Detection of intracellular microRNA using a self-assembling magnetic resonance beacon

Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Gangneung-si, Gangwon-do, Republic of Korea Catholic Kwandong University International St. Mary’s Hospital, Incheon, Republic of Korea Aljeraisy DNA Research Chair, Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia Department of Nucleic Acids Research, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technological Applications, Alexandria, Egypt † These authors contributed equally

MicroRNAs (miRNAs or miRs) are small noncoding RNAs consisting of 18-25 nucleotides.MiRNAs are clinically important because they simultaneously regulate expression of several genes and have crucial roles in proliferation [1] , differentiation [2] , development [3] , apoptosis [4] and diseases [5] .Therefore, the development of a novel miRNA molecular imaging method would be of great value for practical diagnostic and therapeutic applications.

RESEARCH HIGHLIGHT
real-time PCR [7] , RT-PCR [8] , northern blot [9] and in situ hybridization [10] have been used.However, these methods have low sensitivity, are laborious and time-consuming, and require expensive reagents and instruments.The main drawback of these methods is the invasive way of sample preparation and analysis, which requires cell lysis or fixation.During sample preparation, target miRNAs degrade.This makes it impossible to repeat analysis of the same samples or to obtain precise and accurate data from living cells.
For non-invasive miRNA imaging, reporter gene systems have been developed using bioluminescence [11] , radioisotope [12]   , fluorescence [13] and magnetic resonance (MR) [14] techniques.In this system, target miRNA imaging is based on a signal-off system, i.e., the presence of target miRNA reduces the reporter signal.Thus, low target miRNA concentration can produce a false-positive signal from its autofluorescence background.Indeed, low sensitivity and autofluorescence have led to limited in vivo applications of reporter gene systems.
Molecular beacon (MB) has been developed as a signal-on strategy for non-invasive miRNA imaging with high sensitivity.MB utilizes a fluorescent probe and a quencher molecule.The signal-on strategy is useful when the target miRNA expression level is low because of its low autofluorescence.However, imaging is achieved by a fluorescence signal.The critical drawback of fluorescence-based imaging is its low penetration in tissue, which results in low signal-to-noise contrast [15] .Thus, MB is not appropriate for clinical applications.
We have developed a new way of using magnetic resonance imaging (MRI) to detect intracellular miRNA by using self-assembling magnetic nanoparticles [16] .MRI provides high resolution and sensitivity for imaging anatomy and tissues and is widely used clinically.Magnetic nanoparticles were employed as the MRI contrast agent, and the MR signal-on was induced by binding of target miRNAs and subsequent nanoparticle disassembly.The target miRNA binding-responsive contrast change has a high potential as a novel MR imaging tool.For imaging and future clinical applications, this work aimed at detecting miR124a, which is highly expressed during neurogenesis [17] .
For construction of a miR124a detecting MR beacon (miR124a MR beacon), self-assembling magnetic nanoparticles were prepared using 50-nm carboxyl-group functionalized cobalt ferrite magnetic fluorescence nanoparticles.The miR124a MR beacon self-assembly was initiated by incubation of magnetic nanoparticles with an amine-group functionalized 3'-adaptor or 5'-adaptor and a miR linker containing an adaptor-binding sequence and miR124a-binding sequence at a molar ratio of 1:16:16 (Fig. 1).In brief, 3'-or 5'-adaptor conjugated nanoparticles were hybridized together by a miR linker to form self-assembled nanoparticles.In the absence of target miR124a, the self-assembled nanoparticles did not show any conformational change and generated a low T2 value and a weak MRI signal.However, in the presence of miR124a, the self-assembled nanoparticles hybridized with the miR124a binding sequence of the miR linker.The specific hybridization between the target miR124a and the miR linker induced detachment of the miR linker and nanoparticle disassembly, resulting in MRI intensity signal-on and an increased T2 value.
