Palindrome-Embedded Hairpin Structure and Its Target-Catalyzed Padlock Cyclization for Label-Free MicroRNA-Initiated Rolling Circle Amplification

Abstract

Highly sensitive detection of microRNAs (miRNAs) is of great significance in early diagnosis of cancers. Here, we develop a palindrome-embedded hairpin structure and its target-catalyzed padlock cyclization for rolling circle amplification, named PHP-RCA for simplicity, which can be applied in label-free ultrasensitive detection of miRNA. PHP-RCA is a facile system that consists of only an oligonucleotide probe with a palindrome-embedded hairpin structure (PHP). The two ends of PHP were extended as overhangs and designed with the complementary sequences of the target. Hence, the phosphorylated PHP can be cyclized by T4 DNA ligase in the presence of the target that serves as the ligation template. This ligation has formed a palindrome-embedded dumbbell-shaped probe (PDP) that allows phi29 polymerase to perform a typical target-primed RCA on PDP by taking miRNA as a primer, resulting in the production of a lengthy tandem repeat. Benefits from the palindromic sequences and hairpin-shaped structure in padlock double-stranded structures can be infinitely produced during the RCA reaction and provide numerous binding sites for SYBR Green I, a double-stranded dye, achieving a sharp response signal for label-free target detection. We have demonstrated that the proposed system exhibits a good linear range from 0.1 fM to 5 nM with a low detection limit of 0.1 fM, and the non-target miRNA can be clearly distinguished. The advantages of high efficiency, label-free signaling, and the use of only one oligonucleotide component make the PHP-RCA suitable for ultrasensitive, economic, and convenient detection of target miRNAs. This simple and powerful system is expected to provide a promising platform for tumor diagnosis, prognosis, and therapy.

Scheme 1. (A) Molecule Structure of PHP and Functional Region Analysis and (B) the Principle of PHP-RCA for Label-Free Ultrasensitive Detection of Target miRNA

Figure 1

Feasibility of the PHP-RCA-based sensing system for the detection of miR-21. (A) Native PAGE (8%) analysis of various reaction mixtures: (a) miR-21, (b) PHP, (c) PHP + miR-21, (d) PHP + T4 DNA ligase + phi29 polymerase, and (e) PHP + miR-21 + T4 DNA ligase + phi29 polymerase. The sensing system was 40 μL, and the concentrations of the target miR-21, PHP, T4 DNA ligase, phi29 polymerase, and dNTPs are 5 nM, 5 nM, 4 U/μL, 0.1 U/μL, and 10 mM, respectively. (B) Fluorescence spectra of the same reaction mixtures from A. SYBR Green I was used as an indicator.

Figure 2

Effects of (A) the RCA reaction time and (B) the concentration of SYBR Green I on the performance of the PHP-RCA sensing system. The concentration of target miR-21, PHP, T4 DNA ligase, phi29 polymerase, and dNTPs are 5 nM, 5 nM, 4 U/μL, 0.1 U/μL, and 10 mM, respectively. Error bars represent the standard deviations (SD, n = 3). The signal-to-noise ratio (SNR) refers to the ratio of the fluorescence peak generated by the target to the background fluorescence. “N” and “P” denote negative samples without miR-21 and positive samples with miR-21, respectively.

Figure 3

Capability of the PHP-RCA for target miR-21 detection. (A) Fluorescence spectra of the PHP-RCA system upon addition of different concentrations of target miR-21, ranging from 0 to 5 nM. Inset: fluorescence spectra in the presence of a low target concentration of the miR-21. (B) Dependence of the peak fluorescence response on the concentration of target miR-21 in a range from 0.1 fM to 5 nM. Linear response of the sensing system at (C) the low and (D) high target concentration ranges. Error bars are standard deviations from three repetitive experiments.

Figure 4

(A) Fluorescence signal induced by other miRNA (miR-141, miR-155, miR-31, miR-122, and miR-26a) and (B) target miR-21 with one, two, and three mutation points (MT1, MT2, and MT3, respectively) against miR-21 at a concentration of 5 nM. The fluorescence signal-to-noise ratio was calculated by F/F₀ where F and F₀ represent the fluorescence intensity of PHP-RCA in the presence and absence of target miR-21, respectively. The error bar represents the standard deviation (SD) estimated from three replicate measurements. ^****P < 0.0001, independent-sample t-test.

Figure 5

Performance of PHP-RCA for miR-21 detection in the (A) serum and (B) plasma. (C, D) Performance of PHP-RCA for miR-21 detection in real samples. F and F₀ represent the fluorescence intensity of PHP-RCA in the presence and absence of the target, respectively. The error bar represents the standard deviation (SD) estimated from three replicate measurements. ^****P < 0.0001, independent-sample t-test.