PinMol: Python application for designing molecular beacons for live cell imaging of endogenous mRNAs

Abstract

Molecular beacons are nucleic acid oligomers labeled with a fluorophore and a quencher that fold in a hairpin-shaped structure, which fluoresce only when bound to their target RNA. They are used for the visualization of endogenous mRNAs in live cells. Here, we report a Python program (PinMol) that designs molecular beacons best suited for live cell imaging by using structural information from secondary structures of the target RNA, predicted via energy minimization approaches. PinMol takes into account the accessibility of the targeted regions, as well as the inter-and intramolecular interactions of each selected probe. To demonstrate its applicability, we synthesized an oskar mRNA-specific molecular beacon (osk1236), which is selected by PinMol to target a more accessible region than a manually designed oskar-specific molecular beacon (osk2216). We previously demonstrated osk2216 to be efficient in detecting oskar mRNA in in vivo experiments. Here, we show that osk1236 outperformed osk2216 in live cell imaging experiments.

Cup is essential foroskarmRNA translational repression during earlyDrosophilaoogenesis

“…We analyzed osk mRNA colocalization with Bru1-GFP in both the nurse cells’ cytoplasm and the ooplasm at mid stages after nurse cell microinjections with five different osk -specific molecular beacons (Fig. 1B) (Bratu et al 2003; Bayer et al 2018). Using the object-based image analysis software Icy (de Chaumont et al 2012), we found that 57±3% (n=5) of osk -containing particles associated with Bru1-GFP in the nurse cells, and 43±4% (n=6) in the oocyte (Figs 1B, S1B).…”

“…Egg chambers were microinjected with a solution containing a cocktail of five osk -specific molecular beacons at a concentration of 200 ng/µl of each probe. Molecular beacons were designed, synthesized, labeled and purified as we previously described (Supplemental_Methods_S2) (Bayer et al, 2018; Bratu et al, 2003). Imaging was initiated within seconds after microinjection, and data acquisition was performed at room temperature on a LeicaDMI-4000B inverted microscope (Leica Microsystems, Buffalo Groove, IL) mounted on a TMC isolation platform (Technical Manufacturing Corporation, Peabody, MA), with a Yokogawa CSU10 spinning disc head and Hamamatsu C9100–13 ImagEM EMCCD camera.…”

“…Egg chambers were microinjected with a solution containing a cocktail of five osk-specific molecular beacons at a concentration of 200 ng/µl of each probe. Molecular beacons were designed, synthesized, labeled and purified as we previously described (Supplemental_Methods_S2) (Bayer et al, 2018;Bratu et al, 2003) Acquisition software: Leica LAS-X. Optical Z-stacks with a slice thickness ranging from 0.3 to 0.5 µm were acquired using a manual XY-stage with piezo-Z (PerkinElmer), or automated XYZ-piezo stage, respectively.…”

Recent Efforts and Milestones for Simulating Nucleic Acid FRET Experiments through Computational Methods

“…A new tool for the computational generation of RNA molecular beacons has been developed by Bayer et al 48 called PinMol. This aids with complex molecular beacon designs of up to 26 nucleotides and at least 18 nucleotides, energetically optimized through free energy optimization approaches.…”

Recent Advances in the Molecular Beacon Technology for Live-Cell Single-Molecule Imaging

“…To date, accurate prediction of the structure of an RNA as well as its variations with space and time within the native cellular environment has been a formidable challenge. For simplicity, selection of potential target RNA regions has conveniently been achieved based on structures predicted by computational algorithms ( Zuker, 2003 ; Bayer et al., 2019 ). However, such algorithms could only provide somewhat reliable predictions and are particularly not accurate if the RNA of interest is greater than 400 nucleotides in length ( Reeder et al., 2006 ).…”