Single palindromic molecular beacon-based amplification for genetic analysis of cancers

Highlights

• A novel palindromic fragment-incorporated molecular beacon (P-MB) was proposed.

• P-MB-based assay is the simplest SDA platform where only one probe is involved.

• The P-MB-based assay displays high sensitivity and excellent selectivity.

• The sensing system can detect different cancer-related genes simultaneously.

Abstract

The detection of biomarkers is of crucial importance in reducing the morbidity and mortality of complex diseases. Thus, there is a great desire to develop highly efficient and simple sensing methods to fulfill the different diagnostic and therapeutic purposes. Herein, using tumor suppressor p53 gene as model target DNA, we developed a novel palindromic fragment-incorporated molecular beacon (P-MB) that can perform multiple functions, including recognition element, signal reporter, polymerization template and primer. Upon specific hybridization with target DNA, P-MBs can interact with each other and are extended by polymerase without any additional probes. As a result, hybridized targets are peeled off from P-MBs and initiate the next round of reactions, leading to the unique strand displacement amplification (SDA). The newly-proposed enzymatic amplification displays the detection limit as low as 100 pM and excellent selectivity in distinguishing single-base mutation with the linear response range from 100 pM to 75 nM. This is the simplest SDA sensing system so far because of only involving one type of DNA probe. This impressive sensing paradigm is expected to provide new insight into developing new-type of DNA probes that hold tremendous potential with important applications in molecular biology research and clinical diagnosis.

Introduction

The occurrence and prognosis of many complex diseases are associated with the changes in various molecules, such as DNA, RNA, proteins and small molecules, many of which can serve as diagnostic biomarkers and drug targets (Gormally et al., 2007, Pasternak et al., 2013, Percy et al., 2013). Thus, the development of efficient methods to detect crucial biomarkers is highly desirable for early diagnosis and targeted therapy (Liu et al., 2014, Ziegler et al., 2012), yet remains a great challenge. Although some traditional biomolecular assays, including PCR to screen nucleic acids and ELISA to report protein and small molecules (Ke and Zhang, 2012; Yu et al., 2009), have been proposed for sensitive biomarker detection, the applications of existing classical nucleic acid amplification methods are limited to some extent. Because of their own intrinsic advantages and disadvantages depending on target species and detection environments, and no single method has gained supremacy (Elghanian et al., 1997). Efforts are still to be made to find better strategies to meet the increasing diagnostic and therapeutic needs.

Among the existing methods, molecular beacon (MB), proposed by Tyagi and Kramer in 1996 (Tyagi and Kramer, 1996), has been engineered to efficiently recognize and detect target nucleic acids, proteins, small molecules and even whole cells (Zheng et al., 2015). Conventional MB is single-stranded oligosequence doubly labeled with fluorophore and quencher group on two terminals and can fold into hairpin structure. The stem-loop structure keep chemically attached groups in close proximity, and thus the fluorophore is quenched in the absence of target. Upon binding to the target, MB can change its molecular configuration and separate the fluorophore from quencher, generating a detectable fluorescence signal. However, the detection sensitivity of MB is only partially successful because this probe reports its target DNA in a one-to-one manner. To improve the sensitivity of MB-based biosensing system, signal amplification strategies have been extensively used, such as nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), nicking endonuclease signal amplification (NESA), exonuclease-aided target recycling, non-covalent DNA catalytic reactions, hybridization chain reactions (HCR) and so on. The characteristics and possible applications of these sensing strategies have been summarized in published reviews (Yan et al., 2014; Zhao et al., 2015). Unfortunately, these amplification methods involved two or more oligonucleotides and often require relatively complex handling, costly equipment and/or labor-intensive and time-consuming process. Some of them also run the risk of cross-contamination from amplicons, and false-positive results are frequently obtained (Xu et al., 2011; Zou et al., 2011). These drawbacks have, to some extent, limited their application in the basic research into human diseases and medical diagnosis and treatment. To overcome these limitations, there is a real need for the development of a simpler and more cost- effective alternative for the detection of target DNA.

To simplify the signal transduction process without compromising the signal amplification effect, a novel palindromic molecular beacon (P-MB) was proposed in the present study, where the palindromic fragment was designed at the 3′-end. Palindromes, known as inverted duplications of nucleotide sequences, are found naturally in prokaryotic and eukaryotic genomes, many of which are present in functionally important regions and play important roles with respect to the function of these regions (Cain et al., 2001, Gruss and Novick, 1987, Lupski and Weinstock, 1992, Rocco et al., 2010). The characteristics of palindromic fragments are that two strands with the same base sequences can form double-stranded nucleic acids by the intermolecular self-hybridization (namely, involving only one type of nucleic acids). Utilizing the P-MB probe, we developed the first real single probe-based SDA sensing system.

In this work, the tumor suppressor gene p53, a molecular marker for personalized medicines, was chosen as the model target. The hybridization between P-MB and target gene unfolds the P-MB hairpin structure, opening the originally locked palindromic fragment in P-MB. All the released palindromic fragments of P-MB/target duplexes hybridize in pairs and could serve as polymerization primers. Subsequently, the enzymatic replication occurs and makes the hybridized target DNAs fall off from P-MBs. The displaced target DNAs are available for next hybridization/polymerization cycle, leading to the SDA effect. Despite impressive assay simplicity and operation convenience, high sensitivity and wide response range are achieved. The signal amplification mechanism of P-MB is illustrated, the signal amplification effect is confirmed and the assay feature of sensing system is evaluated in comparative manner.