Polymerase-based Nucleic Acid Amplification Technology For MicroRNA Detection

MicroRNAs(miRNAs) are a class of non-coding-protein, short endogenous RNAs that act as important post-transcriptional regulators of gene expression. Recently, increasing evidence has revealed that miRNAs play key roles in many biological processes, such as cell development, metabolism, apoptosis and tumorgenesis. The nucleic acid amplification detection techniques are mainly carried out by producing abundant copies of targeted sequences or transforming the target into other particular nucleic acid sequences or signal reporting molecules to gain the good results. Such target or signal amplification processes normally render the detection more sensitive and selective. However, their experimental design is very sophisticated, difficult to manipulate and the fluorescence-labeled probe or special enzyme used in this method would make detection quite expensive, which can limit their wide range of applications. Therefore, amplification strategies for convenient, low-cost and sensitive detection of miRNAs are in urgent need. In this thesis, the polymerase-based nucleic acid amplification detection techniques are studyed, and we have designed a series of new routes to detect mi RNAs in a simple, rapid, sensitive and economic pattern for early diagnostic, therapeutic intervention, as well as for drug discovery in cancers. The main work of my graduation thesis will be written in details in the sections below.1. According to the contents of this paper, miRNAs, terminal deoxynucleotidyl transferase(TdTase) and nucleic acid amplification techniques for miRNA detection are reviewed. First, an overview of the origin, mechanism of action and biological functions of miRNAs, and the important role and significance of miRNAs in cell growth and differentiation, the immune system and cancer, and as a tumor suppressor or carcinogenic gene is accomplished. Then, the importance and classification of DNA polymerase is briefly described, and TdTase polymerase activity analysis is emphasized. Finally, a review of several commonly used miRNA detecting nucleic acid amplification technology, including polymerase chain reaction(PCR) detection technology, rolling circle amplification(RCA) detection technology, strand displacement amplification(SDA) detection technology, duplex-specific nuclease(DSN) amplification detection technology and hybridization chain reaction(HCR) amplification detection technology is achieved.2. A novel, simple and common branched cascade enzymatic amplification(BCEA) system for the rapid and sensitive analysis of miRNAs is unveiled. Through target miRNA and capture probes DNA directly hybridize, the proposed signal amplification strategy is designed to initiate a branched cascade of enzymatic polymerization reactions and a large amount of double-stranded amplification branches by introducing the second primer to improve the efficiency, in order to detect, amplify and measure a specific miRNA using SYBR Green ?(SG)as the fluorescence signal. Moreover, the fluorescence intensity of SG improve with the increasing concentration of miRNA(1 fM to 10 pM), and the detection limit is estimated to be down to 0.1 fM. And the BCEA method not only can efficiently discriminate between single-nucleotide polymorphisms, but also can be directly used for sensitive miRNA detection of cancer cell extracts. The strategy has multifaceted excellences: Firstly, it reduces the complexity and cost of the experimental design and operation; secondly, the low detection limit and high selectivity of the method can be directly used for the highly sensitive detection of miRNAs in crude cellular extracts of cancer cells, holding great potential for application in clinical diagnosis; and finally, the proposed BECA method is simple, efficient and versatile, as the concept of the BCEA method can be applied to other sensing technologies.3. A directly detection of miRNAs is achieved based on the Hg2+-assistant dual polymerase isothermal nucleic acid amplification(Hg2+-DPINA) technology. This method depends on the hybridization of target miRNA and the template DNA, which can induce the synthesis of the ploy-thymine sequence catalytically generated by polymerase Klenow Fragment exo-(KFexo-) and TdTase. And, by introducing the Hg2+ to this system, the T-Hg2+-T double-stranded structure can be formed. Then, SG as the fluorescence dye, which is commonly used as a reporter, can preferably bind to the duplex via intercalation, leading to strong fluorescent signals, and the fluorescence enhancement level of SG is positively correlated with concentration of miRNAs, which realizes the sensitive detection of miRNAs. The miRNA detection is successfully achieved with a good linearity between 10 pM and 10 nM and a detection limit of 5 pM. The strategy has multifaceted excellences: First of all, by the introduction of Hg2+, double-stranded structure can be directly formed, avoiding the mismatch or strand displacement due to the hybridization reaction, to improve the binding efficiency of the double-stranded; secondly, the strategy relies on the fluorescence “turn-on” sensing mode, which offer more sensitivity and reduce the likelihood of a false positive signal. Moreover, this strategy is practical for the assay of target miRNA from cell lysates.4. A new strategy based on enzymatically engineered primer extension poly-thymine(EPEPT) and nanomaterials in situ generation technology is reported for the rapid and sensitive analysis of miRNAs by exploiting polyT-CuNPs nano-dye in situ synthesized. It is found that the short miRNA as a primer can be efficiently converted into a long poly-thymine(polyT) sequence by common catalyzing of the polymerase KFexo- and TdTase, which acts as a template in the formation of fluorescence copper nanoparticles(polyT-CuNPs) nano-dye in situ synthesized by addition of Cu2+ and sodium ascorbate, emitting intense red fluorescence. And the fluorescence intensity of polyT-CuNPs is in log-linear correlation with the concentration of miRNA, which realizes the low background and high sensitivity miRNA detection, over a range from 1 pM to 1 nM with the detection limit of 100 fM. And the detection method was used for miRNA detection in a variety of cell lysates with satisfactory results. More importantly, as the polyT-CuNPs can emit red fluorescence and has the excellent property of Stokes shifting, this strategy can avoid interference from complex mediums and realize visual detection under the UV lamp, holding the detection potential in real complex mediums and offering a potential tool in a wide range of potential applications, such as fluorescence imaging, clinical diagnostics and biological analysis fields.5. Based on the optical properties of the ligand-sensitized Tb3+ which has excellent Stokes shift and strong and sharp emission spectra, a novel analysis method is constructed for sensitive detection of mi RNAs, based on G-rich DNA-sensitized luminescence of terbium(III). First, the polymerization reaction is introduced by target miRNA, to form a long G-rich DNA sequence which can greatly enhance the fluorescence of Tb3+ by resonance energy transfer as the fluorescence signal for miRNA detection. With the increase of the target mi RNA concentration(0-1 nM), the fluorescence spectrum intensity of Tb3+(Em = 545 nm) is gradually increased, and miRNAs are detected with the detection limit of 100 fM. The advantage of detection method is that based on the energy resonance transfer effect between G-rich nucleotide sequence and Tb3+ applied to miRNA detection, a novel rare earth ion fluorescence enhancement-based miRNA detection technique is proposed, providing a credible and sensitive identification of miRNAs in a complex sample system. Based on above method, Tb3+ and semiconductor QDs-based dual-emission ratiometric fluorescence sensing platform is constructed for sensitive detection of miRNAs. First, QDs are coupled with capture probe(pDNA) to form QDs-pDNA complex, as the internal reference. Then the polymerization reaction is introduced by target miRNA, to form a long G-rich DNA sequence which can greatly enhance the fluorescence of Tb3+ by resonance energy transfer as the fluorescence signal for miRNA detection. According to the fluorescence spectral intensity ratio(I545/I610), miRNAs are detected. Dual-emission ratiometric fluorescence sensing technology can aviod the influence of external factors, such as sample environment, instruments and manual operation, to ensure the reliability of experimental results.