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Intracellular gene transcription factor protein-guided MRI by DNA aptamers in vivo - PubMed

Intracellular gene transcription factor protein-guided MRI by DNA aptamers in vivo

Christina H Liu et al. FASEB J. 2014 Jan.

Abstract

The mechanisms by which transcription factor (TF) protein AP-1 modulates amphetamine's effects on gene transcription in living brains are unclear. We describe here the first part of our studies to investigate these mechanisms, specifically, our efforts to develop and validate aptamers containing the binding sequence of TF AP-1 (5ECdsAP1), in order to elucidate its mechanism of action in living brains. This AP-1-targeting aptamer, as well as a random sequence aptamer with no target (5ECdsRan) as a control, was partially phosphorothioate modified and tagged with superparamagnetic iron oxide nanoparticles (SPIONs), gold, or fluorescein isothiothianate contrast agent for imaging. Optical and transmission electron microscopy studies revealed that 5ECdsAP1 is taken up by endocytosis and is localized in the neuronal endoplasmic reticulum. The results of magnetic resonance imaging (MRI) with SPION-5ECdsAP1 revealed that neuronal AP-1 TF protein levels were elevated in neurons of live male C57black6 mice after amphetamine exposure; however, pretreatment with SCH23390, a dopaminergic receptor antagonist, suppressed this elevation. As studies in transgenic mice with neuronal dominant-negative A-FOS mutant protein, which has no binding affinity for the AP-1 sequence, showed a completely null MRI signal in the striatum, we can conclude that the MR signal reflects specific binding between the 5ECdsAP1 aptamer and endogenous AP-1 protein. Together, these data lend support to the application of 5ECdsAP1 aptamer for intracellular protein-guided imaging and modulation of gene transcription, which will thus allow investigation of the mechanisms of signal transduction in living brains.

Keywords: AP1 knockout; attention; monoamine oxidase A; stress; transgenic mice.

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Figures

Figure 1.
Figure 1.

Aptamer affinity. A) We tested the in vitro binding of dsAP1 aptamers with various ECs to rh AP1 protein in the presence of 5 or 0.5 mM of DTT. B) Specificity of all aptamers for the rh NF-κβ protein by traditional gel shift assays. C) Protocol times for our studies.

Figure 2.
Figure 2.

Uptake and distribution of aptamers. To examine for cell-specific uptake of AP-1 aptamer, we delivered FITC-5ECdsAP1 (120 pmol/kg, i.c.v. injection). We then administered saline (SAL; A–D) or amphetamine (AMPH; 4 mg/kg, i.p. injection; E–I) 3 h later (n=2 each); frozen brain samples were obtained 4 h after saline or amphetamine (15). A, E) Brain tissue was stained for FITC-5ECdsAP1 detection. B, F) Brain tissue was stained for nuclei detection. C, G) Brain tissue was treated with fresh 4% paraformaldehyde to label GFAP expressed by astroglia [Cy3-labeled antibodies against GFAP (ab7260; Abcam), and Hoechst (purple) stain for nucleic acids]. D, H) Merged images. I) FITC-5ECdsAP1 was located near the neuronal nuclei (∼10 μm in diameter) stained with propidium iodide. Scale bar = 10 μm.

Figure 3.
Figure 3.

Subcellular distribution of aptamers: SPION-5ECdsAP1 or AU-5ECdsAP1 was delivered at a dose of 40 μg Fe/kg (A–C) or 40 μg Au/kg (D–E), i.c.v. injection). We administered amphetamine (n=2 each) as described in Materials and Methods; at 4 h after delivery, we obtained brain samples from the NAc and, in preparation for TEM, fixed the tissue for full staining (A) as well as for staining without osmium and uranyl acetate (B–D) and lead (E), as described in Materials and Methods. Arrows in panels B and D show that several EDNs were identified in the ER, at the site of protein translation. Arrows in panels D and E show that EDNs are not artifacts related to the lead stain. G, glia; N, neuronal nucleus based on diameters.

Figure 4.
Figure 4.

Target-guided MRI with SPION-5ECdsAP1 (40 μg Fe/kg or 120 pmol dsAP1/kg, i.c.v. injection). A) MRI acquisition protocol and data processing methods. B) Increase in MR signal intensity in subtraction R2* maps. C–E) Frequency of the drop in MRI signal (R2* or 1/T2* ms × 1000) is shown as percentage increase in ΔR2* maps in 3 groups of mice: saline (SAL) vs. amphetamine (AMPH) (C), or SCH23390 vs. AMPH (D, E). Bar graphs show means ±

sem

(t test, vs. SAL controls) for SPION retention in various ROIs in live brains following SPION-5ECdsAP1. F) Anatomic ROIs from which we obtained MRI data.

Figure 5.
Figure 5.

Distribution of SPION-5ECdsAP1 in living brains. Subtraction maps of AP-1 TF protein-guided whole-brain MRI after 1 typical treatment of amphetamine vs. saline (A) or SCH23390 with AMPH vs. saline with AMPH (B).

Figure 6.
Figure 6.

Binding specificity of SPION-5ECdsAP1 (4 mg Fe/kg or 12 nmol dsAP1/kg, i.p./i.c.v. injection). A) Homogenously elevated R2* values measured before (Pre), as well as 2 and 4 h after delivery of SPION-5ECdsAP1. B, C) EDNs were detected in the perivascular space by TEM under full stain (B) as well as under partial stain (C). Solid arrow in panel B (left image) indicates an EDN transporting across the membrane; broken arrows indicate naked EDNs; asterisks indicate EDNs enclosed by membrane. Arrow in panel C inset indicates END from artifacts. D) Representative aptamer uptake for SPION-5ECdsAP1 in C57black6 mice (n=2 each; panels 1 and 2) or SPION-5ECdsAP1 or SPION-5ECdsNF-kb in double-transgenic A-FOS mutant mice (n=3; panels 3 and 4) is shown as ΔR2* maps; increase is calculated according to the result shown in Fig. 5A. E) Signal increase of SPION-5ECdsAP1 in the striatum, shown in panel D; this ROI is defined in panel A. Difference in striatal ΔR2* of panel D3 and SPION-5ECdsRan above the baseline was not significant.

Figure 7.
Figure 7.

Mechanism of molecular MRI for intracellular TF proteins. A) The 3 major components of the nanoparticle-dsDNA aptamer: short dsDNA aptamer, avidin-biotin linkage or direct linkage, and MR contrast agent or microscopy agent. B) Proposed endocytosis pathway of DNA-mediated nanoparticle uptake. C) Once internalized, DNA aptamer binds to TF protein, whereby the nanoparticles remain within the unique cells that express the TF protein. C1–C3) Potential applications after target binding. D) If dsDNA does not bind to TF protein, the nanoparticles are excluded from the cell, and no image data can be acquired (Fig. 6D3). When the dose is sufficient to compete for intracellular TF protein, modulation by the TF protein is prevented (C3).

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