Nano-flares for mRNA Regulation and Detection

Abstract

We build off the previously described concept of a nano-flare to develop an oligonucleotide gold nanoparticle conjugate that is capable of both detecting and regulating intracellular levels of mRNA. We characterize the binding rate and specificity of these materials using survivin, a gene associated with the diagnosis and treatment of cancer, as a target. The nanoconjugate enters cells and binds mRNA, thereby decreasing the relative abundance of mRNA in a dose-and sequence-dependent manner and resulting in a fluorescent response. This represents the first demonstration of a single material capable of both mRNA regulation and detection. Further, we investigate the intracellular biochemistry of the nanoconjugate, elucidating its mechanism of gene regulation. This work is important to the study of biologically active nanomaterials such as the nano-flare and is a first step towards the development of an mRNA responsive 'theranostic'. KeywordsNanoparticle; oligonucleotide; mRNA; detection; gene regulation; theranostic Over the past decade, researchers have investigated the conjugation of biomolecules to inorganic nanomaterials, which has led to the development of hybrid materials with new activities. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] One important class of hybrid nanomaterial is composed of a gold nanoparticle functionalized with a dense monolayer of oligonucleotides. These polyvalent nanoconjugates have many interesting properties, including distance-dependent optical features, 21,22 enhanced nucleic acid binding, 23 resistance to degradation, 24 and the ability to enter cells without use of transfection agents. 25 These remarkable properties have enabled controlled assembly of materials, 26-28 molecular diagnostics, [29][30][31][32] and intracellular studies. [33][34][35] Materials with both regulation and detection capabilities are of growing interest for use in personalized medicine. 36 These 'theranostic' materials have the potential to both treat and diagnose disease and are useful for investigating intracellular events (i.e. target recognition and control of biological function). Traditionally, antisense oligonucleotides and molecular beacons have been used to regulate and detect intracellular mRNA, respectively. Antisense oligonucleotides regulate gene expression by binding target mRNA and preventing translation. 37 Molecular beacons detect nucleic acids by coupling a binding event with a signal transduction mechanism, such as the separation of a fluorophore-quencher pair. 38 Given that cell entry and mRNA binding are the first steps in both processes, it should be possible to design a single material for both regulation and detection. To the best of our knowledge, however, a material capable of both mRNA regulation and detection has not been reported. 39 Such materials must be readily taken up by cells, stable in intracellular environments, capable of binding nucleic acids, and possess a switchable signal that can be conveniently detected. NIH Public Access RES...

Aptamer-based biosensors for biomedical diagnostics

“…Despite these difficulties, one such technology called 'nano-flares', has been commercialized to detect mRNA. 124,125 Nano-flares refer to a special fluorescent DNA-AuNP conjugate as shown in Figure 3C, except that the quencher is replaced by AuNPs. Compared to molecular dark quenchers (such as Dabcyl), AuNPs have better quenching efficiency and longer quenching range.…”

Quantification and real-time tracking of RNA in live cells using Sticky-flares

“…Previously our group introduced the concept of the Nanoflare, a spherical nucleic acid (SNA) conjugate capable of quantifying RNA concentration in live cells with single cell resolution (17)(18)(19)(20)(21)(22). The Nanoflare consists of a 13-nm gold nanoparticle core functionalized with a densely packed, highly oriented shell of oligonucleotides designed to be antisense to a target RNA transcript.…”

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

“…If these controls turn out satisfactory one can expect to obtain reliable specific fluorescent signals using nanoparticles specific for other target genes. The concentration of the applied nanoparticles may also be critical as they have been shown to down-regulate the target sequence 24 . However, this effect requires much higher concentrations ( 5 nM) than the concentrations used in the experiments presented here (400 pM).…”