Supplementary Information

Aaron Frank

Aleksandra Walczak

eLife Assessment
This study presents a method for expressing single-stranded DNA fluorescent aptamers in E. coli using a retron-based strategy. The evidence supporting the successful expression and folding of DNA aptamers is solid, with clear demonstration of fluorescence after extraction, though the aptamers do not function in living cells. The method represents an important technical advance that will likely become standard for DNA aptamer expression in bacterial systems.

anonymous

Reviewer #1 (Public review):
Summary:
The authors use an interesting expression system called a retron to express single-stranded DNA aptamers. Expressing DNA as a single-stranded sequence is very hard - DNA is naturally double stranded. However, the successful demonstration by the authors of expressing Lettuce, which is a fluorogenic DNA aptamer, allowed visual demonstration of both expression and folding, but only after extraction in cells, but not in vivo (possibly because of the low fluorescence of Lettuce, or perhaps more likely, some factor in cells preventing Lettuce fluorescence). This method will likely be the main method for expressing and testing DNA aptamers of all kinds, including fluorogenic aptamers like Lettuce and the future variants / alternatives.
Strengths:
This has an overall simplicity which will lead to ready adoption. I am very excited about this work. People will be able to express other fluorogenic aptamers or DNA aptamers tagged with Lettuce with this system.
Weaknesses:
Some things could be addressed/shown in more detail, e.g. half-lives of different types of DNA aptamers and ways to extend this to mammalian cells.

Reviewer #2 (Public review):
Summary:
This manuscript explores a DNA fluorescent light up aptamer (FLAP) with the specific goal of comparing activity in vitro to that in bacterial cells. In order to achieve expression in bacteria, the authors devise an expression strategy based on retrons and test four different constructs with the aptamer inserted at different points in the retron scaffold.
The initial version of this manuscript made several claims about the fluorescence activity of the aptamers in cells, and the observed fluorescence signal has now been found to result from cellular auto-fluorescence. Thus, all data regarding the function of the aptamers in cells have been removed.
Negative data are important to the field, especially when it comes to research tools that may not work as many people think that they will. Thus, there would have been an opportunity here for the authors to dig into why the aptamers don't seem to work in cells.
In the absence of insight into the negative result, the manuscript is now essentially a method for producing aptamers in cells. If this is the main thrust, then it would be beneficial for the authors to clearly outline why this is superior to other approaches for synthesizing aptamers.

Intracellular Expression of a Fluorogenic DNA Aptamer Using Retron Eco2

Figures and data

Schematic of the Eco2 gene locus, which encodes the non-coding RNA (ncRNA, blue) and the Eco2 reverse transcriptase gene (Eco2RT, green).

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