A direct selection strategy for isolating aptamers with pH-sensitive binding activity
Preprint posted on 1 November 2018 https://www.biorxiv.org/content/early/2018/11/01/460063
Article now published in ACS Sensors at http://dx.doi.org/10.1021/acssensors.8b00945
Who has excellent target binding and sensitive pH-switching? Sensitive pH guys! A strategy to isolate pH-sensitive aptamersSelected by Zhang-He Goh
Background of preprint
Aptamers are molecules consisting of short chains of nucleotides or amino acids that are popularly used as affinity reagents. They are especially valuable because they are easily functionalised to yield molecules that are used in a broad spectrum of applications. However, despite the ease with which researchers can manipulate the physicochemical properties and functionalities of these aptamers, a few major challenges remain.
One such difficulty lies in introducing the modality of pH-sensitivity into these aptamers. The popularity of pH-sensitive aptamers derives from their multiple uses, especially in cell biology and in drug formulations. Unfortunately, while a toolbox of pH-sensitive motifs has been characterised and successfully introduced into aptamers, researchers in this field currently face two limitations. First, active binding motifs within existing aptamers must be characterised before pH-response elements can be added. This logic predicates on the notion that the addition of these pH-response elements to the binding site should not reduce binding affinity. In fact, this idea relates to the second limitation. Because there are not many known pH-sensitive motifs, researchers’ options in incorporating these motifs into the aptamer are limited. This complicates the optimisation process of the aptamer even after the motif is introduced successfully into the aptamer at the relevant site.
The research gap therefore necessitates a method to directly select pH-sensitive aptamers with desirable properties. Gordon et al. engaged in this process through a direct selection technique of aptamers while characterising them using two factors: target binding properties, as well as pH response sensitivity.
Key findings of preprint
Gordon et al. undertook three main procedures in this preprint. The first procedure involved the use of a three-step screening process for aptamers, from which the authors derived two aptamers, S3 and S8, for further study. Next, the authors focussed on S8, and characterised it using microscale thermophoresis (MST), an alternative method. Third, the authors aimed to identify the factors that contributed to pH sensitivity in S8.
(A) Three-step screening process for aptamers
These aptamers synthesised from the screening process were compared to SBA29, an aptamer isolated, characterised, and labelled as St-2-1 by Bing et al. . After the screening process, Gordon et al. selected the top ten aptamers based on either copy number or enrichment, and sequenced them. This led them to focus on S3 and S8, two sequences that showed the largest decrease in binding between pH 7.4 and pH 5.2. Specifically, the authors used the equilibrium dissociation constant Kd to estimate the binding affinity of S3 and S8, where a lower Kd signifies a stronger binding affinity. The authors found that binding affinity of both S3 (Kd = 24.2 ± 3.4 nM) and S8 (Kd = 112 ± 19 nM) for streptavidin was strong at pH 7.4. The binding affinity of both aptamers decreased significantly at pH 5.2, though the exact dissociation constants lay outside the range that could be assayed. Based on their analysis of S3 and S8, Gordon et al. concluded that their screening process is effective at creating pH-responsive derivatives of existing aptamers.
(B) Characterisation of the binding affinity of S8 using MST
Gordon et al. subsequently characterised S8 in greater detail, choosing S8 over S3 for S8’s strong pH-sensitivity. This plays on the idea of triangulation, in which different methodologies are combined to answer the same research question, so that the strengths of one method can overcome the weaknesses of another. When MST was used to characterise binding affinity, the authors found reasonable agreement between the two measurement techniques conducted.
(C) Identifying factors that contributed to pH sensitivity in S8
Finally, Gordon et al. identified the factors that contributed to pH sensitivity. In this step, the authors introduced three changes in S8: point mutations predicted to modulate the stability of the structure at lower pH; other mismatches at the predicted pH-dependent mismatch site; and replacement of specific G-C pairs with C-A or G-A mismatches in the stem of the structure. Based on this final series of experiments, the authors postulated that the unusual G-A pairing at site 62 in the randomised domain may be responsible for the pH-responsiveness of the aptamer by modulating its stability and conformation at different pH conditions.
