A minimal hybridization chain reaction (HCR) system using peptide nucleic acids

Abstract

The HCR represents a powerful tool for amplification in DNA-based circuitry and sensing applications, yet requires the use of long DNA sequences to grant hairpin metastability. Here we describe a minimal HCR system based on peptide nucleic acids (PNAs). A system comprising a 5-mer stem and 5-mer loop/toehold hairpins was found to be suitable to achieve rapid amplification. These hairpins were shown to yield >10-fold amplification in 2 h and be suitable for the detection of a cancer biomarker on live cells. The use of γ-peg-modified PNA was found to be beneficial.

Fig. 1. Schematic representation of different inputs and outputs of the HCR.

Fig. 2. (A) Basic design of the PNA HCR and structures of γ-modified PNA backbones used in this study. (B) 5% PAGE analysis of the HCR using FITC-labelled γ-serine modified-PNAs. Conditions for the reaction: pH 7.5 0.1× PBS, 0.02% tween-20, 1 μM of each component, 20 μL, r.t., and 2 h of incubation. (C) SDS-PAGE analysis of the HCR using γ-serine modified-PNAs, BCy3serI, serH1, and serH2. Conditions for the reaction: pH 7.5 0.1× PBS, 0.02% tween-20, 1 μM of each component, 15 μL, r.t., and 3 h of incubation.

Fig. 3. (A) 5% PAGE analysis of the HCR using FITC-labelled γ-peg modified-PNAs. Conditions for the reaction: pH 7.5 0.1× PBS, 0.02% tween-20, 500 nM of each component, 20 μL, r.t., and 2 h of incubation. (B) SDS-PAGE analysis of the HCR using γ-peg modified-PNAs, Cy3pegI, pegH1, and pegH2. Conditions for the reaction: pH 7.5 0.5× PBS, 0.02% tween-20, 500 nM of each component, 15 μL, r.t., and 2 h of incubation.

Fig. 4. Quantification of the elongation yield for the PNA HCR. (A) Scheme and result of streptavidin bead fluorescence pulldown assay. Reaction conditions: pH 7 1× SSC, 0.02% tween-20, 15 pmol of BpegI, 1 μM (150 pmol) of pegH1 and Cy3pegH2, and 4 h of reaction. (B) Fluorescence microscopy analysis for the estimation of HCR elongation. Reaction conditions: pH 7 1× SSC, 0.02% tween-20, 10 pmol of FBpegI or BpegI was loaded, 1 μM (200 pmol) of pegH1 and Cy3pegH2, and 4 h of reaction. The elongation fold reflects the number of hairpins incorporated into the HCR product relative to the initiator (elongation fold = (H1 + H2)/I).

Fig. 5. Design of PNA sequences for the kinetic analysis of the (A) first initiation step and (B) elongation step of the PNA HCR calculated using a pseudo-first order reaction kinetic model. On-rate constant (k_on) is reported.

Fig. 6. PNA HCR on CA IX expressing cells. (A) Sequence information and general scheme of the PNA-HCR system for the visualization of CA IX. CA IX was visualized by 1 : 1 ratio imaging with LpegI, PNA HCR (LpegI + pegH1 + Cy3pegH2), or only hairpins (pegH1 + Cy3pegH2). (B) Fluorescence pictures of HT-29 cells treated with LpegI; LpegI + pegH1 + Cy3pegH2; pegH1 + Cy3pegH2 under hypoxic conditions or treated with LpegI + pegH1 + Cy3pegH2 under normoxic conditions. Scale bar: 20 μM. (C) Fluorescence intensity profiles recorded along a line intersecting the cell and quantification of fluorescence on cell membranes. Error bars represent standard deviation. Statistics were calculated using an unpaired t-test. ****p < 0.0001.