Label-free luminescent detection of LMP1 gene deletion using an intermolecular G-quadruplex-based switch-on probe

Highlights

• A series of luminescent iridium(III) complexes as the split G-quadruplex probes.

• Simple and selective gene deletion detection platform was developed.

• This platform could detect down to 10 nM of the target gene in aqueous solution.

Abstract

We have synthesized a series of luminescent iridium(III) complexes and investigated their ability to act as luminescent split G-quadruplex probes. After screening, the iridium(III) complex 1 [Ir(2-phenylquinoline)₂(3,4,7,8-tetramethyl-1,10-phenanthroline)]PF₆ was validated as a highly-selective G-quadruplex probe and was utilized to construct a label-free intermolecular G-quadruplex-based assay for the selective and sensitive detection of LMP1 gene deletion. This “mix-and-detect” assay is simple and selective, and could detect down to 10 nM of the target gene in aqueous solution with a linear range from 10 to 500 nM. We also investigated the performance of our split G-quadruplex-based sensing platform for LMP1 gene deletion in the presence of cellular debris, demonstrating the robustness of this sensing system in biological samples. Comparative assays were also performed using either organic dyes or labeled oligonucleotides as signal-transducing agents.

Introduction

Nasopharyngeal carcinomas (NPC) occur with high frequency in Southeast Asia (Parkin et al., 1999), and are associated with infection with the Epstein–Barr virus (EBV). The evidence for this relationship stems from the increased antiviral antibody titers and the presence of the viral genome in NPC patients, as well the monoclonal nature of the viral episome in NPC tumor cells. While EBV infection is ubiquitous in the world, the incidence of NPC is region-dependent, suggesting that NPC may be related to a subtype of EBV (Abdel-Hamid et al., 1992). Latent membrane protein 1 (LMP1) of EBV is the most likely candidate virus gene that participates in the transformation of the epithelium (Wang et al., 1985), and hence NPC-associated polymorphism of LMP1 has attracted much attention as a disease indicator. Most LMP1 genes derived from Chinese NPC biopsies are marked with a deletion of 10 amino acids (346–355 aa) or a 30-bp deletion (168287–168256 nt) in the carboxyl terminus (del-LMP1), the loss of the Xho I site in the amino terminus, and multiple base substitutions in the coding region (Hu et al., 1991). Among 187 cases of NPC biopsies in Asia in a study, 86% of them exhibited the del-LMP1 mutation (Edwards et al., 1999). This result suggests a potential association between del-LMP1 and NPC. Furthermore, del-LMP1 protein has greater potential to transform established rodent fibroblasts and immortalize human keratinocytes in vitro (Li et al., 1996), and to confer weaker immunogenicity that allows the tumor cells to escape immunosurveillance (Hu et al., 2000). Therefore, the development of a sensitive and efficient method to detect the deletion in the LMP1 gene is of paramount importance.

To date, numerous methods have been developed for the selective detection of DNA, including capillary electrophoresis (Kleparnik and Bocek, 2007), polymerase chain reaction (Saiki et al., 1988) and rolling circle amplification (Hourcade et al., 1973). However, those methods tend to be time-consuming, and involve tedious sample preparation, complex operation and/or the use of costly instrumentation (Zhu et al., 2008). The use of DNA for the recognition and detection of analytes has attracted much attention due to the low cost, stability and versatility of DNA oligonucleotides.

The G-quadruplex motif is a non-canonical DNA secondary structure consisting of square-planar arrangements of guanine nucleobases stabilized by Hoogsteen hydrogen bonding and monovalent cations (Zhao et al., 2004). The rich structural polymorphism of the G-quadruplex motif (Krishnan and Simmel, 2011) has stimulated the development of numerous G-quadruplex-based platforms for the detection of metal ions (Qu et al., 2012, Xu et al., 2010, Zhu et al., 2009; He et al., 2013) DNA (Su et al., 2013, Wen et al., 2012, Xu et al., 2013), small molecules (Peng et al., 2009), protein (Yin et al., 2012) and enzyme activity (Leung et al., 2013a, Lin et al., 2014, Su et al., 2012; Ma et al., 2013). G-quadruplexes that are formed by the association of two separate oligonucleotides can be termed bimolecular or “split” G-quadruplexes. Previous research has demonstrated that the hybridization of G-quadruplex flanking regions with target oligonucleotides can result in the formation of a split G-quadruplex structure that could be utilized for the specific detection of nucleic acid sequences (Deng et al., 2008).

Luminescent metal complexes have attracted growing interest in optoelectronic devices, molecular imaging, chemosensing and as structural probes for sensing (Gill and Thomas, 2012, Liu et al., 2011, Xu et al., 2014, Yu et al., 2008; Ma et al., 2014). Luminescent metal complexes can show selective binding affinities for specific DNA conformations. Luminescent metal complexes possess the following advantages that make them attractive as G-quadruplex probes: (i) the properties of metal complexes can be easily tuned by adjustment of the auxiliary ligands, (ii) their relatively large Stokes shifts help to prevent self-quenching, (iii) metal complexes can be synthesized by simple synthetic protocols (Liu et al., 2011, Metcalfe and Thomas, 2003, Yang et al., 2012), and (iv) organic fluorophores tend to have nanosecond lifetimes, can be hard to distinguish from background autofluorescence. On the other hand, the long lifetime of triplet metal-to-ligand charge transfer (³MLCT) phosphorescence in the visible region can enhance image signal stability and reduce interference from background autofluorescence.

Previously, we have developed a luminescent G-quadruplex-based assay for the detection of one type of gene deletion using an iridium(III) complex (He et al., 2012). We anticipated that the sensitivity of the assay for the detection of gene deletion could be significantly improved by screening a series of iridium(III) complexes as selective G-quadruplex probes.