WO2019078722A1

WO2019078722A1 - Coacervate-based detection of a compound of interest

Info

Publication number: WO2019078722A1
Application number: PCT/NL2018/050690
Authority: WO
Inventors: Louis REESE; Willem Kasper SPOELSTRA; Stan Johan Jozef BROUNS
Original assignee: Technische Universiteit Delft
Priority date: 2017-10-20
Filing date: 2018-10-19
Publication date: 2019-04-25
Also published as: NL2019775B1

Abstract

The present invention relates to the field of biotechnology, more specifically to the field of molecular diagnostics; to a method for coacervate-based detection of the presence of a compound of interest in a sample, wherein upon recognition of the compound of interest by the target recognition moiety, coacervate phase separation is either resolved or induced, wherein the extent of phase separation is a measure for the presence of the compound of interest.

Description

Coacervate-based detection of a compound of interest Field of the invention

The present invention relates to the field of biotechnology, more specifically to the field of molecular diagnostics; to a method for coacervate-based detection of the presence of a compound of interest in a sample, wherein upon recognition of the compound of interest by the target recognition moiety, coacervate phase separation is either resolved or induced, wherein the extent of phase separation is a measure for the presence of the compound of interest. Background of the invention

There are numerous methods and assays available for detecting a compound of interest in a sample, following are non-limiting examples. A protein may be detected by an antibody assay such as ELISA and Western blot. A cell of interest may be detected by flow cytometry and ELIspot. A nucleic acid may be detected by Southern blot, Northern blot, sequencing and PCR. In all said assays, the compound of interest (target) is recognized by a target recognition moiety. Detection takes place either directly, e.g. by a coloured label attached to the target recognition moiety or indirectly when the target recognition moiety, upon recognition starts (e.g. an enzymatic reaction) which results in a detectable entity or when the target recognition moiety is e.g. detected by another target recognition moiety. The assays mentioned are known to the person skilled in the art and many are listed in e.g. Molecular Cloning, a Laboratory Manual (2012) Green and Sambrook, Cold Spring Harbor Laboratory Press.

There is a continuing urge to simplify the read-out of detection assays. For instance an ELISA- based laboratory pregnancy test has over the years evolved into an easy to use at home dipstick. Novel detection assays have recently been developed. Some of these are based on certain genome/gene editing enzymes and complexes related to the prokaryotic immune system CRISPR- Cas. These mechanisms have been shown to be highly sensitive in recognizing specific sequences of DNA (Cas9, Cpfl) and RNA (C2c2/Cas13a). More specifically, such mechanisms include a guide RNA (crRNA) and Cas proteins (CRISPR associated proteins) with endonuclease activity which together form a genome/gene editing complex and recognize sequences complementary to crRNA. Upon sequence recognition, the Cas proteins cut the nucleotide chain at the recognized site. Various variants Cas proteins with endonuclease activity have been identified or engineered, such as Cas proteins that upon recognition only nick (cut a single-strand instead of the double strand or do not cut at all (e.g. dCas9). Recently, it was demonstrated that the protein Cas13a, upon recognition of its target, undergoes a conformational change into a state of collateral cleavage, in which it cleaves any RNA sequence it encounters. It hereby becomes an unspecific RNase, rather than a specific one. The loss of specificity upon target recognition of nucleases like Cas13a provides a possibility for sensitive nucleic acid detection, as the collateral cleavage activity of Cas13a can be converted into a measurable signal (J. S. Gootenberg et al, "Nucleic acid detection with CRISPR- Cas13a/C2c2," Science, vol. 356, no. 6336, pp. 438-442, 2017; J.S. Abudayyeh et al, "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.," Science, vol. 353, no. 6299, p. aaf5573, 2016).

In Blocher and Perry ("Complex coacervate-based materials for biomedicine," Wiley Interdiscip. Rev. Nanomedicine Nanobiotechnology, pp. 76-78, 2016), complex coacervate-cased materials for Biomedicine are presented, and it is suggested that coacervates may inter alia be used for the detection of DNA. Blocher and Perry reference to Knaapila et al ("Polyelectrolyte complexes of a cationic all conjugated fluorene-thiophene diblock copolymer with aqueous DNA. J. Phys. Chem. B 2015, 1 19 (7), 3231-3241 DOI: 10.1021/jp51 10032). In Knaapila et al, detection is performed use specific copolymers to form nanoparticles containing DNA; depending on the amounts of DNA, the electronic properties of the nanoparticles change, and photoluminescence is quenched.

Altogether, there is still a need to simplify detection assays and specifically read-out of detection assays; especially for the detection of a nucleic acid of interest, sequencing and fluorescence-based assay described by Blocher and Perry are laborious and require highly specialized measurement equipment and trained personnel.

