RNA Mimics of Green Fluorescent Protein

Paige, Jeremy S; Wu, Karen; Jaffrey, Samie R.

doi:10.1126/science.1207339

Cited by 1,149 publications

(1,476 citation statements)

References 43 publications

Supporting

Mentioning

1,420

Contrasting

Unclassified

Order By: Relevance

“…The aptamers were selected on the basis of their brightness with respect to GFP to ensure the visual detection of the target. 24 Successful multiplexing of target detection proved the potential of relay PCR to generate multiple signals with aptamers having distant emission spectra (Supporting Information Table S2). …”

Section: ■ Results and Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Sequence-Specific Biosensing of DNA Target through Relay PCR with Small-Molecule Fluorophore

Yasmeen

Zhao

et al. 2016

ACS Chem. Biol.

View full text Add to dashboard Cite

Polymerase chain reaction coupled with signal generation offers sensitive recognition of target DNA sequence; however, these procedures require fluorophorelabeled oligonucleotide probes and high-tech equipment to achieve high specificity. Therefore, intensive research has been conducted to develop reliable, convenient, and economical DNA detection methods. The relay PCR described here is the first sequence-specific detection method using a smallmolecule fluorophore as a sensor and combines the classic 5′−3′ exonuclease activity of Taq polymerase with an RNA mimic of GFP to build a label-free DNA detection platform. Primarily, Taq polymerase cleaves the 5′ noncomplementary overhang of the target specific probe during extension of the leading primer to release a relay oligo to initiate tandem PCR of the reporting template, which encodes the sequence of RNA aptamer. Afterward, the PCR product is transcribed to mRNA, which could generate a fluorescent signal in the presence of corresponding fluorophore. In addition to high sensitivity and specificity, the flexibility of choosing different fluorescent reporting signals makes this method versatile in either single or multiple target detection.H ighly sensitive and selective DNA detection methods are important scientific tools in molecular biology and medical research. Many analytical methods for specific nucleic acid quantification have been developed. 1−9 PCR is widely used because of its remarkably high rapidity, precision, and reproducibility. Although the monitoring of PCR products formation can be carried out by gel electrophoresis, this technique is slow and laborious for high throughput applications. Real time PCR amplification, a modified procedure, has been achieved by adding DNA intercalating dyes, such as ethidium bromide and SYBR Green. 10−12 The main disadvantage of the application of DNA intercalators in PCR detection is their nonspecific binding with amplification products as well, which cannot be distiguished from the true amplicons. Alternatively, dual-labeled oligonucleotide probes have been used for sequence specific detections. The TaqMan approach where PCR-induced probe cleavage coupled with fluorescent signal generation was one of the first sequence specific methods to allow real-time PCR monitoring. It utilizes the 5′−3′nuclease activity of Thermusaquaticus(Taq) polymerase 13 to achieve high specificity and sensitivity (achieving detection of 10−100 DNA copies), but the TaqMan method is expensive and requires specially modified fluorogenic oligonucleotide probes and sophisticated real-time PCR machines. To decrease the high cost of labeled oligonucleotides in sequence dependent detection techniques, several new strategies have been developed such as the mediator probe method. 14−23 Nonetheless, fluorophore-modified oligonucleotide probes are still required for this methodology, though the cost is lower than that of a TaqMan probe. This study is the first report of a sequence-specific PCR system based on an unlabeled oligonucleotide,...

show abstract

Section: ■ Results and Discussionmentioning

confidence: 99%

“…24 This RNA−fluorophore complex displays fluorescence properties similar to GFP, but unlike most fluorescent proteins it is resistant to photobleaching. Further, DFHBI is fluorescent only upon binding to RNA, and it is not activated by the other components in the reaction.…”

mentioning

confidence: 99%

Sequence-Specific Biosensing of DNA Target through Relay PCR with Small-Molecule Fluorophore

Yasmeen

Zhao

et al. 2016

ACS Chem. Biol.