What I like about this preprint
I chose this preprint for two reasons. First, Gordon et al. tackled a good scientific challenge. The research gap bridged by the authors is especially interesting because the authors compared the behaviours of the aptamers between the physiological pH of 7.4 and the acidic pH of 5.2. Given that the delivery of drugs to physiologic sites have proven to be difficult in various diseases, the ability of an aptamer that would be able to distinguish between environments with these two pHs is physiologically relevant. Two such examples are the acidity of the microenvironments of tumours  and burn wounds .
Second, the strategy adopted by Gordon et al. shows how far synthetic biologists have come from the use of conventional systematic evolution of ligands by exponential enrichment (SELEX). In this work, Gordon et al. cleverly apply the same set of principles from SELEX in using positive and negative selection to isolate aptamers that are both specific and sensitive to different pH environments, but couple this with fluorescence-activated cell sorting (FACS) to boost enrichment rates.
The work that Gordon et al. have described in this preprint shows how the authors’ selection strategy can be used to better identify, isolate, and characterise novel aptamers. In particular, this preprint provides an approach that can be applied to synthesising aptamers for molecular targets other than streptavidin that was used in this study, especially when we do not have prior structural knowledge of either the aptamer or its target.
Future directions may branch from this preprint in three potential ways:
Investigating a wider range of pH environments. This preprint has demonstrated the possibility of using an aptamer that changes conformation in acidic environments with a gradual pH response. The next step may involve inventing a new generation of aptamers that may have one or more of the following properties:
- Having a single-step, rather than a gradual pH response, possibly through a cascade-type reaction
- Changing conformations in targeted alkaline conditions above physiologic pH
- Changing conformations in response to physiologic stimuli other than pH, such as in hypoxic or oxidative conditions
Incorporating these aptamer technologies into drug formulation strategies. Having a new suite of aptamers that can adapt to their immediate chemical environment would be immensely useful in drug release technologies. These aptamers may be used to encapsulate drugs and be targeted to release them only at the desired sites where the chemical environment permits.
Identifying and applying general trends observed for the pH-sensitivity of these aptamers to accelerate aptamer discovery. Two decades ago, Lipinski et al. came up with “Lipinski’s Rule of 5”, five general guidelines that undergird the identification and development of potential drug molecules . In the same vein, a similar set of guidelines may be developed to aid in the discovery and development of new aptamers. This trend may even be expedited in an age of big data, in which artificial intelligence may assist in the establishment of new patterns that propel the field forward.
Questions for authors
- In this study, the binding affinities of these aptamers were characterised. Are the avidities of these aptamers likely to follow the same trends? Are any significant differences expected between the affinities and the avidities of these aptamers?
- Other limitations that prevent aptamers from being more widely utilised as drugs is their in vivo Compared to biologics, which are generally stable against proteases, aptamers are susceptible to various pharmacokinetic problems, including extremely high rates of metabolism and excretion . How might this strategy be used to improve the in vivo stability of these aptamers?
 Bing T, Yang X, Mei H, Cao Z, Shangguan D, Conservative secondary structure motif of streptavidin-binding aptamers generated by different laboratories, Bioorganic & medicinal chemistry 18(5) (2010) 1798-1805.
 Danhier F, To exploit the tumor microenvironment: Since the EPR effect fails in the clinic, what is the future of nanomedicine?, Journal of Controlled Release 244 (2016) 108-121.
 Bennison LR, Miller CN, Summers RJ, Minnis AMB, Sussman G, McGuiness W, The pH of wounds during healing and infection: a descriptive literature review, Wound Practice & Research 25(2) (2017) 63-69.
 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings1PII of original article: S0169-409X(96)00423-1. The article was originally published in Advanced Drug Delivery Reviews 23 (1997) 3–25.1, Advanced Drug Delivery Reviews 46(1) (2001) 3-26.
 Zhou J, Rossi J, Aptamers as targeted therapeutics: current potential and challenges, Nature Reviews Drug Discovery 16 (2016) 181.
Posted on: 14 November 2018 , updated on: 29 September 2019
doi: https://doi.org/10.1242/prelights.5550Read preprint
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