When designing therapeutic constructs and diagnostic kits and reagents, a key consideration is stability. Aptamers have several key advantages over more traditional antibody-based reagents. They are more thermally stable, less immunogenic, and can be easily conjugated without a loss in aptamer function. Their small size can better facilitate infiltration of tumor tissue and entry into cells. Chemical synthesis ensures continued access to new batches of material without the batch-to-batch variability issues associated with bioproduction. Despite these advantages, a key concern surrounding aptamers has been stability, perhaps most specifically the stability of small strands of DNA and RNA in the presence of nucleases ever-present in circulation and in serum and plasma samples.2,7

Resisting Nuclease Degredation

Aptamer researchers have developed several techniques that can be applied during library design and at amplification steps in the selection process to improve aptamer resistance to nucleases. While DNase and RNase are stable enzymes that are very effective at degrading nucleic acids, understanding their method of recognition and function has enabled the development of several protective aptamer modifications.

Have additional questions about aptamer selection and techniques for extending stability? Base Pair aptamer specialists can explain the various options, along with associated time and cost implications, to help identify the modifications best suited to your application.

Submit a question or request a free project proposal today.

2’ – Sugar Modifications

Because natural RNA is almost instantly degraded in serum, researchers selecting RNA aptamers for in vivo use routinely incorporate a small modification at the 2’-hydroxyl of the ribose sugar of the RNA aptamer. One of the most common modifications is replacement of the 2’-OH group with a 2’-fluorine. Researchers at Albert Einstein College of Medicine evaluated DNA and RNA constructs with several modifications. The DNA construct had a half-life of five hours in fresh human serum, while 2’-fluoro RNA had a half-life of 10 hours. 2’-O-methyl RNA and RNA with 2’-fluoro at G bases and 2’-O-methyl at A, C, and U bases displayed an estimated half-life of >240 hours. Due to an observed decrease in potency of serum nucleases over time, extended half-life was estimated based on results for the first 24 hours.3 Researchers at Archemix found that an RNA aptamer with some 2’-O-methyl substitutions was stable for >24 hours in 95% rat plasma and an aptamer with full 2’-O-methyl substitutions was stable for up to 96 hours.2

3’ – Conjugation to Biotin or Inverted dT

Attaching a biotin to the 3’-end of an aptamer is one method of blocking the activity of 3’ exonucleases. Researchers at the University of Hong Kong demonstrated protective effects of 3’-biotinylation with an aptamer to the SARS Coronavirus incubated in DMEM with 10% serum.7,10 Studies at the University of Hong Kong showed greater protective effects for 3’-inverted dT (deoxy-thymidine) than 3’-biotinylation. In both cases, the conjugated aptamer maintained selectivity and inhibitory activity.10 Researchers at Albert Einstein observed a 2- to 3-fold improvement in stability in human serum upon addition of a 3’-inverted dT residue to a DNA aptamer and 2’-fluoro RNA aptamer with paired bases at the 3’ end. Other groups have observed 20- to 50-fold improvement in aptamer stability when working with aptamers that are unpaired at the 3’ end.3

Base Pair performed a study of the stability of unmodified and modified DNA aptamers with a specified length of 32 nucleotides in human serum. Base Pair’s fibronectin aptamer, catalog number ATW0008, was evaluated at 37°C over a period of 14 days. As demonstrated by the representative Figures 1 and 2 corresponding to unmodified DNA versus 5’-biotin-3’-inverted dT, modification extended DNA aptamer half-life from hours to days.

Evading Renal Clearance

The reticuloendothelial system (RES) is a network of cells and tissues involved with the phagocytosis and clearance of foreign particles and microbes.4 Evading this natural defense system and improving distribution to target tissues and cells is another challenge in the design of aptamers for therapeutics and in vivo imaging.


Polyethylene glycol (PEG) is a water soluble, non-biodegradable polymer. 5’conjugation to polyethylene glycol, or PEGylation, is a commonly-used method for extending the half-life of aptamers and aptamer complexes in vivo. Conjugation to PEG offers protection from reticuloendothelial cells and proteolytic enzymes designed to eliminate foreign molecules and microbes. It can also increase solubility. Targeted delivery of PEGylated drugs through aptamer complexes increases bioavailability and reduces systemic toxicity.6

Researchers at Archemix studied the pharmacokinetics and biodistribution of an RNA aptamer with 2’-F and 2’-O-Me modifications and a 3’-inverted-dT cap both with and without PEGylation in a mouse model. The modified  aptamer without PEGylation had a half-life in circulation of five hours, showing the successful protective effects of the RNA modifications. Addition of a 20kDa or 40kDa PEG increased the half-life to 7 hours and 12 hours, respectively. The biggest change was seen in tissue distribution and calculated bodily exposure to drug after administration (AUC or area under the curve). The AUC for the unconjugated, modified RNA aptamer was 3573 ng*h/mL. Values following conjugation to 20kDa or 40kDa PEG were 146,344 ng∙h/mL and 110,009 ng∙h/mL, respectively. Modified aptamer conjugated to 20kDa PEG showed the highest concentration of aptamer in highly perfused organs.2

PEG up to 20kDa is excreted through the renal system while PEG >20kDa is eliminated by fecal excretion.6 Though PEG is generally thought to be biocompatible, PEGylated nanoparticles have been shown to promote complement activation and production of anti-PEG antibodies. Chemical modifications to PEG have successfully reduced this immune response. Interestingly, 25% of patients possess pre-existing anti-PEG antibodies due to exposure to PEG through consumer products.11 Careful selection of molecular weight and structural modification of PEG can maximize serum stability and minimize immunogenicity.6 The broad use of PEGylation and the approval of several PEGylated drugs makes this a very attractive option for extension of in vivo stability and improved distribution.

