Using Distal-Site Mutations and Allosteric Inhibition To Tune, Extend, and Narrow the Useful Dynamic Range of Aptamer-Based Sensors

Abstract

Here we demonstrate multiple, complementary approaches by which to tune, extend or narrow the dynamic range of aptamer-based sensors. Specifically, we have employed both distal site mutations and allosteric control to tune the affinity and dynamic range of a fluorescent aptamer beacon. We show that allosteric control, achieved by using a set of easily designed oligonucleotide inhibitors that competes against the folding of the aptamer, allows to rationally and finely tune the affinity of our model aptamer across three orders of magnitude of target concentration with greater precision than that achieved using mutational approaches. Using these methods we generate sets of aptamers varying significantly in target affinity, which we then combined to recreate several of the mechanisms employed by nature to both narrow and broaden the dynamic range of biological receptors. Such ability to finely control the affinity and dynamic range of aptamers may find many applications in synthetic biology, drug delivery and targeted therapies, fields in which aptamers are of rapidly growing importance.

Real-time measurement of small molecules directly in awake, ambulatory animals

“…Created using well-established in vitro selection methods (16,17), aptamers can be generated that bind a wide range of analytes (18) and can be rationally reengineered such that they undergo a large-scale conformational change upon binding these analytes (19) over arbitrarily broad (20,21) or narrow (20,22) concentration windows. E-AB sensors use this conformational change to generate an easily measureable electrochemical signal without the need for the target to undergo a chemical transformation (23).…”

Specificity and Ligand Affinities of the Cocaine Aptamer: Impact of Structural Features and Physiological NaCl

“…These results are consistent with the reports of others. 5,7,8 To test for independent effects of cocaine on 2AP fluorescence, the structures of the 38COC1-2AP derivatives were distorted by annealing them with a complementary 20 nt oligonucleotide (cDNA (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)) that covers the presumptive cocaine binding pocket. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 These hybrid molecules showed no change in 2AP fluorescence upon the addition of cocaine, cocaine metabolites, or neomycin-B ( Figure S5).…”

A modular clamp-like mechanism to regulate the activity of nucleic-acid target-responsive nanoswitches with external activators

“…Pioneer works from the groups of Breaker and Famulok have recreated in-vitro allosteric regulation to control ribozymes 52 and nucleic acid target-responsive elements. 53 Other applications of allosteric regulation include sensing [53][54][55][56][57] and those in the emerging field of synthetic biology. [58][59] Here we have expanded on this theme and have rationally designed novel clamp-like target-responsive DNA-based nanoswitches that, by using a strategy similar to that of allosterically controlled intrinsically disordered proteins, can be activated by a partial folding induced by the binding of an external activator.…”

“…Compared to other methods where modulation of the activity of DNA-based receptors [54][55][56][57] has been achieved with allosteric approaches our strategy appears particularly advantageous for several reasons. Because it takes advantage of a triplex-forming nucleic acid recognition module that can be triggered in a highly specific and sensitive way by natural DNA strands, 44 our approach appears far more suitable for sensing and synthetic biology applications than those based on triplex-forming portions triggered by non-natural targets (i.e.…”