Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection

Abstract

Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5' and 3' termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

Figure 1. Results of the reverse validation of selected PF-aptamers (PF20N-RO-MARAS-84-1) with positive (CRP) and negative controls (N1, N2, and N3).

(a) Before NSS, (b) After NSS, to provide nonspecific suppression.

Figure 2. Standard calibration curve of CRP concentration using PF-aptamer as the capture probe.

Figure 3. Recovery rate of spiked pure CRP of concentration 2000 nM in binding buffer, serum-1, -2, and -3 using PF-aptamer as the capture probe.

Figure 4. Comparison of the measured CRP concentration using PF-aptamer-based q-PCR method and monoclonal antibody based nephelometry method for blind serum samples.

(a) The correction of CRP concentration between the two measurement methods; (b) and (c) Bland-Altman plot analyses of the CRP concentrations of nephelometry and “Before NSS” and “After NSS”, respectively.

Figure 5. Schematic illustration of the PF-MARAS procedure.

The target and negative serum-MNPs (e.g., 1, 2, and 3) were prepared. After the formation of secondary structures, the 20-nt primer free library (5′-N₂₀-3′: PF library) was incubated with the target-MNPs. The unbound PF library was removed through magnetic separation. The bound mixture was re-dispersed and subjected to a MARAS selection process. Another magnetic separation was performed to remove the detached oligonucleotides. The bound mixture was re-dispersed and the oligonucleotides were eluted from MNPs through a heating process and then purified. The obtained oligonucleotides were incubated with negative serum-MNPs (1) then a magnetic separation was performed to remove the bound mixture containing oligonucleotides bound to negative MNPs (1) and the supernatant was collected. The collected supernatant containing unbound oligonucleotides was incubated with the next negative serum-MNPs (2). The negative selection process was repeated. After a final run of the negative selection, the final supernatant containing unbound oligonucleotides (PF-aptamer: 5′-S₂₀-3′) were obtained, in which, i is a free index starting from 0.

Figure 6. Schematic illustration of the cloning process for primer free aptamers.

The 5′-S₂₀-3′ was ligated to ds-5′-p-stem-loop-3′ primer by T4 RNA ligation. After purification, an extension was performed by incubated DNA polymerase and dNTP with ligated product. A CIP reaction was performed to remove the 5′-phosphorylation of un-ligated ds-5′-p-stem-loop-3′ primer. After another purification, the ds-5′-S₂₀-stem-loop-CCCCC-cS₂₀-A-3′ and ds-5′-stem-loop-CCCCC-A-3′ were ligated to TA-vector, and subjected to PCR amplification. A gel electrophoresis was performed for the PCR product, and the gel with correct size of PCR product was cut and used for cloning and sequencing to identify the sequence of PF-aptamer.

Figure 7. Schematic diagram illustrating the experimental procedure of reverse transcription for determining binding specificity of selected PF-aptamers toward target.