Summary of the invention

The invention provides for a method for coacervate-based detection of the presence of a compound of interest in a sample, comprising contacting the sample with a target recognition moiety and a coacervation agent, wherein upon recognition of the compound of interest by the target recognition moiety, coacervate phase separation is either resolved or induced, wherein the extent of phase separation is a measure for the presence of the compound of interest.

The invention further provides for a method of diagnosing a condition in a subject, comprising: a) obtaining a sample from the subject;

b) performing a method according to the invention; and

c) relating the presence or absence of the compound of interest with the presence or absence of the condition in the subject.

The invention further provides for a kit of parts comprising:

a) a target recognition moiety capable of detecting a compound of interest and capable of inducing coacervation upon recognition of the compound of interest; and/or

b) a coacervation agent.

The invention further provides for a device, preferably a mobile device, for performing a method according to the invention, comprising a reaction container and means for detecting coacervation- associated phase separation.

The invention further provides for the use of a coacervation agent as defined in any one of the previous claims for the detection of a compound of interest. Description of the figures

Fig. 1 : Schematic description of the invention for the specific cases of RNA and Cas13a protein. Left: A sample RNA is added to a solution of Cas13a protein which carries the crRNA recognition sequence and collateral RNA as a reporter polymer. Middle: There are two possible outcomes, if the target is recognized, the Cas13a protein collaterally cleaves RNA (sample RNA, as well as reporter RNA); if the target is not recognized, RNA molecules remain uncleaved/intact.

Right: Demonstration of turbidity change due to coacervation. The effect was tested here in the presence of Cas13a, crRNA, and target (top, clear solution; homogeneously mixed state) and the absence of crRNA and target (bottom, turbid solution; coacervate state).

Fig. 2: Illustration of the theoretical basis of the coacervation method, based on the presented modelling. The spinodal curve shifts to larger σ when the polymers are cut into smaller pieces. Here plots of (φ) are shown for polymer lengths 400 (Fig. 2A) and 100 (Fig. 2B). Where a solution at the indicated point P is in the coacervate region for polymers of length 400. In the case of polymers of length 100, the point P lies outside the coacervate region and the solution is mixed.

Fig. 3: Schematic of the lattice model for polyelectrolytes. The plus and minus signs indicate lattice sites occupied by parts of polycations (red) and polyanions (blue), respectively. All empty lattice sites are occupied by solvent molecules. Panel A shows a homogeneously mixed lattice gas, whereas panel B shows a snapshot of the phase separated (coacervate) regime of the lattice model. Fig. 4: Increasing the temperature "moves down" the spinodal curve σ_Ν(φ, Τ). Points in parameter space which are initially in the mixed region can enter the coacervate region. This transition is reversible.

Fig. 5: Upper row of tubes: reaction mixture containing 1.0 wt% spermine, and Cas13a activated by target and crRNA after 0 minutes and after 60 minutes.

Lower row or tubes: reaction mixture containing 1 .0 wt% spermine and inactive Cas13a; not containing target and crRNA after 0 minutes and after 60 minutes. Coacervates are visible by turbidity at 0 minutes in the presence of both activated Cas 13a and inactive Cas13a (coacervate state). At 60 minute, no coacervates are visible in the presence of active Cas13a (homogeneously mixed non-coacervate state).

Fig. 6: PAGE gel showing the reaction product of activated Cas12a cleaving poly(dT) length 60

(T60) at 1 , 15, 30, 45 and 60 minutes after start of the reaction.

Fig. 7: DNaseAlert fluorescence assay to test for collateral DNA cleavage. DNase Alert (IDT) is a fluorophore-quencher pair linked by ssDNA, and the fluorophore becomes fluorescent upon cleavage of the ssDNA linkage. Black line shows cleavage of collateral DNA upon presence of a target sequence, gray line shows collateral DNA cleavage without the presence of a target sequence.

Fig. 8: Liquid-liquid phase separation of poly(dT) and pLL depends on the poly(dT) length. Long (>40 nts) poly(dT) robustly phase separates with 5 mg/mL pLL in buffer (20 mM Tris-HCI, 66.6 mM KCI, 5 mM MgCI2, 1 mM DTT, 5% (v/v) glycerol, 50 pg/mL heparin, pH 7.5), whereas short (<20 nts) poly(dT) does not (at equal volume fraction). The dT-monomer concentration was kept constant (1 .2 mM dT) to ensure that all tubes contain an equal volume fraction of poly(dT), except for poly(dT) length 0, which did not contain any poly(dT) and serves as a control.

Fig. 9: Naked eye detection of Cas12a activity. Using the collateral cleavage activity, Cas12a degrades poly(dT) upon target recognition thereby preventing complex coacervation (upper row). In absence of target DNA, there is no collateral cleavage activity and complex coarcevation takes place (lower row).

