Determination of minimal sequence for binding of an aptamer. A comparison of truncation and hybridization inhibition methods

Abstract

Nucleic acid aptamers are attracting increasing interest as sensing and therapeutic molecules as a consequence of their high affinity and specificity for their target molecules. Aptamers are selected from large random libraries and where structural data are available it appears that only a small fraction of the sequence is actually involved in direct contact with the target. As there are many advantages to minimizing the size of the aptamer a rapid method that can determine those parts of the sequence critical for the target binding would be very useful. In this paper we describe mapping the effective binding region of an aptamer selected against electric eel acetylcholinesterase. As originally selected this aptamer is 77 nucleotides in length and was shown to bind the target with a high affinity (K d ¼ 174 AE 27 pM). Truncation to 39 nucleotides enhanced affinity for its target by an order of magnitude (K d ¼ 14 AE 1 pM). To further probe the relationship between sequence and affinity, we used two approaches: truncation and hybridization inhibition. Binding assays were performed with a number of truncated variants to determine a minimal binding sequence. A similar set of measurements for hybridization inhibition was also performed to allow a comparison of the two approaches. In general hybridization inhibition resulted in comparable conclusions to those found by truncation. The exception to this was where the former resulted in steric clashes between double stranded DNA regions and the target. In this case the effect on affinity was less pronounced than with truncation.

Inkjet Printed Nanopatterned Aptamer‐Based Sensors for Improved Optical Detection of Foodborne Pathogens

“…Finally, a group of test strips was blocked with 20 µL of complementary DNA solution (10 × 10 −6 m ) in order to discard the possibility of nonspecific interaction between the primary aptameric sequence immobilized on the test line and E. coli O157:H7. The results confirmed the high specificity of the detection events, demonstrating that the hybridization with the cDNA resulted in destabilization of the secondary conformation and further inhibition of the physical interaction with the target as consistently reported before for truncated aptameric sequences …”

Label‐Free Electrochemiluminescence Aptasensor for Highly Sensitive Detection of Acetylcholinesterase Based on Au‐Nanoparticle‐Functionalized g‐C3N4 Nanohybrid

“…The DNA aptamer sequence was ordered from Sangon Biotech (Shanghai, China). The AChE aptamer sequence information is as follows: 5′‐GGT TGA CTG TAG CTC TGG CAG ACG TAG TGT GAA GGT ACC‐(CH 2 ) 3 ‐SH‐3′.…”

Aptamer Sensors for Drugs of Abuse and Medical Biomarkers: Design, Engineering and Application in Complex Samples

“…Generally, most techniques rely on similar trial-and-error mechanisms until a short aptamer sequence is identified. 17,100,101 For example, the cocaine aptamer was previously engineered to destabilize the target-free structure, with aptamer folding only occurring when cocaine was bound to the aptamer. 102 The signaling occurred through a fluorescent label that was attached to one end of the aptamer and a quenching molecule attached to the opposite end.…”