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Jennifer B Breen, Alexandria J Trujillo, Mark Christian Butler, Sriganesh Ramachandra Rao, Jack M Sullivan; RNA Mango - Finding a Needle in a Haystack: Tracking Rhodopsin mRNA using RNA Aptamers. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4495.
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© ARVO (1962-2015); The Authors (2016-present)
Specific 3′UTR mRNA sequences (e.g. zip codes) traffic RNA out of the nucleus and into the cytoplasm for protein production, and this knowledge is critical for success of post-transcriptional gene silencing agents (e.g. ribozymes) as co-localization is essential to enzymatic efficacy. We hypothesize a newly developed RNA Aptamer “MANGO”, conjugated with a small molecule fluorescent dye “TO1-Biotin”, can label a target mRNA, track the path/distribution, and aid in biotechnological applications (e.g. SELEX) to develop more efficient hammerhead ribozymes.
Two MANGO aptamers (M and M-C4) were inserted in the 1410 nt stem/loop of a 1532 nt human rhodopsin (hRHO) cDNA and in vitro transcribed. The 1410 nt region was chosen based on RNA structure algorithm predictions of accessibility and maintenance of hRho overall structure. Equal molar concentrations of TO1-Biotin were added, and fluorescence observed on a gel-imaging box. Controls include WT hRho, buffer, and dye alone. Cells transfected with hRho M and M-C4 cDNA constructs were incubated with TO1-Biotin, Alexa647-1D4 antibody and DAPI and visualized on a confocal microscope (EX/EM 488/535nm). Fluorescence intensity was quantified using ImageJ, and analyzed by two-tailed Student’s t-test. HEK293S cells alone, cells with WT hRho serve as controls.
With an intrinsic 260nm excitation, visualization of TO1-Biotin binding to in vitro transcribed hRho M, hRho M-C4 and WT hRho RNA at 100nM is seen with RFUs at 66.2, 50.2, 24.5 respectively. Using ImageJ, WT hRho mRNA (n=12) Corrected Total Cell Fluorescence (CTCF) for TO1-Biotin binding is 4605.96 (p=0.94); while hRho M-C4 (n=11) had a CTCF of 43158.36 (p=1.91E-3), which indicates specificity to the MANGO aptamer in mammalian cells. Images show DAPI (blue) stained cells containing Alexa647 (red) bound to hRho proteins, overlaid with clusters of TO1-Biotin (green), which indicates that hRho MANGO mRNA is present during and does not disrupt protein production.
Preliminary data suggests that MANGO aptamer may be used to track hRho mRNA produced and shuttled within the cell and cytoplasm. High-resolution information for mRNA localization requires further optimization. Spatial assessment of mRNA hRho trafficking may allow optimal hammerhead ribozyme delivery to co-localize within photoreceptors of the eye.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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