Detailed description of the invention

It has been established by the inventors that, surprisingly, coacervation agents can be used in detection assays, specifically in the read-out of detection assays. The inventors have inter alia established that the phase separation of coacervation agents can be used as a detection mechanism, specifically of presence of a compound of interest such as an RNA, such as a viral RNA. The phase separation in the invention is binary, e.g. there is either (increased) turbidity resulting from phase separation (coacervate state) or none (homogeneously mixed, non-coacervate state); this allows for easy read-out (e.g. by the naked eye or a straight-forward device that is capable to discriminate turbid from non-turbid solutions) and for reduction of errors since false positives and negatives are reduced. In addition, the method according to the invention is substantially cheaper than prior art methods.

Accordingly, in a first aspect the invention provides for a method for coacervate-based detection of the presence of a compound of interest in a sample, comprising contacting the sample with a target recognition moiety and a coacervation agent, wherein upon recognition of the compound of interest by the target recognition moiety, coacervate phase separation is either resolved or induced, wherein the extent of phase separation is a measure for the presence of the compound of interest.

The invention can thus make use of the formation of coacervates (induction) as well the disappearance of coacervates (resolving).

The invention thus provides for a method for coacervate-based detection of the presence of a compound of interest in a sample, comprising contacting the sample with a target recognition moiety and a coacervation agent to form a mixture wherein phase separation of the coacervation compound occurs, wherein the mixture upon recognition of the compound of interest by the target recognition moiety goes from turbid coacervate state to homogeneously mixed non-coacervate state wherein the extent of phase separation is a measure for the presence of the compound of interest.

The invention thus also provides for a method for coacervate-based detection of the presence of a compound of interest in a sample, comprising contacting the sample with a target recognition moiety and a coacervation agent to form a mixture wherein no phase separation of the coacervation compound occurs, wherein the mixture upon recognition of the compound of interest by the target recognition moiety goes from homogeneously mixed non-coacervate state to turbid coacervate state wherein the extent of phase separation is a measure for the presence of the compound of interest. As an example ATP can be formed by the target recognition moiety, the formed ATP inducing coacervation in the presence of e.g. poly lysine. In such case, the target recognition moiety may already be present in the sample.

The person skilled in the art will comprehend that the coacervate state, not all coacervation agent will be in the coacervate state (phase), some of the coacervation agent may be in the homogeneously mixed non-coacervate state (phase) and vice versa; this is acceptable for the purpose of the invention as long as a binary read-out (turbid versus clear solution) is possible. In an embodiment, the coacervation agent comprises or consists of the compound of interest. This may be the case when the coacervation agent and the compound of interest are RNA. The person skilled in the art will comprehend that when the coacervation agent comprises or consists of the compound of interest, still an extraneous coacervation agent that does not comprise or consist of the compound of interest may be added to the reaction mixture.

Preferably, the phase separation is binary, i.e. is such that there is either (complete) phase separation resulting in a turbid, coacervate state or that there is no phase separation at all and the mixture remains in a homogeneously mixed state. Preferably, in the coacervate state (phase) of the mixture at least 70, 75, 80, 85, 90, 91 , 92, 93, 94 ,95 ,96, 97, 98, or preferably at least 99% of the coacervation agent is present. Preferably, in the homogeneously mixed state (phase), at most 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or preferably at most 1 % of the coacervation agent is present. The compound of interest may be any compound of interest. A preferred compound of interest is a molecule of at least a minimum specific size. A further preferred compound of interest is a metabolite, preferably a primary or secondary metabolite, or is a polymer, preferably a biopolymer, more preferably a polynucleotide such as a DNA or an RNA, or a polypeptide. When the compound of interest is a polynucleotide, the polynucleotide may be single-stranded or double-stranded, preferably single stranded, more preferably a single-stranded RNA or a single-stranded DNA, even more preferably a double stranded DNA. The RNA or DNA may be an RNA or DNA from a pathogen such as from a bacterium or from a virus. The polynucleotide from a pathogen such as a viral RNA may have be purified or isolated from a bodily fluid such as blood, urine, plasma et cetera. The person skilled in the art knows how to perform such purification or isolation. The viral RNA may be any viral RNA such as from HIV, Zika en Dengue virus.

When the compound of interest is a polynucleotide, the method according to the invention can conveniently be used for the identification of a polynucleotide comprising a sequence of interest, but also for measurement of or monitoring polymerization reactions of (polynucleotides such as a PCR reaction or other amplification reaction. In addition, other enzymatic activities or chemical reactions which depend on the presence, absence, or synthesis of nucleic acid chains or other chain molecules which undergo coacervation can be measured or monitored.

