Measurement

One of the main aims within our project was to develop a detection method to detect the presence of specific RNA sequences without the use of any complicated laboratory equipment. Furthermore, this method should be cheap and widely available to everyone. We indeed succeeded in developing such method and this page is dedicated to describe how this novel measurement method was implemented in our project. It is based on structures called 'coacervates'.
Coacervates are polymer-rich regions in solutions of mutually attractive polymers. The process of mutually attractive polymers phase-separating into a polymer-rich and polymer-poor phase is known as coacervation. This process can under some circumstances be observed by the naked eye, as coacervates generally cause solutions to be more turbid. A key physical property of coacervates is that they require polymers of a certain length to form. In general, only polymers that are ‘long enough’ form coacervates. The underlying reason for this can be explained theoretically and experimentally. These latter facts directly imply that (changes in) polymer length can be visualized to the naked eye, which we utilized to design a novel detection method coined CINDY Seq. However as we will argue in greater detail below, the method has potential to serve as a far broader method to characterize existing and future BioBricks, and the activity of many enzymes that show synthesis or degradation of any (coacervating) polymer.

• The coacervation method in our project

  In our project we developed the Coacervate Inducing Nucleotide Detection of Your Sequence (CINDY Seq) method for visualizing the activity of Cas13a into a readout visible to the naked eye. When Cas13a binds to its RNA target it undergoes a conformational change and engages in a state of collateral cleavage. In this state, it non-specifically cleaves the RNA it encounters (Abudayyeh et al. 2016; Gootenberg et al. 2017; Liu et al. 2017). Long sequences of RNA have been demonstrated to form coacervates visible to the naked eye with spermine (Aumiller et al. 2016). The RNA sequences will likewise be cleaved by the activated Cas13a. After cleavage, the previously long ‘collateral’ RNA is no longer able to form coacervates, and thus the method allows the naked eye detection of target recognition of Cas13a. In absence of the RNA target, Cas13a will remain inactive and the collateral RNA remains at its original length at which it still coacervates with spermine. The solution will show increased turbidity. This difference in turbidity (see Figure 2) shows whether Cas13a has been activated or not. This indicates the presence or the absence of the RNA target. We were in fact able to demonstrate that coacervation can be employed as a reliable method. More elaborate experimental results can be found on the detection results page.

  

  Figure 2: Schematic outline of the CINDY Seq method. CINDY Seq is a method that can allows detection of specific RNA sequences with the use of Cas13a. If the added RNA sample contains the Cas13a target, the long RNA polymers will be collaterally cleaved by the active Cas13a. When there is no target present in the sample, Cas13a remains inactive and will not cleave the RNA polymers. These RNA molecules will form coacervates.

• Perspectives for characterizing existing and future biobricks

  The nature of CINDY Seq is such that, in principle, it can be used to visualize any significant change in polymer length. As a result, this provides a promising outlook to characterize existing and future biobricks that involve the polymerization or degradation of coacervate-forming polymers (see figure soepkip). Examples of such enzymes are: nucleases, proteases and amylases. Furthermore, it has been demonstrated that even the molecule ATP can form coacervates (Jia et al. 2014), so that one can even imagine to demonstrate energy consumption or ATP synthesis.
  An obvious example of a BioBrick that can directly be characterised using the coacervation detection method is BBa_K1923001, encoding for a variant of Cas13a closely related to the one we have used in our project. With the coacervation detection method any crRNA and RNA target combination can be tested, for activating Cas13a, and even binary tests for off-target effects can be done by introducing mutations in the target RNA.

  

  Figure 2: Schematic outline of the CINDY Seq method. Proteins that can be characterised by CINDY Seq are involved in the assembling of degradation of polymers.

  Another example of a BioBrick that could be characterized using coacervates is T5 Phage Exonuclease (BBa_K676006). This part encodes for a DNase that can denature linear ssDNA and dsDNA, but will not cleave supercoiled plasmids, which makes it ideal when manufacturing plasmids. It has been demonstrated that coacervates can also be made using ssDNA and dsDNA (Jain & Vale 2017). The cleaving activity of this (and other) nuclease can therefore be tested using the coacervation detection method. One more Nuclease that can characterised in a similar way is the coding sequence for Nuclease regulated by T7-promoter (BBa_K2144000).

• Further improvements

  Several optimization and broadening steps for the coacervation method can be thought of. Trivial optimization steps are those in which the coacervate forming polymers and their concentrations are optimized to achieve maximum visual difference between coacervates and homogenous solutions. Furthermore, there exist dyes that partition into coacervates rather than in the polymer-poor phase, and by centrifugation the dyed coacervates can be spun down to the bottom of a tube (Aumiller et al. 2016; Aumiller & Keating 2015). For more quantitative measurements with the coacervation method, absorbances can be measured in a UV/Vis spectrophotometer, as we did for several experiments as well.

References:
1. Abudayyeh, O.O. et al., 2016. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science (New York, N.Y.), 353(6299), p.aaf5573.
2. Aumiller, W.M. et al., 2016. RNA-Based Coacervates as a Model for Membraneless Organelles: Formation, Properties, and Interfacial Liposome Assembly. Langmuir, 32(39), pp.10042–10053.
3. Aumiller, W.M. & Keating, C.D., 2015. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles. Nature Chemistry, 8(2), pp.129–137.
4. Gootenberg, J.S. et al., 2017. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 356(6336), pp.438–442.
5. Jain, A. & Vale, R.D., 2017. RNA phase transitions in repeat expansion disorders. Nature, 546(7657), pp.243–247.
6. Jia, T.Z., Hentrich, C. & Szostak, J.W., 2014. Rapid RNA Exchange in Aqueous Two-Phase System and Coacervate Droplets. , pp.1–12.
7. Liu, L. et al., 2017. The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a. Cell, 170(4), p.714–726.e10.