Graphene oxide and molecular beacons-based multiplexed DNA detection by synchronous fluorescence analysis

We have developed a multiplexed DNA detection method based on graphene oxide （GO） and molecular beacons （MBs） by synchronous fluorescence analysis, demonstrated it by an oligonucleotide sequence of wild-type HBV （T1） and a re- verse-transcription oligonucleotide sequence of the RNA fragment of HIV （T2） as a model system. In the absence of targets DNA, FAM-tagged free MB probes （PHBv） and ROX-tagged free MB probes （PHIv） are adsorbed on GO via π-π interactions between DNA nucleobases and nucleosides, and the π-rich GO brings the fluorophores of MB and GO into close proximity. And then, the fluorescence of fluorophores is quenched by GO. But in the presence of targets DNA, PHBV and PHW hybridize with their targets DNA resulting in the formation of double-stranded DNA （dsDNA）, causing the separation of PHBV and PHW from the surface of GO and the recovery of the fluorescence of fluorophores （FAM and ROX） simultaneously. The simultane- ous detection of T1 and T2 can be realized by measuring fluorescence signals of FAM and ROX, respectively. Under the op- timum conditions, the fluorescence intensities of two dyes all exhibit good linear dependence on their target DNA concentra- tion in the range of 5×10-11-5×10 9 M. The detection limit of T1 is 3×10-11 M （3σ）, and that of T2 is 2×10-11 M. Compared with other methods for DNA detection based on GO, the proposed method has some advantages including higher selectivity and shorter analytical time.

Mechanics of molecular beacons for mRNA detecting

Decoding genetic information is crucial for gene therapy and cancer diagnosis,which has attracted growing interest in the field of clinical medicine and life science.In this study,we conducted a comprehensive exploration to obtain the detection mechanism of molecular beacons from a mechanics point of view.The potential energy function of molecular beacon/target system is established firstly,based on which the profile of molecular beacons is solved by genetic algorithm optimization.The length of stem and the total energy are further calculated when the target is hybridized with loop and stem.The results show that the hybridization between target and stem is energetically favorable compared with that between target and loop,indicating a new detection strategy.These analyses may cast light on understanding the mechanism of molecular beacons detection,and further help to design novel molecular beacons with high resolution and quantification.

Real-time fluorescent quantitative immuno-PCR method for determination of fluoranthene in water samples with a molecular beacon

A reliable and sensitive competitive real-time fluorescent quantitative immuno-PCR （RTFQ-IPCR） assay using a molecular beacon was developed for the determination of trace fluoranthene （FL） in the environment.Under optimized assay conditions,FL can be determined in the concentration range from 1 fg/mL to 100 ng/mL,with y=0.194x ＋ 7.859,and a correlation coefficient of 0.967 was identified,with a detection limit of 0.6 fg/mL.Environmental water samples were successfully analyzed,recovery was between 90% and 116%,with intra-day relative standard deviation （RSD） of 6.7%-12.8% and inter-day RSD of 8.4%-15.2%.The results obtained from RTFQ-IPCR were confirmed by ELISA,showing good accuracy and suitability to analyze FL in field samples.As a highly sensitive method,the molecular beacon-based RTFQ-IPCR is acceptable and promising for providing reliable test results to make environmental decisions.

REAL-TIME DETECTION OF SURVIVIN mRNA EXPRESSION IN CERVICAL CANCER CELL LINES USING MOLECULAR BEACON IMAGING

Objective To detect the expression of survivin mRNA in cervical cancer cell lines using molecular beacon imaging technology. Methods Human cervical cancer cells (HeLa and SiHa) and human fetal lung fibroblast HFL-I were cultured in vitro. After adding 100nmol/L survivin mRNA molecular beacon, the fluorescent signals were observed under fluorescent microscope. The expressions of survivin in cervical cancer cells and HFL-I cell were examined by immunocytochemical streptravidin-biothin peroxidase (SP) assay at the same time. Results Two kinds of survivin mRNA molecular beacon, with different color fluorescence, had strong fluorescent signal in cervical cancer cell lines, and the signal in SiHa cell line was stronger, but these signals were not found in HFL-I ; Immunocytochemical staining of positive survivin was located in the cytoplasm of cervical cancer cell lines HeLa and SiHa, whereas, no expression of survivin was detected in HFL-I cell line. Conclusion The technology of molecular beacon imaging can be used to detect the expression of survivin mRNA in viable cells successfully, and may provide a new approach to the diagnosis of early stage cervical cancer and the following-up in the clinic.