The coacervation agent may be any coacervation agent known to the person skilled in the art that preferably is capable of binary phase separation. The coacervation agent preferably is a polymer or a biopolymer such as a polypeptide or a polynucleotide. A preferred polynucleotide is RNA, preferably a single-stranded RNA such as polyuridylic (polyU) RNA. More preferably, the coacervation agent is a single-stranded RNA molecule, preferably with CUG and/or CAG repeats. Preferably, a polyU RNA is at least 60, 70, 80, 90, 100, 150, or at least 200 bases in length. Preferably, an RNA comprising CUG and/or CAG repeats is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, or at least 200 repeats in length. Another preferred polynucleotide is DNA, preferably single-stranded DNA such as poly(dT), preferably at least 60, 70, 80, 90, 100, 150, or at least 200 bases in length. Preferably, the single-stranded DNA molecule is rich in adenine and thymine (A T-rich), preferably the A/T content is at least 60, 65, 70, 80, 85, 90, 95, 100%.

Preferably, the coacervation agent is a compound that forms coacervates by electrostatic interaction, by cation-pi interaction, by polar/hydrophobic interaction and/or by entropic interaction. The person skilled in the art will comprehend that two or more different coacervation agents may be used and/or that additional agents may be required to facilitate or enhance the process. This may also including physical manipulation of the coacervation reaction, such as heating, or exposing to electric or magnetic fields, or to light. Preferred additional coacervation agents are the coacervation agents as defined herein. Preferred additional coacervation agents are spermine and spermidine, which are preferably used in a concentration as used in the examples herein. Other preferred coacervation agents are agents that comprise compounds selected from the group consisting of ATP, poly-lysine, poly-arginine, dextran, and polyethylene glycol. When the coacervation agent is ATP, another agent is preferably further present, preferably this further agent is poly-lysine. In such case, ATP can conveniently be the compound of interest.

In an embodiment, the coacervation agent is a polypeptide, part of a polypeptide or a fusion polypeptide that coacervates in the presence of a polynucleotide. Preferably, in this embodiment, the polypeptide binds to and/or interacts with the polynucleotide, preferably with single-stranded RNA or single-stranded DNA. Preferred polypeptides have low structural complexity, are rich in Tyrosine and/or Phenylalanine as well as Glycine and Serine. A preferred example of such polypeptide is the FUS protein or the Tau protein.

Preferably in this embodiment, the polypeptide comprises :

a) amino acids selected from the group consisting of: Ser, Thr, Asn, Glu, Gly, and/or

b) comprises amino acids selected from the group consisting of: Pro, Phe, Tyr, and Trp, or c) comprises less than 20 % amino acids selected from the group consisting of: Pro, Phe, Tyr, and Trp, and comprises more than 10% amino acids selected from the group consisting of Arg, Lys, Asp, and Glu.

Preferably, the polypeptide comprises repeats selected from the group consisting of:

a) Arg-Arg-Ala-Ser-Leu

b) Arg-Gly-Gly

c) Arg-Gly

d) Ser-Arg

e) Arg-Pro.

f) Phe-Gly

g) Tyr-Gly

Other preferred repeats are Gly-Tyr-Gly, Gly-Tyr-Ser, Ser-Tyr-Gly, Ser-Tyr-Ser, Gly-Phe-Gly, Gly- Phe-Ser, Ser-Phe-Gly, Ser-Phe-Ser, and combinations thereof. The target recognition moiety may be any target recognition moiety known to the person skilled in the art that is able to, upon recognition of the compound of interest, is capable of (binary) phase separation of the coacervation compound, e.g. from homogeneous state to coacervation state or from coacervation state to homogeneous state. Preferably, the target recognition moiety is a molecular machine, a protein, an enzyme, an enzymatic network (such as presented in Science. 2017 Aug 1 1 ;357(6351 ):605-609. doi: 10.1 126/science.aao0100. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Kazlauskiene et al and Nature. AUG 2017; 548(7669):543-548. doi: 10.1038/nature23467. Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Niewoehner et al) or a genome editing enzyme complex. A preferred genome editing complex is the well-known CRISPR-CAS system with all its variants known to date to the person skilled in the art (reviewed in e.g. (1) CRISPR-Cas: Adapting to change Simon et al, Science 07 Apr 2017: Vol. 356, Issue 6333, eaal5056 DOI: 10.1 126/science.aal5056; (2) Current Opinion in Systems Biology Volume 5, October 2017, Pages 9-15 Applications of CRISPR-Cas for synthetic biology and genetic recording Schmidt&Platt). A preferred genome editing enzyme within the genome editing enzyme complex is Cas13a. Another preferred genome editing enzyme within the genome editing enzyme complex is Cas12a. The person skilled in the art will comprehend that a genome editing enzyme complex comprises a genome editing enzyme and a guide RNA (may also referred to as crRNA or guide-RNA). The genome editing enzyme complex recognizes a specific target sequence in a target polynucleotide (the compound of interest) and in its active conformation becomes an a-specific nuclease instead of a target specific nuclease.

