FaptaSyme: A Strategy for Converting a Monomer/Oligomer-Nonselective Aptameric Sensor into an Oligomer-Selective One

Abstract

Aptameric sensors can bind molecular targets and produce output signals, a phenomenon that is used in bioassays. In some cases, it is important to distinguish between monomeric and oligomeric forms of a target. Here, we propose a strategy to convert a monomer/oligomer-nonselective sensor into an oligomer-selective sensor. We designed an aptazyme that produced a high fluorescent output in the presence of oligomeric α-synuclein (a molecular marker of Parkinson's disease) but not its monomeric form. The strategy is potentially useful in the design of point-of-care tests for the diagnosis of neurodegenerative diseases.

Keywords: aptamers; aptazymes; biosensors; deoxyribozymes; split probes.

Figure 1.

Design of split aptazyme (SAptaz) sensor for the detection of α-synuclein (α-syn). A) A catalytic DNA oligonucleotide, DZ-10–23, can cleave a fluorophore- and a quencher-labeled fluorogenic substrate (F_sub). A position for splitting the DZ as discovered by Mokany et al.^[10] is indicated by the red arrow. B) Split aptazyme design strategy: both DZ and the aptamer DNA strands are split into two parts. Each half of the aptamer is linked to one-half of DZ to form SAptz-1a and SAptz-1b strands, which form the catalytic DZ core when aptameric portions bind α-syn (see Figure S1 for more details). C) Selectivity of α-syn recognition by the split SAptaz-1 sensors. All samples contained 200 nM F_sub, 2 nM SAptz-1a, 50 nM SAptz-1b, and either no protein target (Noise) or 100 nM either oligomeric or monomeric α-syn, or 26 μM of thrombin (Negative control).

Figure 2.

Full-Aptamer-Split-DNAzyme (FaptaSyme) sensor design and performance. A) Design strategy: only the DZ sequence is split in half; each segment is linked to a full aptamer sequence. FaptaSyme-a and FaptaSyme-b strands bind α-syn by their aptameric portions and form a DZ catalytic core followed by fluorescent signaling. In both cases, DZ and aptameric portions were linked by oligoethylene glycol linkers (dashed lines). B) Selectivity of α-syn recognition by the FaptaSyme sensor. All samples contained 200 nM F_sub, 2 nM FaptaSyme-a, 50 nM FaptaSyme-b, and either no protein target (Noise) or 100 nM oligomeric or monomeric α-syn, or 26 μM of thrombin (Negative control). The concentrations of FaptaSyme were optimized, as detailed in Figure S2.