Real-time Monitoring of Nucleotide Excision of Molecular Beacon Catalyzed by Klenow Fragment

Klenow fragment（KF） uses the activity of a separate exonuclease to excise nucleotide, which is a crucial step in DNA replication and repair. Here is a novel sensitive and convenient method introduced for real-time monitoring nucleotide excision by KF with a molecular beacon as a detecting probe in a homogeneous solution. This method, which overcomes the drawbacks of traditional methods such as discontinuity, time consuming and low sensitivity, was used to assay KF activity and the detection limit reached up to 0.4 U/mL. In addition, the method was applied to investigating the effects of metal ions and chemical drugs on the reaction. The results demonstrate that it is a potential high-throughput assay for screening inhibitors and activity analysis of KF in vitro.

Development and Application of Detection Methods for Capture and Transcription Elongation Rate of Bacterial Nascent RNA

Objective Detection and quantification of RNA synthesis in cells is a widely used technique for monitoring cell viability,health,and metabolic rate.After exposure to environmental stimuli,both the internal reference gene and target gene would be degraded.As a result,it is imperative to consider the accurate capture of nascent RNA and the detection of transcriptional levels of RNA following environmental stimulation.This study aims to create a Click Chemistry method that utilizes its property to capture nascent RNA from total RNA that was stimulated by the environment.Methods The new RNA was labeled with 5-ethyluridine(5-EU)instead of uracil,and the azido-biotin medium ligand was connected to the magnetic sphere using a combination of“Click Chemistry”and magnetic bead screening.Then the new RNA was captured and the transcription rate of 16S rRNA was detected by fluorescence molecular beacon(M.B.)and quantitative reverse transcription PCR(qRT-PCR).Results The bacterial nascent RNA captured by“Click Chemistry”screening can be used as a reverse transcription template to form cDNA.Combined with the fluorescent molecular beacon M.B.1,the synthesis rate of rRNA at 37℃is 1.2 times higher than that at 15℃.The 16S rRNA gene and cspI gene can be detected by fluorescent quantitative PCR,it was found that the measured relative gene expression changes were significantly enhanced at 25℃and 16℃when analyzed with nascent RNA rather than total RNA,enabling accurate detection of RNA transcription rates.Conclusion Compared to other article reported experimental methods that utilize screening magnetic columns,the technical scheme employed in this study is more suitable for bacteria,and the operation steps are simple and easy to implement,making it an effective RNA capture method for researchers.

R11 peptides can promote the molecular imaging of spherical nucleic acids for bladder cancer margin identification

One of the critical problems in bladder cancer(BC)management is the local recurrence of disease.However,achieving the accurate delineation of tumor margins intraoperatively remains extremely difficult due to the lack of effective tumor margin recognition technology.Herein,survivin molecular beacon(MB)and R11 peptide-linked spherical nucleic acids(SNAs)were synthesized as nanoprobes(AuNP-MB@R11)for sensitive detection of BC margins.Physicochemical properties proved that R11 peptides and survivin MB were successfully loaded onto the surface of SNAs.AuNP-MB@R11 had good stability against nuclease activity and high sensitivity and specificity to detect survivin single strand DNA(ssDNA)in vitro.According to cytology,R11 peptides could increase the BC targeting ability and membrane penetrability of SNAs.Notably,R11 peptides significantly promoted the disintegration of lysosomes and the release of SNAs to enhance fluorescence imaging quality.Further RNA sequencing proved that some genes and pathways related to endocytosis and lysosomes were significantly regulated,such as AGPAT5,GPD1L,and GRB2.In orthotopic BC models and a clinical sample from a patient with BC,AuNP-MB@R11 showed a more legible cancerous fluorescence margin and offered remarkably improved detection compared to those achieved by SNAs.R11 peptide-linked SNAs present promising potential to identify BC margin,which may help to improve the R0 resection rate in surgery and improve patients’quality of life.

Engineering Novel Molecular Beacon Constructs to Study Intracellular RNA Dynamics and Localization

With numerous advancements in novel biochemical techniques, our knowledge of the role of RNAs in the regulation of cellular physiology and pathology has grown significantly over the past several decades. Nevertheless, detailed information regarding RNA processing, trafficking, and localization in living cells has been lacking due to technical limitations in imaging single RNA transcripts in living cells with high spatial and temporal resolution. In this review, we discuss tech- niques that have shown great promise for single RNA imaging, followed by highlights in our recent work in the development of molecular beacons （MBs）, a class of nanoscale oligonucleotide-probes, for detecting individual RNA transcripts in living cells. With further refinement of MB design and development of more sophisticated fluorescence microscopy techniques, we envision that MB-based approaches could promote new discoveries of RNA functions and activities.