The sample may be any sample comprising a compound of interest. The sample may be a fresh sample or may be a sample that has been stored, e.g. stored cold or frozen. Preferably, the sample has been extracted from a plant, bacterium, fungus, animal or a human. Preferably, the sample is, or is derived from, a tissue, a bodily fluid such as blood, plasma, saliva, exudate, excrement, urine, stool. When the sample is 'derived from' it may have been processed, such as purified, to remove compounds that could inhibit detection of the compound of interest.

Detection of the phase separation may be performed by any means in the art. It may straightforward be performed using the naked eye, such as in the examples herein or by a detection technology. Detection may be performed using a device, such as a device that can measure turbidity. Such device may be any type of device, including, but not limited to a chip or biochip. Preferably, detection does not involve or require fluorescence technology.

It will be clear to the person skilled in the art that the method according to the invention can conveniently be used in a method of diagnosis. Accordingly, in a second aspect, the invention provides for a method of diagnosing a condition in a subject, comprising:

a) obtaining a sample from the subject;

b) performing a method according to the first aspect of the invention; and

c) relating the presence or absence of the compound of interest with the presence or absence of the condition in the subject. All features of this aspect are preferably those as defined in the first aspect of the invention. The subject may be any subject, e.g. a plant, bacterium, fungus or animal. The subject preferably is a vertebrate, more preferably a mammal, more preferably a human.

The condition may be any condition, preferably a genetic condition and/or preposition. A genetic condition and/or preposition may be any condition and/or preposition that is caused or enhanced by a genetic aberrance. In such case the compound of interest is the gene with (or without) the genetic aberrance. Detection of the presence or absence of the genetic aberrance can be correlated with the presence or absence of the condition and/or preposition.

The person skilled in the art will comprehend that the method according to the invention allows multiplex determination of and this distinguishing between two or more compounds of interest. Especially when using a genome editing enzyme complex such as a Cas13a-based complex or a Cas12a based complex, the presence of multiple possible targets may be detected using distinct, specific crRNAs. In addition, in this embodiment, the method according to the invention can be used to distinguish between e.g. two viral strains such as Dengue and Zika. As such, the invention thus provides for a method of diagnosing a condition in a subject, as described previously herein, wherein the condition is infection with a pathogen such as a bacterium or a virus and diagnosis may comprise distinguishing between e.g. two viral strains such as Dengue and Zika.

It will be clear to the person skilled in the art that the components of the methods according to the first and second aspect of the invention may be conveniently be provided in a kit. Accordingly, in a third aspect the invention provides for a kit of parts comprising:

b) a coacervation agent.

Preferably, at least two coacervation agents are present in the kit, preferably these agents are a single-stranded polynucleotide, preferably a polyU RNA or an RNA comprising CUG and CAG repeats, and spermine or spermidine, preferably spermine. These at least two compounds may be present in separate containers or in a single container. In another embodiment, the single-stranded polynucleotide is preferably poly(dT) or an AT rich DNA molecule.

The features of this aspect of the invention are preferably those as defined in the first and second aspect of the invention.

It will be clear to the person skilled in the art that the read-out of the methods according to the first and second aspect of the invention may be straightforwardly performed by the naked eye, as described previously herein, but may also be performed by a device, preferably a mobile device, such as a chip or a biochip.

Accordingly, in a fourth aspect, the invention provides for a device, preferably a mobile device, for performing a method according to the first and second aspect of the invention, comprising a reaction container and means for detecting coacervation-associated phase separation. The invention further relates to the use of a coacervation agent as defined herein for the detection of a compound of interest as defined herein. In an embodiment of the invention, the compound of interest is not an RNase. Background on the phenomenon of coacervation

When two or more attracting polymer species are mixed in solution and the right conditions are provided, phase separation into a polymer-rich and polymer-poor region occurs. The polymer-rich region in such a solution is coined the 'coacervate' phase. This paragraph is dedicated to explaining this physical phenomenon, and how it can be used in a method to detect the cleavage of RNA by unspecific RNases (such as activated Cas13a). It will become clear that such a method allows naked-eye detection and additionally, has the prospective of being more sensitive as well as very low-cost over the prior art.

What are coacervates?

As stated above, coacervates are regions in a solution rich in mutually attracting polymers. We will focus on combinations of positively and negatively charged polymers as an illustrative example (polycations and polyanions respectively). Such polyelectrolytes attract each other by electrostatic interactions. However, different mutual interactions between the polymers fall into the same class of coacervation phenomena (including cation-pi interactions, dipole-dipole interactions due to polar molecules, hydrophobic interactions, as well as entropic interactions).

