Overview

Part 1 - Imaging using Foldscope

This part of the homework is a choose-your-own-adventure in DIY microscopy.

1. Assemble your FoldScope and verify its function using one of the included slides. For more resources on FoldScope see https://www.foldscope.com/user-guide
2. Explore! We are providing students with several types of living organisms: 1) Spirulina major. A marine cyanobacteria (blue-green algae) culture that contains spiraled trichomes. 2) Hypsibius exemplaris, strain Z151. Tardigrades sometimes known as water bears. You are also encouraged to collect samples from your environment (home, apartment, garden, pet, nearby park, whatever!). The goal is to acquire, using a smartphone, the most beautiful microscopic image you can acquire. Make sure you pay special attention to Focus & Field of view – choose a sample you can get entirely in focus (or not if that’s your artistic whim, but at least achieve a good focus on part of the image). Microscopy is a useful tool in materials science, but since this is a synthetic biology class, please image something biological .
3. Collect your experimental results:
   1. Why did you choose this sample?
   2. What did you observe?
   3. What was the final magnification and sampling frequency of your image? (I.e., Approximately how large is each pixel in physical space?) You’ll need to use the final magnification and the sensor size (number of pixels) of your camera to calculate this. Be a good scientist (even though it’s art) and please include a scale bar in your image.
   4. What is the approximate resolution of your microscope? (Note – this is a harder and more open-ended question than it seems! Please read the FoldScope paper, and supplement on Microscopy.)
   5. In what ways is FoldScope different from a typical scientific microscope? Discuss at least one difference in optical design. (see supplement on Microscopy)
   6. Submit your best image(s)!
4. Challenge Option: Design & conduct a foldscope experiment with a control and at least one experimental condition. For example, try to incubate your specimen at different temperatures, or exposing them to an environmental challenge.
   1. What is the hypothesis for how the experimental condition affected the specimen?
   2. What was the observed result?

Part 2 - Design of smFISH / Spatial Sequencing Assay

The goal of this homework was to design a FISH or a spatial transcriptomics analysis assay.

1. Choose a problem. As described in the lecture, a “perfect” measurement experiment can mean a lot of things, but in this case, a perfect assay is one that’s well suited to your biological question. So first, you’ll need to think of an RNA imaging application. Describe why imaging measurement is useful for this problem.

One of my project ideas includes the design of Arc/Arg3.1-like vesicles for drug/gene therapy-delivery applications. One interesting property of the Arc/Arg3.1 protein (from now on Arc) is that it can selfassemble to a capsid and transport its own mRNA extracellularily, from neuron to neuron.

I thought that if I were to choose this project, I would like to have an in-vitro test that confirms that my designed vesicles do indeed transport nucleotides (mRNA) extracellularily.

After doing a bit more research it seems that many questions surrounding the Arc vesicle haven't been answered as well; Where and when is the vesicle formed? What is the process of packaging? How does the extracellular transport look like? How and where is the vesicle disassembled?

Arc capsid assembly, extracellular mRNA transport and disassembly. Image reference: https://www.arigobio.com/files/editor/images/Arc capsid.jpg

A (time-dependent sm)FISH assay would be a good way to approach at least some of these questions.

1. Pick your target genes. What genes would be relevant to this problem? You can choose any number of genes.

   I could imagine to do multiple tests to evaluate the function of designer capsids, but testing whether they can form and transfer mRNA into different cells is the most critical one. I would use Arc mRNA as proof of concept. Also I'm curious about using smFISH to investigate Arc mRNA location in brain tissue

   • Main target, depending on the assay: human Arc mRNA, mouse Arc mRNA. Arc would be used as proof-of-concept. Later, this can be replaced by the designed protein-based capsids.
   • Control: GFP, or any other reporter. Fluorescent reporters allow to check for protein expression in the capsid receiving cell as well.

2. What is the assay? Would you use multi-color fluorescence? Multiple cycles? Barcoding? Describe why this readout approach is a good fit.

   I could imagine to do multiple tests to evaluate the function of designer capsids, but testing whether they can form and transfer mRNA into different cells is the most critical one. I would use Arc mRNA as proof of concept. Also I'm curious about using smFISH to investigate Arc mRNA location in brain tissue

   1. Test, whether capsids can be formed in and taken up by human(-like) cells (HEK). This can be tested with smFISH, to quantify the uptake. This assay is inspired by a previous protocol (Pastuzyn et al., 2018). For this, I would transfect HEK cells with

   a) Arc alone (does Arc/designed capsid forming protein transport its own mRNA?)

   b) GFP and Arc (does Arc/designed protein transport mRNA other than its own?)

   c) GFP alone (are there ways of extracellular mRNA transport other than Arc/designed protein?)

   The genes of interest would be controlled by a strong promoter, since Arc capsids were shown to incorporate mRNAs that were present in high quantities.

   I imagine the protocol to look roughly like this:

   The cells transfected with either a), b), or c) would then be allowed to recover for 6 h, after which the medium would be changed, and the cells grown for another 18 h. The resulting medium, cleared from the transfected cells and ideally containing formed capsids, would be then transferred to naive HEK cells. Following some incubation time, the cells could then be fixed to perform smFISH on Arc and/or GFP. Arc and GFP would each get differently colored fluorescence labels for smFISH.

   This test can be also performed using neuronal cells taken from Arc KO mice, with appropriate adaptation to the protocol.