Transmission electron microscopy (TEM) images showed the target miR124a concentration-dependent disassembly of the miR124 MR beacons (Figure 2A).In the presence of miR124a (500 and 1000 pmol), the miR124a MR beacons disassembled due to the specific hybridization of the target miR124a to the miR linker and subsequent miR linker detachment from the miR124a MR beacons.However, the miR124a mt MR beacon, a negative control containing a mutated miR124a binding sequence in the miR linker, did not show nanoparticle disassembly.This demonstrates the higher specificity of the designed miR124a binding sequence in the miR124a MR beacon.The T2-weighted MR images of the miR124a MR beacons further demonstrated a miR124a concentration-dependent MR signal increase (Figure 2B).In contrast, the MR signal of the miR124a mt MR beacon showed no response to different miR124a concentrations.These results suggest high specificity of the miR124a MR beacon for detecting miR124a by MR imaging.
The in vitro MR imaging of endogenous miR124a expression during neuronal differentiation was confirmed using the mouse embryonic teratocarcinoma P19 cell line.Neuronal differentiation of P19 cells was induced by treatment with retinoic acid (RA).Magnetofection was used to efficiently transfect the miR124a MR beacons into the P19 cells.In undifferentiated P19 cells, the transfected miR124a MR beacons were highly aggregated.During neuronal differentiation, the expression of endogenous miR124a triggered disassembly of the miR124a MR beacons via selective hybridization to the miR124a binding sequence of the miR124a MR beacons.The T2-weighted images showed a gradual increase of the MR signal in P19 cells transfected with the miR124a MR beacons (Figure 3A).In contrast, P19 cells transfected with the miR124a mt MR beacons showed no change in MR signal during neuronal differentiation.
For in vivo MR imaging of endogenous miR124a, P19 cells (1 x 10 6 ) transfected with the miR124a MR beacons or the miR124a mt MR beacons were incorporated within a poly-L-lactic acid (PLLA) scaffold and subcutaneously implanted into both thighs of nude mice.The right thigh was treated with retinoic acid to induce neuronal differentiation, and the left thigh was used as a untreated control.The T2-weighted MRI signals in both thighs were dark right after implantation of P19 cells (Figure 3B).During neuronal differentiation, the MRI signal of the miR124 MR beacons in the right thigh was increased, whereas those of the miR124a MR beacons in the left thigh and of the miR124a mt MR beacons in both thighs were constant.This demonstrated that the specific MRI signal change in the right thigh came from endogenous miR124a expression in P19 cells during  There is a growing need for molecular imaging methods that are noninvasive and have high sensitivity.Reporter gene and MB based imaging systems have successfully been used as imaging tools for miRNA research.However, these methods employ fluorophores and are therefore limited by poor tissue penetration.Thus, there is still a need for a better, highly sensitive method of detecting miRNAs.Our study demonstrates the potential of MRI to track or monitor many miRNAs of interest during differentiation in vivo.This is the first report of an in vivo MRI method to image intracellular miRNAs during cellular development.Previous MRI methods have been limited with regards to imaging intracellular gene expression because of nonspecific binding of targeting probes, difficulty of target discrimination from surrounding molecules, and phagocytosis of magnetic nanoparticles.Our findings suggest that the self-assembling magnetic resonance beacon method can lead to a new way of miRNA imaging for in vivo studies and clinical applications.

Figure 1 .
Figure 1.Schematic diagram of the self-assembling miR124a MR beacon for detecting intracellular miR124a expression by MRI.Reprinted with permission [16].

Figure 2 .
Figure 2. Characterization of the miR124a MR beacons in test tube.(A) TEM images and (B) T2-weighted MR images of the miR124a MR beacons and the miR124a mt MR beacons incubated with different concentrations of exogenous miR124a.Reprinted with permission [16].

Figure 3 .
Figure 3.In vitro and in vivo MR detection of endogenous miR124a expression during neuronal differentiation in P19 cells using the miR124a MR beacons.T2-weighted MRI of the miR124a MR beacons and the miR124a mt MR beacons (A) prior to and during neuronal differentiation of P19 cells and (B) in an undifferentiated state (Undiff.)and 3 days after neuronal differentiation of P19 cells.The P19 cell-scaffold complexes were treated with (+) or without (-) RA to induce neuronal differentiation.Reprinted with permission [16].