How can coacervates be used to detect RNA cleavage of Cas13a?

An important condition for the formation of coacervates is that polymers need to be 'long' to coacervate. The role of polymer length is as critical as the role of the polymer-polymer interactions. This can be intuitively explained by the fact that longer polyelectrolytes can be attached to parts of multiple other oppositely charged polyelectrolytes, which enhances the formation of the coacervate. RNA is a negatively charged polymer, and multiple efforts have already shown that it can be combined with polycations to form coacervates (Aumiller et al. 2016). Taken together the length and charge dependence of coacervation is the basis of the detection assay: if large strands of RNA are present, and under suitable conditions, the RNA strands coacervate with polycations, whereas if this RNA is chopped up into smaller RNA fragments, the coacervation will no longer occur. The length dependence of coacervation/phase separation can be combined with a target recognition moiety, here Cas13a, which collaterally cleaves RNA and thus turns a solution that forms coacervates into a solution that does not form coacervates.

How can coacervates be used to detect DNA cleavage of Cas12a?

The technique described herein can be used to detect the presence of single-stranded DNA or double-stranded DNA in a solution. Similarly to the RNA cleavage described above, DNA is negatively charged and can be combined with polycation to form coarcervates (see Figure 8) and, again similarly to RNA, the role of the polymer length is critical. When large strands of DNA are present, the DNA strands can coarcevate with polycations under suitable conditions. However, when smaller strands of DNA are present the coarcevation will no longer occur. It has been shown that that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA cleavage activity by the target recognition moiety Cas12a (also known as Cpfl) that completely degrades ssDNA molecules (Chen et al 2018). This degradation only takes place when a the target sequence (that is recognized and bound by Cas12a) is present in the solution. If the target sequence is not present, Cas12a will not degrade the single-stranded DNA so coarcevation will occur (see Fig. 7).

Thus, Cas12a is capable of cleaving DNA into smaller DNA fragments so that coarcevation will no longer occur thereby turning a solution that forms coacervates into a solution that does not form coacervates.

How are coacervates detected?

The formation of coacervates can turn a solution turbid, and this turbidity can be observed by the naked eye or a suitable device. For example absorbance of 500 nm light is one possibility.

Length dependence of coacervate formation

To understand the physics behind the coacervation method requires a brief introduction into the theory of polymer solutions. Consider a solution of positively and negatively charged polymers (polycations and polyanions respectively) of equal valence Z, and that no salts are present in the solution. It will be implicit that throughout all that follows, temperature T and volume of the solution are fixed. For simplicity, we assume that the solution is symmetric, which means that the volume fraction of the polycations φ+ equals the volume fraction of the polyanions φ-, so that φ

Φ₊ = Φ- = 2

Note that this does not imply that the polymers are of equal length. What follows is to use a "lattice model" to represent the solution. In this type of model, the solution is represented as an infinitely large, in which each square can be occupied by part of a polycation/polyanion, or by a single solvent molecule of volume v. This v is the basic unit of space of the lattice, and the effective chain length (N) of the polymer is defined as its volume in units of v.

_ ^polymer

V

In the graphical representation of our lattice model below (Fig. 3), the sites occupied by polycations and polyanions are represented with + and - respectively, and the sites occupied by solvent are empty. In the lattice model, one defines interaction energies between all different neighboring pairs of sites, and calculates the energy of any configuration of the system. We are particularly interested in the Helmholtz Free Energy, since we have assumed constant temperature and volume. The full derivation of this energy is beyond the scope of this description, references include Doi 2013, Perry & Sing 2015, and Veis 201 1 .

The Helmholtz free energy per lattice site /($) in units of k_BT for the case considered here is given by the Voorn-Overbeek model (Qin et al. 2014), and reads:

/(0) = · In ( ) + (1 - φ) · ln(l - φ) - α · {σφ)³'² Here is the interaction strength from Debye-Hiickel theory, and σ≡ Z/N is the charge density of the polymer. For aqueous solutions at room temperature, = 4.1 , and calculation of σ is done below.

The system locally minimizes its Gibbs free energy, and since the Gibbs free energy and the Helmholtz free energy are related as: α(φ, τ_{ι Ρ}) = ν - [_Ρ + αφ, τ)ΐ where V and T are constant, minimizing the Gibbs free energy is equivalent to minimizing the Helmholtz free energy. What remains to be seen however, is if the energy minimum of the system is indeed a phase separated state, or a homogeneously mixed state. The system has a homogenous solution if and only if the mixing criterion is satisfied, and this mixing criterion states that:

d²f

(See appendix)

There will only be coacervation if this criterion is not satisfied in system. It is now possible to plot d²f

the curve for which— - = ί"(φ) = 0, which is known as the spinodal curve, and identify regions in this plot in which coacervation does, or does not happen. From that it will be immediately clear why coacervation happens. Taking the derivative of / twice with respect to φ yields that:

This means that the condition Γ'(φ)=0 can also be written as:

σ_Ν(φ)

If the charge density of the polymers σ is larger than the right hand side of this equation, coacervation will occur, whereas if it is less, homogeneous mixing will occur. The value of σ_Ν ) can be interpreted as the minimum charge density that a polymer duo of length N and volume fraction φ need to have in order to coacervate.

