Part:BBa_K5102073:Experience

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Applications of BBa_K5102073

The part has been used for a proof-of-concept to show the principle of molecular recording on RNA-level. The part has been used in: 1. NanoLuciferase tape-switching assay After obtaining tape sequences, we tested REPAIR’s ability to switch from state zero to one. State zero allows protein translation in the first reading frame, while state one shifts the frame by deaminating the first adenosine in the START codon. To validate this, we used a NanoLuciferase assay with NanoLuc CDS in the second reading frame. Since in state 0, NanoLuc is out of frame with the first START codon, successful REPAIR conversion from state 0 to 1 should increase luminescence, indicating restored NanoLuciferase activity.

To design this experiment, we cloned our tapes into the desired plasmid containing GSG_T2A (BBa_K5102113) and the nano-luciferase (BBa_K5102056). For each of our five tapes, we designed and synthesized one in state 0 and one in state 1 that would serve as positive control. Tape switching was tested with the Nano-Glo® Luciferase Assay System (Promega, N110), which uses a reporter system that quantitatively detects the presence of NanoLuciferase, allowing us to assess whether the state switching has occurred as intended. <img src="" style="background-color: transparent; width: 100% ;display: block; margin: 0 auto;"/>

Due to inconclusive results with the NLuc Assay, we recognized its limitations for testing tape-switching efficiency in our system. Due to time and availability constraints, we opted to shift to testing with XFPs to explore whether their inherent readout output correlates better with successful tape-switching events in future attempts.

2. Tape switching with XFPs

To demonstrate the functionality of our RNA-based molecular recording system, we designed a proof-of-concept experiment that validates the system’s ability to switch between different recorded states using our tapes 2.0 and 3.0.

The experiment aimed to test whether sequential RNA edits, guided by overlapping gRNAs, could reliably induce specific modifications and produce distinct fluorescent readouts in living cells. For that, each tape has three possible states depending on its ORF.

State	Fluorescent Protein	Reading Frame (ORF)	Adenosine Modification in START Codons
0	miRFP670nano3	ORF1	No modifications
1	mScarlet-3	ORF2	First adenosine modified
2	mTagBFP2	ORF3	First and second adenosines modified

To validate the recording of our tapes, HEK293T cells were transfected at 50-60% confluency with three plasmids in a 1:5:5 molar ratio, as described in the table below.

Plasmid	Name	Contents
Plasmid 1	Recording Tape	Tape 2.0 or 3.0 and XFPs
Plasmid 2	Editor	REPAIR v2 editing system
Plasmid 3	pU6 plasmid	gRNAs

Transfection efficiency was evaluated by assessing XFP expression using a CX7 imager. Initial observations at 24 hours post-transfection revealed detectable fluorescence levels, indicating successful construct expression. Subsequent imaging was performed every 24 hours for a total of 72 hours. The figure below presents a compilation of the fluorescence imaging results.

In both tapes, we are able to detect signals from states 0 and 1 (miRFP670nano3 and mScarlet3, respectively). Notably, we also appreciate a blue signal that appears only at 72 hours. This could be due to minimal basal ribosome slipping, suggesting a low likelihood of leaky expression but only over extended periods of time.

Additionally, the composite part has been folded using ViennaRNA to display the secondary structure of the RNA transcript. The image was then displayed via VARNA.

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