Dual FRET molecular beacons for mRNA detection in living cells

Santangelo, Philip J.; Nix, Brent; Tsourkas, Andrew; Bao, Gang

doi:10.1093/nar/gnh062

Cited by 353 publications

(308 citation statements)

References 35 publications

Supporting

Mentioning

298

Contrasting

Unclassified

Order By: Relevance

“…25 Nonspecific opening is a particular problem when MBs are used for mRNA imaging in living cells because of the many biological components of the cell (e.g., proteins) that are capable of complexing with and opening the MBs. 26 BPs, like MBs, are hybridization probes, but instead of having a single probe sequence, they are composed of two independent probe sequences, each with a fluorophore molecule, that are brought together upon hybridization with adjacent sections of the target (Figure 1d). 9 BPs rely mostly on FRET for target detection.…”

Section: Introductionmentioning

confidence: 99%

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

Martí

Jockusch

et al. 2007

Tetrahedron

View full text Add to dashboard Cite

We report the design, synthesis and characterization of binary oligonucleotide probes for mRNA detection. The probes were designed to avoid common problems found in standard binary probes such as direct excitation of the acceptor fluorophore and overlap between the donor and acceptor emission spectra. Two different probes were constructed that contained an array of either two or three dyes and that were characterized using steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy and fluorescence depolarization measurements. The three-dye binary probe (BP-3d) consists of a Fam fluorophore which acts as a donor, collecting light and transferring it as energy to Tamra, which subsequently transfers energy to Cy5 when the two probes are hybridized to mRNA. This design allows the use of 488 nm excitation, which avoids the direct excitation of Cy5 and at the same time provides a good fluorescence resonance energy transfer (FRET) efficiency. The two-dye binary probe system (BP-2d) was constructed of Alexa488 and Cy5 fluorophores. Although the overlap between the fluorescence of Alexa488 and the absorption of Cy5 is relatively low, FRET still occurs due to their close physical proximity when the probes are hybridized to mRNA. This framework also decreases the direct excitation of Cy5 and reduces the fluorescence overlap between the donor and the acceptor. Picosecond time-resolved spectroscopy showed a reduction in the fluorescence lifetime of donor fluorophores after the formation of the hybrid between the probes and target mRNA. Interestingly, BP-2d in the presence of mRNA shows a slow rise in the fluorescence decay of Cy5 due to a relatively low FRET rate, which together with the reduction in the Alexa488 lifetime provides a way to improve the signal to background ratio using time-resolved fluorescence spectra (TRES). In addition, fluorescence depolarization measurements showed complete depolarization of the acceptor dyes (Cy5) for both BP-3d (due to sequential FRET steps) and BP-2d (due to the relatively low FRET rate) in the presence of the mRNA target.

show abstract

Section: Introductionmentioning

confidence: 99%

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

Martí

Jockusch

et al. 2007

Tetrahedron

View full text Add to dashboard Cite

show abstract

“…The exonuclease activity of DNA 10 polymerases will cleave the MBs and disconnect the donor/quencher from the acceptor dye/dye, 11 whereupon a fluorescent signal can be detected and correlated to an increase in amplified DNA 12 109,112 . In live-cell imaging, MBs are popular for tracking intracellular RNA [113][114][115] …”

Section: Molecular Beacons 24mentioning

confidence: 99%

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

2015

View full text Add to dashboard Cite

7Live-cell imaging has provided the life sciences with insights into the cell biology and 8 dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In 9 this review, we focus on the current fluorescent labeling strategies for nucleic acids, and in 10 particular messenger RNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad 11 range of scientific fields. By giving a background of the available techniques and an evaluation 12 of the pros and cons, we try to supply scientists with all the information needed to come to an 13 informed choice of nucleic acid labeling strategy aimed at their particular needs. 14

show abstract

“…In fact, several groups have already demonstrated the feasibility of simultaneously imaging multiple genes in single living cells with molecular beacons [69,70]. To further reduce background signal, especially that due to degradation of beacons by endonucleases in living cells, a novel dual FRET-molecular beacons approach was developed; it utilizes a pair of molecular beacons each labeled with a donor and acceptor fluorophore as illustrated in Figure 3B [65,66]. The probe sequences are chosen such that the molecular beacons hybridize to adjacent regions on a single nucleic acid target, similar to the dual FRET-linear probes.…”

