In vitro selection of structure-switching, self-reporting aptamers

Abstract

We describe an innovative selection approach to generate self-reporting aptamers (SRAs) capable of converting target-binding events into fluorescence readout without requiring additional modification, optimization, or the use of DNA helper strands. These aptamers contain a DNAzyme moiety that is initially maintained in an inactive conformation. Upon binding to their target, the aptamers undergo a structural switch that activates the DNAzyme, such that the binding event can be reported through significantly enhanced fluorescence produced by a specific stacking interaction between the active-conformation DNAzyme and a small molecule dye, N-methylmesoporphyrin IX. We demonstrate a purely in vitro selection-based approach for obtaining SRAs that function in both buffer and complex mixtures such as blood serum; after 15 rounds of selection with a structured DNA library, we were able to isolate SRAs that possess low nanomolar affinity and strong specificity for thrombin. Given ongoing progress in the engineering and characterization of functional DNA/RNA molecules, strategies such as ours have the potential to enable rapid, efficient, and economical isolation of nucleic acid molecules with diverse functionalities.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

The structure and function of SRAs. (A) The DNA library design features a central DNAzyme segment between a pair of 17-base random sequences, flanked by two 20-base PCR primer sites. (B) Specific binding between the SRA probe and its target protein induces the release of the antisense strand that maintains the DNAzyme moiety in an inactive state. Once unmasked, the DNAzyme moiety is able to form an active G-quadruplex structure and, upon binding to NMM, emits an enhanced fluorescence signal to report the specific binding event.

Fig. 2.

Overview of the SRA selection process. (A) Preparation of the DNA library. (B) Incubation of the DNA library with a 12-base biotinylated antisense DNAzyme strand and PCR primer site-blocking strands. (C) Immobilization of the duplex library on streptavidin-coated magnetic beads. (D) Library-bead assemblies are challenged with human α-thrombin target, resulting in elution of SRAs from beads. (E) Magnetic separation of eluted SRAs from bead-bound DNA in the MMS chip. (F) PCR amplification of the eluted SRAs with biotinylated reverse primers, followed by purification on streptavidin-coated magnetic beads. (G) Single-stranded DNA is generated from double-stranded PCR amplicons. (H) Incubation of enriched SRAs with biotinylated antisense DNAzyme strand and PCR primer site-blocking strands in preparation for the next round of selection.

Fig. 3.

Gel electrophoresis image showing the affinity and specificity of the selected SRAs (100 bp product) for thrombin after eight rounds of selection. Lanes: 1, 100 bp ladder; 2, DNA mass standard (intensity of the 100 bp band is equivalent to 10 ng DNA); 3, DNA sample eluted with the selection buffer overnight; 4, DNA sample eluted with the selection buffer for 2 h; 5, DNA sample eluted with 1 μM streptavidin; 6, DNA sample eluted with 500 nM thrombin; 7, DNA sample eluted with 1 μM thrombin. Thrombin and streptavidin incubations were each performed for 2 h.

Fig. 4.

The dependence of SRA elution on thrombin concentration after 15 rounds of selection. (A) Gel image of the PCR-amplified DNA samples eluted by different concentrations of thrombin ranging from 25 nM to 1 μM (lanes 3–8). Lane 1 is 100 bp ladder, and lane 2 is selection buffer only; (B) The dissociation constant (Kd) of the selected SRA pool, calculated from the intensity of the sample bands, is 70 ± 14 nM.

Fig. 5.

SRA_1 functions as an effective fluorescent reporter of target-binding events. (A) Fluorescence measurements of antisense-hybridized SRA_1 upon the addition of thrombin at varying concentrations. The intensity of fluorescence increased monotonically with increasing thrombin concentration. A 700% increase in fluorescence was observed with 100 nM thrombin compared to the sample without target. (B) Calibration curve of SRA_1 molecule derived from the fluorescence signal shows a Kd of 77 ± 14 nM.

Fig. 6.

SRA_1 shows high specificity toward thrombin target. (A) Fluorescence measurements of target specificity of SRA_1. In comparison to the strong fluorescent signal from antisense-hybridized SRA_1 challenged with thrombin (red), we observed only limited signal in negative control experiments with NMM only (black), antisense-hybridized SRA_1 only (purple), or antisense-hybridized unselected library molecules either alone (blue) or challenged with thrombin (cyan). (B) SRA_1 duplexes challenged with 1 μM streptavidin exhibited 27% of the fluorescence signal obtained with 1 μM thrombin.

Fig. 7.

SRA_1 and SRA_2 show specific affinity toward thrombin with nM dissociation constants and negligible binding to streptavidin. RT-PCR determination of dissociation constants for SRA_1 (A) and SRA_2 (B) yields Kd values of 99.3 ± 12.9 nM and Kd = 93.6 ± 14.2 nM, respectively.

Fig. 8.

Fluorescence measurements of antisense-hybridized SRA_1 upon addition of varying concentrations of thrombin in (A) Hepes buffer or (B) 50% FBS. The relatively larger background in serum caused by the presence of thrombin in the undoped serum. The background fluorescence of the undoped serum is subtracted from the three serum samples in B.