Visual detection of single-base mismatches in DNA using hairpin oligonucleotide with double-target DNA binding sequences and gold nanoparticles

Abstract

We describe a hairpin oligonucleotide (HO) with double-target DNA binding sequences in the loop and 11-base in the stem for visual detection of single-base mismatches (SBM) in DNA with highly specificity. The thiol-modified HO was immobilized on gold nanoparticle (Au-NP) surface through a self-assembling process. The strategy of detecting SBM depends on the unique molecular recognition properties of HO to the perfect-matched DNA and SBM to generate different quantities of duplex DNA on the Au-NP surface, which are captured on the test zone of lateral flow test strip via the DNA hybridization reaction between the duplex DNA and preimmobilized DNA probe. Accumulation of Au-NPs produces the characteristic red bands, enabling visual detection of SBM. It was found that the ability of HO to differentiate perfect-matched DNA and SBM was increased dramatically by incorporating double-target DNA binding sequences in the loop of HO. The signal ratio between perfect-matched DNA and SBM was up to 28, which is much higher than that of conventional HO or molecular beacon. The approach was applied to detect the mutation sites, Arg142Cys and Gly529Ile, of transglutaminase 1 gene in autosomal recessive congenital ichthyosis. The results presented here show that the new HO is a potential molecular recognition probe for the future development of nucleic acid-based biosensors and bioassays. The approach can be used for point-of-care diagnosis of genetic diseases and detecting infectious agents or warning against bio-warfare agents.

Figure 1

(A) Typical photo images of the LFSs after analysis of 5-nM SBM, 5-nM wild-type DNA, 50-nM noncomplementary DNA and 0-nM DNA with the new HO and traditional HO. Assay time: 25 min; Running buffer: 4×SSC buffer containing 4% BSA. (B) Histogram of the intensities of the red bands on the test zone of LFSs.

Figure 2

Histogram of the signal ratio between SBM and wild-type DNA with the new HO (blue bar) and traditional HO (red bar) at different DNA concentration levels.

Figure 3

Typical photo images of the LFSs after analysis of 0-nM SBM, 50-nM wild-type DNA, 5-nM R142C DNA, 5-nM G529I DNA, and the mixture of 5-nM R142C DNA+5-nM G529I DNA with the two HO probes.

Scheme 1

(A) The structure of traditional HO with short stem-loop. (B) the structure of new HO with two SBM binding sequences in loop and 11-base in the stem. The bases in black are the sites mismatched with wild-type DNA, and the double bold lines in the loop are used to indicate the beginning and ending binding sites of SBM. (C) the structure of formed HO-SBM complex. (D) Schematic illustration for capturing gold nanoparticles on the test zone of lateral flow strip. (E) Schematic illustration for capturing gold nanoparticles on the control zone of lateral flow strip. (F) Schematic illustration for capturing gold nanoparticles on the test zone of lateral flow strip with traditional HO in the presence of wild-type DNA. SA: streptavidin; Au: gold.