Section: Molecular Beaconsmentioning

confidence: 99%

“…Dual FRET-molecular beacons, therefore, combine the low-background signal and high specificity of molecular beacons with the ability of two-probe FRET assays to differentiate between true target recognition and false-positive signals. It has been demonstrated that upon hybridization to nucleic acid targets, dual FRET-molecular beacons can provide a better signal-to-background ratio than the single molecular beacon approach when appropriate FRET pairs are selected [65,66].…”

Section: Molecular Beaconsmentioning

confidence: 99%

“…Dual FRET molecular beacons have been used to detect K-Ras, surviving and oct4 mRNAs in HDF, Miapaca-2, and H1 cells, respectively [66,72]. Interestingly, k-Ras mRNAs seemed to localize with a filamentous pattern in HDF cell whereas survivin mRNAs seemed to localize in a non-symmetrical pattern within Miapaca-2 cells, often to one side of the nucleus of the cell.…”

Section: Molecular Beaconsmentioning

confidence: 99%

See 1 more Smart Citation

Engineering imaging probes and molecular machines for nanomedicine

Tong

Cradick

et al. 2012

Sci. China Life Sci.

View full text Add to dashboard Cite

Nanomedicine is an emerging field that integrates nanotechnology, biomolecular engineering, life sciences and medicine; it is expected to produce major breakthroughs in medical diagnostics and therapeutics. Due to the size-compatibility of nano-scale structures and devices with proteins and nucleic acids, the design, synthesis and application of nanoprobes, nanocarriers and nanomachines provide unprecedented opportunities for achieving a better control of biological processes, and drastic improvements in disease detection, therapy, and prevention. Recent advances in nanomedicine include the development of functional nanoparticle based molecular imaging probes, nano-structured materials as drug/gene carriers for in vivo delivery, and engineered molecular machines for treating single-gene disorders. This review focuses on the development of molecular imaging probes and engineered nucleases for nanomedicine, including quantum dot bioconjugates, quantum dot-fluorescent protein FRET probes, molecular beacons, magnetic and gold nanoparticle based imaging contrast agents, and the design and validation of zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs) for gene targeting. The challenges in translating nanomedicine approaches to clinical applications are discussed. Nanomedicine is an emerging field that integrates nanotechnology, biomolecular engineering, biology, and medicine [1]. It focuses on the development of engineered nano-scale (1100 nm) materials, structures and devices for better diagnostics and highly specific medical intervention in curing disease or repairing damaged tissues. As a basis for nanomedicine, nanotechnology is the science, engineering, and technology related to the understanding and control of matter at the nano-scale, and the development of materials, devices, and systems that have novel properties and functions due to their nano-scale dimensions or components. Nanotechnology also provides new abilities to measure, control and manipulate matter (including soft matter) at the nano-scale what was unthinkable with conventional tools. Owing to the size-compatibility of nano-scale structures with proteins and nucleic acids in living cells, nanomedicine approaches have the potential to provide unprecedented opportunities for achieving a better control of biological processes, and drastic improvements in disease detection, therapy, and prevention, thus revolutionizing medicine. Over the last ten years or so, significant efforts have been made in the US, China, Europe and elsewhere to develop nanomedicine. For example, the US National Institutes of Health has developed a nanomedicine centers network, and invested a significant amount of research funding to nanomedicine development. Just in FY 2009 (Oct. 1, 2008Sept. 30, 2009, the total NIH funding in nanotechnology/ nanoscience projects was more than 410 million US dollars. SPECIAL TOPIC

show abstract

Dual FRET molecular beacons for mRNA detection in living cells

Cited by 353 publications

References 35 publications

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

Engineering imaging probes and molecular machines for nanomedicine

Contact Info

Product

Resources

About