View full text Add to dashboard Cite

show abstract

“…Fig. 3 a FACS analysis of E. coli cells from rounds 1, 3, and 6. b Proposed secondary structures of aptamers ZT-26 and ZT-324, mutation domains are indicated in gray shadow, and Spinach structure comes from Paige et al (2011 …”

Section: Discussionmentioning

confidence: 99%

“…Recently, Jaffrey and colleagues have engineered an RNA aptamer, denoted ''Spinach,'' that mimics the function of green fluorescent protein (GFP): when bound with 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), Spinach becomes brightly fluorescent. It has been reported that Spinach/DFHBI displays fluorescence properties similar to GFP (*50 % brightness of EGFP), and therefore, this system represents a useful tool to examine RNA activities in cells (Paige et al 2012(Paige et al , 2011Song et al 2014;. The same group has also engineered another aptamer, named ''Broccoli,'' that can also bind and activate DFHBI (Filonov et al 2014).…”

Section: Introductionmentioning

confidence: 99%

Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein Using Fluorescence-Activated Cell Sorting

Zou

Huang

et al. 2015

J Mol Evol

View full text Add to dashboard Cite

Fluorescence-activated cell sorting (FACS) was exploited to isolate Escherichia coli cells that were highly fluorescent due to the expression of RNA aptamers that induce fluorescence of 3,5-difluoro-4-hydroxybenzylidene imidazolinone. Two different aptamers, named ZT-26 and ZT-324, were identified by this method and compared to the fluorescence-signaling properties of Spinach, a previously reported RNA aptamer. Aptamer ZT-26 exhibits significantly enhanced fluorescence over Spinach only in vitro. However, aptamer ZT-324 is 36 % brighter than Spinach when expressed in E. coli. The FACS-based selection strategy presented here is attractive for deriving fluorescent RNA aptamers that function in cells as it directly selects for cells with a high level of fluorescence due to the expression of the RNA aptamer.

show abstract

“…If inserted to 3′ end of RNA molecules including lncRNAs, the localization of target RNAs can be revealed upon addition of the fluorophore to the cell. One RNA mimic of eGFP referred to as Spinach binds DFHBI [(Z)‐4‐(3,5‐difluoro‐4‐hydroxybenzylidene)‐1,2‐dimethyl‐1H‐imidazol‐5(4H)‐one] was inserted to the 3′ end of 5S rRNA and successfully revealed its subcellular distribution 42. Therefore, the GFP RNA mimics can be applied to lncRNA research so as to enhance and simplify the attempt to map subcellular localization of these transcripts and how they might change in cancer disease.…”

Section: Methods Used To Localize Lncrnas Inside the Cellmentioning

confidence: 99%

State of the art technologies to explore long non‐coding RNAs in cancer

Salehi

Taheri

Azarpira

et al. 2017

J Cellular Molecular Medi

View full text Add to dashboard Cite

Long non‐coding RNAs (lncRNAs) comprise a vast repertoire of RNAs playing a wide variety of crucial roles in tissue physiology in a cell‐specific manner. Despite being engaged in myriads of regulatory mechanisms, many lncRNAs have still remained to be assigned any functions. A constellation of experimental techniques including single‐molecule RNA in situ hybridization (sm‐RNA FISH), cross‐linking and immunoprecipitation (CLIP), RNA interference (RNAi), Clustered regularly interspaced short palindromic repeats (CRISPR) and so forth has been employed to shed light on lncRNA cellular localization, structure, interaction networks and functions. Here, we review these and other experimental approaches in common use for identification and characterization of lncRNAs, particularly those involved in different types of cancer, with focus on merits and demerits of each technique.

show abstract

RNA Mimics of Green Fluorescent Protein

Cited by 1,149 publications

References 43 publications

Sequence-Specific Biosensing of DNA Target through Relay PCR with Small-Molecule Fluorophore

Sequence-Specific Biosensing of DNA Target through Relay PCR with Small-Molecule Fluorophore

Selection of Intracellularly Functional RNA Mimics of Green Fluorescent Protein Using Fluorescence-Activated Cell Sorting

State of the art technologies to explore long non‐coding RNAs in cancer

Contact Info

Product

Resources

About