A Label-free and Functional Fluorescent Oligonucleotide Probe Based on a G-Quadruplex Molecular Beacon for the Detection of Kanamycin

A label-free and turn-off fluorescent method for the quantitative detection of kanamycin based on a funtional molecular beacon was developed. The molecular beacon consists of two hairpin structures with a split G-rich oligonucleotide in the middle. The kanamycin＇s aptamer formed the loops portion for recognizing kanamycin, and the G-quadruplex bound by Thioflavin T（ThT） was employed as the reporter. In the absence of target, the molecular beacon folded into double stem-loops and the splited G-rich oligonucleotid came close to form a G-quadruplex. When ThT bound to the G-quadruplex, the fluorescence intensity of the solution increased. Upon the addition of kanamycin, the function between kanamycin and aptamer unfolded the hairpin and disassembled the G-quadraplex structure, resulting in a significant decrease in the fluorescence intensity. A good linear relationship ranging from 0.7 nmol/L to 10 nmol/L was achieved and the limit of detection was 0.37 nmol/L. Besides, it could efficiently recognize kanamycin in real samples.

Inhibited aptazyme-based catalytic molecular beacon for amplified detection of adenosine

Combining the inhibited aptazyme and molecular beacon （MB）, we developed a versatile sensing strategy for amplified detection of adenosine. In this strategy, the adenosine aptamer links to the 8-17 DNAzyme to form an aptazyme. A short sequence, denoted as inhibitor, is designed to form a duplex spanning the aptamer-DNAzyme junction, which blocks the catalytic function of the DNAzyme. Only in the presence of target adenosine, the aptamer binds to adenosine, thus the inhibitor dissociates from the aptamer portion of the aptazyme and can no longer form the stable duplex required to inhibit the catalytic activity of the aptazyme. The released DNAzyme domain will hybridize to the MB and catalyze the cleavage in the presence of Zn2＋, making the fluorophore separate from the quencher and resulting in fluorescence signal. The results showed that the detection method has a dynamic range from 10 nmol/L to 1nmol/L, with a detection limit of 10 nmol/L.

Ligase-based ultrasensitive detection of DNAzyme cleavage product using molecular beacon

Detection for deoxyribozyme （DNAzyme） cleavage usually needs complex and time-consuming radial labeling, gel electro- phoresis and autoradiography. A new approach was reported for detection DNAzyme cleavage product based on molecular beacon （MB）. Part of the loop of MB was designed to complementary to DNAzyme cleavage product. MB was employed to monitor ligation process of RNA/DNA complex and to convert directly cleavage product information into fluorescence signal. Detection limit of the assay is 0.02 nmol/L. The cleavage product of 8-17 DNAzyme against HCV-RNA was detected perfectly based on this assay. The method is fast, simple and ultrasensitive, which might hold great promise in DNAzyme reaction and DNAzyme gene therapy.

The exploration of quantum dot-molecular beacon based MoS_(2) fluorescence probing for myeloma-related Mirnas detection

Highly sensitive and reliable detection of multiple myeloma remains a major challenge in liquid biopsy. Herein, for the first time, quantum dot-molecular beacon (QD-MB) functionalized MoS_(2) (QD-MB @MoS_(2)) fluorescent probes were designed for the dual detection of multiple myeloma (MM)-related miRNA-155 and miRNA-150. The results indicate that the two probes can effectively detect miRNA-155 and miRNA-150 simultaneously with satisfactory recovery rates, and the limit of detections (LODs) of miRNA-155 and miRNA-150 in human serum are low to 7.19 fM and 5.84 fM, respectively. These results indicate that our method is the most sensitive detection so far reported and that the designed fluorescent probes with signal amplification strategies can achieve highly sensitive detection of MM-related miRNAs for MM diagnosis.

Ultrasensitive detection of cancer biomarker microRNA by amplification of fluorescence of lanthanide nanoprobes

Sensitive detection of cancer biomarker microRNAs （miRNAs） is of vital importance for cancer diagnosis and treatment. Nonetheless, the detection sensitivity in the existing miRNA bioassays is severely limited by the structural characteristics of miRNA （including small length and high sequence homology） because most of these methods are based on target amplification. Herein, we report a novel approach to sensitive and specific detection of low-abundance miRNA via a unique strategy of nanoprobe dissolution-enhanced fluorescence amplification, in which a capture probe featuring molecular beacon structure is designed. By means of this strategy, miRNA-21 was detected in a linear range from 10 fM to 100 pM with a detection limit as low as 1.38 fM. High selectivity of the newly developed biosensor was demonstrated by the good discrimination against a target with a single-base mismatch. Furthermore, this assay was used for the detection of miRNA-21 added into fetal bovine serum samples with the recovery in the range of 90.2%--108% and coefficients of variation below 10.1%, indicating its promising applications to RNA immunoassays and early cancer diagnosis.