In vitro selection of DNA aptamers binding pesticide fluoroacetamide

Figures & data

Fig. 1. Structure formula of fluoroacetamide and its analog, N-fluoroacetyl glycine (A) and synthetic steps of N-fluoroacetyl glycine (B).

Table 1. Sequences of the 40nt core regions of the cloned aptamers.

Aptamer ID	Sequences of the 40nt core regions (5′ to 3′)	Identical sequences
Group I
1-1	ACCTGCAGGCGCGAGTTTCAGATCAAAACTTGTCTGGCGT	43
2-3	ACCTGCTTGTGCGAGGTGCAGATCAAAACTTGTCTGGCGT	1
2-4	ACCTGCAGGCGCGAGTTTTAGATCAAAACTTGTCTGGCGT	1
2-16	AACCTGCAGGCGCGAGTTTCAGATCAAAACTTGTCTGGCGT	1
4-4	ACCTGCAGGCGCGTGTTTCAGATCAAAACTTGTCTGGCGT	1
4-20	ACCTGCAGGCGCGAGTTCAGATCAAAACTTGTCTGGCGT	1
Group II
6-1	GGCGTGTGCCCTGCGGATGAAGACTGGTCT	3
Group III
1-17	GCCCTAAATACGAGCAACAGCGGATGAAGACTGGTCT	1
5-3	GGGGTAGTGTGCCCTGCGGATGAAGACTGGTCTG	1
2-8	TTGTATCTGTTATTATTATTCTGTTTTCGTTTGCTATAAC	1

Fig. 2. ¹H NMR spectra confirming the structure of fluoroacetamide analog.

Fig. 3. Schematic representation of aptamer selection procedure.

Fig. 4. Analysis of PCR products from each round of SELEX enrichment. PCR products were sampled after each round of enrichment and run on a 2% agarose gel.

Notes: M: molecular ladder (50–500 bp), Lanes 1-10: PCR products from enrichment rounds 1-10, and lane 11: no template control.

Fig. 5. Affinity of the selected ssDNA to the target in each selection round. The initial ssDNA library was used as reference.

Fig. 6. Multiple sequence alignments of the selected aptamers (without primers).

Notes: All positions with 75% or higher homology were highlighted and represented by lowercase letters in the consensus line. Identity = 66.89%. Numbers on the right-hand side indicate the length of each aptamer sequence. The number of nucleotides in the randomized region may have been changed by PCR mutations.

Fig. 7. Predicted secondary structures of aptamer 1-1, 6-1, 2-8, and 1-17.

Note: The bases with high similarity were marked by square.

Fig. 8. Binding curves of aptamer 1-1 and 6-1 to N-fluoroacetyl glycine examined by SPR.

Fig. 9. Binding curves of aptamer 1-1 and 6-1 in the SPR-based inhibition assay.

Fig. 10. Characterization of specificity of aptamer 1-1 for fluoroacetamide. The binding affinity of aptamer 1-1 to fluoroacetamide and other structurally similar molecules was tested by a dipstick assay based on lateral-flow technology. The fluorescence intensity at test line for each target was measured. The buffer solution without any target was used as blank.