Conjugation to Cholesterol

Researchers in Korea working with a 2’-fluoro RNA aptamer to the Hepatitis C virus explored cholesterol conjugation as a means of extending in vivo aptamer half-life. The 2’-fluoro RNA aptamer with a 3’ inverted dT residue displayed a half-life of 6 hours following IV administration in a mouse model. Adding cholesterol to the 5’ end of the aptamer increased the half-life to 10-14 hours. Addition of cholesterol increased the calculated bodily exposure to drug after administration (AUC or area under the curve) of 100 mg/kg aptamer administered via IV from 46 ± 17 μg∙hour∙ml to 382 ± 40 μg∙hour∙ml, an 8-fold increase. The aptamer-cholesterol conjugate showed a good dose response and no detectable toxicity in both in vitro and in vivo tests.5

Improving Thermal/Conformational Stability

While thermal stability is not considered a primary concern in vivo, improved conformational stability and promotion of double-stranded conformations can offer enhanced nuclease resistance, as single-stranded regions are more susceptible to nuclease attack.8 Thermal and conformational stability of aptamers and aptamer-target complexes is also important for field-based diagnostic assays and biosensing applications.

Post-SELEX G-C Modifications

Working with a structure-switching anti-cocaine RNA aptamer, researchers at York University in Toronto explored several post-SELEX aptamer modifications to improve thermal stability. Switching a single A-T base pair to a G-C base pair in one of three stems increased the melting temperature (TM) by 4.7°C. Alternating the G-C bases in this stem (from G-C, G-C, G-C, G-C to G-C, C-G, G-C, C-G) increased the TM by another 4°C.9

Locked Nucleic Acids

Locked nucleic acids (LNAs) are modified nucleotides in which the 2′-O and 4′-C atoms of the ribose sugar are joined through a methylene bridge, limiting flexibility. LNAs can be inserted into both DNA and RNA aptamers. Oligonucleotides with LNAs have increased melting temperatures and improved stability in a wide range of biofluids.1

Researchers in Berlin, Germany tested a modified RNA aptamer to Tenascin-C to determine if LNA substitution could further improve thermal stability, serum half-life and in vivo distribution. Substitution with additional 2’-O-methyl groups had little effect on melting temperature of the original 2’-deoxy-2’-fluoro / 2’-deoxy-2’-O-Me aptamer, while incorporation of LNAs at positions 1 through 5 doubled the melting temperature from 37°C to 74°C. Incorporation of LNAs at too many sites prevented aptamer binding. All three aptamers (original, 2’O-methyl and LNA) had a strong serum half-life (42 hours, 49 hours and 53 hours, respectively.) LNA aptamer showed a modest increase in tissue distribution vs. the 2’-O-methyl construct and the original 2’-deoxy-2’-fluoro / 2’-deoxy-2’-OMe aptamer. While the 2’-O-Me substitutions offered sufficient serum half-life, LNA incorporation was far more effective at increasing thermal stability.8

Custom Aptamer Discovery and Design

There are a wide range of tools and techniques available for improving aptamer stability. Understanding stability requirements and determining the best options for enhancing stability is a critical part of the project design process. Required stability and potential modifications must be carefully considered. In addition to achieving the desired stability, the best approach will preserve aptamer selectivity and function, while minimizing the cost and time for aptamer production.

Contact Base Pair today with any questions about aptamers, aptamer stability, custom aptamer discovery, or potential applications for aptamers. Base Pair specializes in creative, consultative aptamer discovery and application development.


1. Farrell, Robert E., Ph.D, RNA Methodologies (Fourth Edition), Chapter 18. Elsevier, Inc. 2010

2. Healey, J.M., et al. Pharmacokinetics and biodistribution of novel aptamer compositions. Pharmaceutical Research. 2004. 21(12).

3. Kratschmer, C and Matthew Levy. Effect of chemical modifications on aptamer stability in serum. Nucleic Acid Therapeutics. 2017. 27(6):335-344.

4. Lazer, G., et al. The Role of the Reticuloendothelial System in Natural Immunity. Natural Immunity. 2005. pp. 95-101.

5. Lee, C.H., et al. Pharmacokinetics of a cholesterol-conjugated aptamer against hepatitis C virus (HCV) NS5B protein.

6. Mishra, P. et al. PEGylation in anti-cancer therapy: An overview. Asian Journal of Pharmaceutical Sciences. 2015. 11(3):337-348.

7. Ni, S., et al. Chemical modifications of nucleic acid aptamers for therapeutic purposes. International Journal of Molecular Sciences. 2017. 18:1683.

8. Schmidt, K.S., et al. Application of locked nucleic acids to improve aptamer in vivo stability and targeting function. Nucleic Acids Research. 2004. 32(9):5757-5765.

9. Shoara, A.A., Development of a thermal-stable structure-switching cocaine-binding aptamer. Biochimie. 2018. 145:137-144.

10. Shum, Ka To and Julian A. Tanner. Differential inhibitory activities and stabilization of DNA aptamers against the SARS Coronavirus helicase. ChemBioChem. 2008. 9:3037-3045.

11. Verhoef, J.J.F. and Thomas J. Anchordoquy. Questioning the use of PEGylation for drug delivery. Drug Delivery and nTranslational Research. 2013. 3(6):499-503.