It's tempting to think of DNA solely as a static molecular carrier of information. The long stretches of double-stranded DNA that contain our genetic code tend not to stray from their double helix unless acted upon by external molecular machinery. However, shorter snippets of DNA can break these rules to offer exciting dynamic properties. Given the right sequence, DNA can form aptamers (single-stranded segments that sensitively and specifically bind molecular targets) as well as enzymes, switches, and sensors, all using the same 4-base code. 

In this work, we wanted to push the limits of how "smart" a single strand of DNA can be, by packing in not only target binding and molecular switching, but also a range of pH responsive behaviors, all into short DNA sequences. 

We focused on creating a pH-responsive design because of the potential for improving aptamer-based diagnostics and therapeutics. Local variations in pH are critical in diseases like cancer, where metabolic dysregulation in solid tumors lowers extracellular pH¹. Aptamers designed to bind molecular indicators of cancer specifically at low pH would have enhanced accumulation in tumors, allowing selective delivery of imaging agents. Conversely aptamers designed to bind drug cargoes at high pH and release them at low pH could greatly enhance therapeutic delivery to these cells.

Our approach fuses aptamer-based molecular switches² with pH responsive "motifs"^3,4  – snippets of DNA that undergo large structural changes when protonated or deprotonated by changing pH. By controlling the position of motifs within the sequence and optimizing the resulting designs, we ensure that our designs are: 1) general enough to work with many base aptamers and target a broad range of biomolecules, and 2) tunable so that the pH properties can be adjusted to meet the demands of many biomedical applications.

This strategy yielded effective pH-responsive switches for targeting both high and low pH. We showed that by inserting or mutating just a few bases within the switch's DNA sequence, we could turn an aptamer with no pH-sensitivity into one with over 1000-fold selectivity in binding between low and high pH. Further, we were able to show that with simple, predictable design tweaks, we could adjust the pH response to target pH conditions across the physiologically relevant range. We believe the high generality of this design will greatly expand the pool of possible biological targets and drug cargoes for functional DNA nanodevices. 

But why stop at just high or low pH? My advisor, Dr. Tom Soh, offered an interesting challenge midway through my work on this project: to design a completely new type of pH control. We came up with the "pH window" concept, fusing our low and high pH mechanisms to create aptamers that would bind strongly only within a narrow range and shut off when the pH was either too low or high. This new capability could enable biotechnologies to precisely target steps within the endosome-lysosome pathway, where each step in the trafficking and degradation process is characterized by small pH variations. Further, those looking to improve the control over nanoscale self-assemblies might turn to this design to more finely regulate pH-triggered processes.

In getting to these final, high-performing designs, I learned a great deal about the structure, energetics, and chemistry of DNA aptamers and motifs. For me, it raised the question of how densely we can pack short DNA sequences with function – be it the combination of target-specific structure switching and pH-responsiveness shown in this work, or other important features like allostery, kinetic control, and proofreading mechanisms. The better we can control all these properties in DNA systems, the better we will be at creating "smart" nano-biotechnology to diagnose and treat disease. 

References:

1. Webb, B. A., Chimenti, M., Jacobson, M. P. & Barber, D. L. Dysregulated pH: A perfect storm for cancer progression. Nature Reviews Cancer vol. 11 671–677 (2011).

2. Wilson, B. D., Hariri, A. A., Thompson, I. A. P., Eisenstein, M. & Soh, H. T. Independent control of the thermodynamic and kinetic properties of aptamer switches. Nat. Commun. 10, 5079 (2019).

3. Idili, A., Vallée-Bélisle, A. & Ricci, F. Programmable pH-Triggered DNA Nanoswitches. J. Am. Chem. Soc. 136, 5836–5839 (2014).

4. Fu, W. et al. Rational Design of pH-Responsive DNA Motifs with General Sequence Compatibility. Angew. Chemie Int. Ed. 58, 16405–16410 (2019).