For a fixed charge density, it is now possible to show that longer polymers in solution coacervate, whereas shorter ones don't. In other words, the system will go from a coacervate state into a homogeneously mixed state if the polymers are shortened. A full mathematical treatment lies beyond our scope here, instead a graphical approach is chosen.

We start by studying the function σ_Ν (φ) for a solution containing polymers of effective chain length iV=400 (Fig. 2A). Say that the used polymers have charge density σ=0.135 and are present in a concentration φ=0.008. As can be seen in Fig. 2A, at the point P, the system lies in the region where coacervation occurs. If the polymer length changes from iV=400 to a length of N=100, where the properties of the solution remained unchanged, coacervation disappears:

The polymers still have exactly the same charge density, and the volume fraction is also unaltered, such that the system stays at the exact same point P. However, the same graph of σ_Ν(φ)ίθΓ W=100, is clearly different (see Fig. 2B). The point P suddenly lies outside the coacervate region, and in the region where the solution is homogeneously mixing (Fig. 2B).

The above argument qualitatively explains coacervation from a theoretical point of view. In addition it can easily be shown that this explanation is general for any values of N, and that we did not conveniently choose N = 400 and N = 100 to meet our statement:

We saw in Fig. 2 that the coacervate region in the σ(φ) plot "moved upwards" as we chopped up the polymers. First we rephrase the statement that σ(φ) "moved upwards" with decreasing N as: for a fixed φ, the point σ(φ) will be higher if N is lower. This is equivalent to saying that for arbitrary N > 0,

(¾ < ⁰

The subscript "φ" means that φ is held constant. This reduces the proof to showing that the derivative of σ with respect to N is always negative. After some calculus, one finds:

Although this expression looks complicated, the important result is that it is the product of positive terms with a minus sign in front, and thus we conclude that any point on the curve σ(φ) "moves up" (that is, in the positive σ direction) with decreasing N as it did in Fig. 2.

Turbidity of coacervate solutions cannot be increased by increasing polymer concentration

This can be seen for example in Fig. 2A, taking the system to be at point P and increasing the volume fraction of polymers φ will have the system move out of the coacervate region. Adding more polyelectrolytes to a solution thus does not mean that the system will move further into the coacervate region, but out of it. In fact, having too many polymers will cause coacervates to disappear. We can prove this by showing that σ(φ) only has one single minimum in the region 0 < φ < 1:

First note that the minima of σ are given by:

Taking this derivative and equating it to zero gives the equation:

Of which the exact analytical solutions are is given by:

As we expect N » 2, we can say N∞N + 2 ^ N - l we can approximate our result as:

1 2 1

4 ⁺ N ^~ 2

Because by definition φ cannot be negative, the only physically relevant solution, we define the critical concentration φ to be:

Note that lim [d>_c] = 0 , meaning that as the polymer size increases, the lower the critical polymer

W→co

volume fraction is required to have coacervation (for a fixed charge density). At the same time it proves that there is only one single extremum of σ(φ) for positive φ, and we already know that this is a minimum from examining the graph.

Temperature dependence of coacervate formation

In fact, the model presented here also reveals that coacervation is temperature dependent. Although the free energy expression provided above does not show the explicit dependence on temperature T, it does in fact depend depend on T through the Debye-Hiickel interaction strength .

It can in fact be shown (Qin & De Pablo 2016) that:

(4TT) 1/2

a Where λ_Β is given as the Bjerrum length, which is defined as the length at which the electrostatic (Coulomb) interaction energy between two elementary particles equals the thermal energy. The Bjerrum length therefore plays an important role in (complex) coacervation where electrostatic interactions drive the formation of coacervates. In the following we provide a simple scaling argument to support this picture which is based on the coulomb interaction between on elementary charges.

The Coulomb energy between two elementary charges, as function of the separation r between them, is given as the integral of the Coulomb force:

4πε₀ε_ΓΓ

In this expression, e = 1.602 · 10^~19 C is the elementary charge and ε₀ = 8.85 · 10^~12 Fm^→ is the permittivity of vacuum, and e_r the relative permittivity of the medium. Equating this energy to the thermal energy, k_BT, yields the value for the Bjerrum length λ_Β: ne_n£_rkr>T ^' For an aqueous solution at room temperature, ε_τ ¾ 80, T = 293.15 if, the Boltzmann constant is k_B = 1.38 · 10^~23 m²kg^■ s^~2K^~ . This yields a value for the Bjerrum length of: λ_Β « 0.71 nm³ , such that a « 4.1 .

