen in plasma was estimated at 10 days, while for the second licensed aptamer therapeutic, Iverzay, the half life is 12 days.

Other methods (often used in combination with backbone modifications) include the addition of dTdT, or an inverted nucleotide cap, on one end of the aptamer. These caps inhibit endonucleases as they block the binding region on the 5’ or 3’ end. When used in conjunction with backbone modification is it common to see 5-10 fold increases in stability.

By understanding the different characteristics of oligonucleotides, we are able to design the perfect solution for your application, whatever the challenge.

If you would like more information or would like to discuss how we can develop aptamers for your application then reach out to our team.

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