   

   Assay to test for mRNA transfer via protein vesicles. Created with BioRender.com

   2. I also thought about ways to use smFISH for research purposes: A spatial, analysis of Arc expression in mouse brain tissue (at different time points, after inducing Arc expression) using smFISH might be interesting. It doesn't seem like it has been done before, thus it might provide some precise quantification of Arc mRNA in dependence of time and high resolution information about its dendritic transport.

   Also, considering that Arc capsids were estimated to be 3 nm thick, while cell walls tend to be around 4 nm thick, labelling of capsid mRNA with smFISH probes might be possible, provdided that the probes do not interfere with capsid formation.

   Perhaps one could also check for the primary Arc transcript - which still includes all introns and is localized in the nucleus only. If a cell within the brain tissue contains processed Arc mRNA, but no primary Arc mRNA, it might be a hint that the mRNA was received from a donor cell, since Arc mRNA is already processed at entry into the capsid. However, to reliably conclude a mRNA transport within brain tissue, more proof, as well as a better understanding of the uptake mechanisms by the receiving cell would be needed.

3. Pick one gene and design the homology domains (target sequence domains) for 5 probes.

I used Uniprot to get the Accession number for human Arc/Arg3.1, selecting mRNA NM_015193.4 from the RefSeq database.

I then uploaded the sequence into Benchling and annotated both, the Matrix-domain and the Capsid-domain (CA-domain), as well as the NTD and CTD subdomains within the Capsid-domain. Although NTD contains the oligomerization domain, both, NTD and CTD are critical for Capsid formation.

Therefore, I used the regions more downstream of the gene to (semi-)randomly select five 25 base long probes.

Upon randomly selecting the 5 probes we then were offered to use the "Manual Probe Analyzer" provided in the colab notebook by Dr. Evan Daugharthy. I tweaked the code a little bit so that I can analyze my selected probes all at once, instead of one at a time (and save some image processing time afterwards).

The resulting input and output looked like this:

Overall, the resulting probes showed large differences in their melting temperatures, some of them showing a difference of more than 10 °C (e.g. primer 4 and primers 2 and 3), making them unlikely to work together. Hairpins were found for all probes. While the hairpins of primers 2, 3, 4 and 5 would likely be melted off, the hairpin found in primer 1 has a melting temperature that is above the probe's one, which would likely hinder proper binding of primer 1.

The next step was to use an tool, "PROBE-O-MATIC", again provided in the colab notebook, that automatically designs probes along the full-length mRNA sequence and to select (appropriate) parameters. I kept the target length at 25 bases and the threshholds for probe melting temperature at a maximum of 70°C and a minimum of 60°, since most of the previous, randomly selected probes fell in that range. I also kept the maximal hairpin melting temperature at 45 °C, so that the hairpins are less stable than the probes.

The Arc mRNA is about 2950 bases long. Since my first runs resulted in hundreds of probes, and I wanted to know the exact number to better evaluate the outcome of subsequent parameter changes, I decided to add an index to the probe_o_matic function (indicated by Probe no. in the output) for each resulting probe. Overall I got 869 probes out of my submitted sequence.

I therefore reduced the maximum probe melting temperature to 64 °C next, to get probes with less than 5 °C difference. This reduced the probes number to ~500, so I next checked, whether there were probes with hairpin Tms at 0 °C (by setting the maximum hairpin Tm at 0°C). This resulted in ~80 probes.

Here an excerpt from the output:

A probe length of 25 bases is very common, since larger probes oftentimes either lead to higher GC-contents, thus higher melting temperatures or increased possibilities for hairpin formation. These design constrains were also apparent in my own probes. Increasing the probe length to 30 bases yielded only 20 probes, a probe length of 35 only 6.

I chose probes that were a) outside of the Arc CDS and b) downstream of Arc CDS to avoid truncated transcripts and which c) did not overlap with each other .

A visualization of the selected probes (framed in pink), as well as a summary of the probe properties can be seen in the figure underneath.

I then used BLAST to check for unspecific binding of the first probe, starting at base 1416. I used the refseq_rna database to narrow the hits down to unique entries of RNAs and further limited my search to human (homo sapiens) RNAs. I also set the settings on showing 500 sequences.

BLAST returned 500 sequences, suggesting that there might be even more sequences the probe fits. Most of the hits were predicted RNAs and transcript variants, but there were also many encoding enzymes and transcription factors and other cell machinery. However, only Arc mRNA covered the probe fully and with 100% identity.

A couple of hits showed coverage higher than 60% (more than 15 paired bases). Thus I further analyzed the hits with the highest coverage (and identity) first with the NUPACK tool, to check their melting temperature against the smFISH probe. I used a 2-strand melting model to evaluate the following sequences:

1. Arc mRNA (as control)

2. An uncharacterized long non-coding RNA, which had 88% coverage and 95% identity with the probe

3. Arylsulfatase L (ARSL), transcript variant 4, mRNA, with 92% coverage and 91.3% identity

4. Homo sapiens ERGIC and golgi 3 (ERGIC3), transcript variant 3, mRNA with 60% coverage and 100% identity.

I summarized the generated melt profiles by the NUPACK tool into one plot:

The FISH probe bound less stable to the uncharacterized long non-coding RNA and the ERGIC mRNA variant than Arc mRNA itself, even though to a small degree. However, surprisingly, the probe bound the arylsulfatase L mRNA variants stronger than Arc mRNA, which might make unspecific binding of the probe to that specific mRNA even more likely than Arc mRNA does. Therefore another probe might need to be designed that allows more specificity and allows to discriminate non-specific binding at higher temperatures without melting off the other probes with lower tms as well.