However, we need to note that λ_Β depends on T and the relative permittivity e_r, which is in fact a function of T as well. By direct measurements of the dielectric constant of water at various temperatures (Malmberg & Maryott 1956), it was shown that e_r decreases exponentially with temperature. Our best fit to the data reveals a relative permittivity of:

e_r T) = 302.92 · e-° ^005T ,

Where T is measured in Kelvin.

Taken together the above equations reveal the temperature dependence of and can now be used to extract the desired scaling law in the following way:

_3

a oc (T^■ ε_Γ(Γ)) ²

And by recalling our function of interest

_2

σ_Ν(φ) oc a 3 ,

one finds an exponentially dampened, linear dependence of T:

σ_Ν(φ, Τ) <x T - e-° ^00ST .

In this expression the explicit temperature dependence is included as we have obtained it from our phenomenological fit to the data. By studying this function it can be easily checked that for T > 200 K, it is a decreasing function with increasing temperature. The pre-factor p = 0.005 in the exponential function is thereby indicating a characteristic temperature scale T _har = = 200 K, which is specific to the solvent used, water in this case. The liquid-water regime, is 273.15 < T < 373.15, so we conclude σ_Ν , Τ) is always decreasing with increasing temperature in the relevant regime. Increasing the temperature thus "moves down" the spinodal curve as shown in figure 3. We note that our brief theoretical treatment of this subject is including many assumptions regarding the nature of the polyelectrolyte solution, and a more quantitative assessment would be desirable in the future. However, we would like to stress that a qualitative agreement with our observations could be established starting from the simplest available model.

Typical values of parameters in coacervate formation

Here we will consider the parameters of the polymers that we most frequently used in our coacervate experiments, namely positively charged spermine and negatively charged RNA. Note that he volume of a single water molecule (which is the basic unit of our lattice model), is 0.03 nm³.

This can be calculated by dividing its molar volume (18.02 g/mol) over its density (997 g/L), yielding the volume of a single mole, and then dividing by Avogadro's number N_A = 6.0^■ 10²³ to get the volume per individual water molecule.

The density of spermine is p_sp = 937 g/L and its molecular weight 202.35 g/mol. This means that one molecule of spermine has a volume of ^202,35 ¾ 0.36 nm³ so that:

937-N_A

0.36 nm³

N^{sp =} 0.03 nm^{3 = 12 '}

And given that spermine has a valence of +4 at pH -7.5, its charge density is given by:

sp = 0.33

12

For RNA, the effective chain length in water can roughly be approximated as (Phillips et al. 2010): ^nucleotide ' ⁿ 0-3 nm³ - 71

^N = v_H2o ^¾ 0.03 m^{3 ¾ 10} ' where n is the number of nucleotides in the RNA polymer. Since nucleotides have a single charge, the charge density of RNA is given by:

In

σ^ ^¾ ϊο^ ^{= 0}·¹ · Appendix: derivation of the mixing criterion

For homogeneous mixing to occur in the solution, the system must satisfy the so-called mixing criterion (Doi 2013). To see exactly what the mixing criterion means, let us consider two solutions of arbitrary solutes with volumes and concentrations (Υι, φ^ and (ν₂, φ₂). Mixing these two solutions yields a mixture of volume V₁ + V₂ and final volume fraction of solutes:

Where χ = : If the two solutions mix homogeneously, the free energy of the homogeneously mixed state must be less than the energy of the two solutions separately, or: As can be seen, we are now looking at the total Helmholtz free energy, not per lattice site. This statement can be rewritten as:

+ (1 - χ) ₂) < χ (φ_χ) + (1 - x)/( ₂)

Since by definition 0 < x < 1, and thus φ_χ < φ < φ₂ , this statement is equivalent to saying: to have homogeneous mixing for arbitrary , φ₁ and φ₂ , the point [(φ) is always below the line connecting the points /((^) and (φ₂). This can only be true if there are no "bumps" in / in between (^) and /(φ₂), because if there were a bump, one could draw a line through it connecting two points on the curve, and the above inequality would not hold. Since bumps in a curve by definition imply that the second derivative changes sign twice, and because we are looking for a minimum in /, the homogeneous mixing criterion can simply state:

Homogeneous mixing of two solutions occurs if and only if for ^ < φ < φ₂ :

d²f

> 0

δφ²

References

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.

Chen, Janice S., et al. "CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity." Science 360.6387 (2018): 436-439.

Doi, M., 2013. Soft Matter Physics, Oxford University Press.

Malmberg, C.G. & Maryott, A. a., 1956. Dielectric constant of water from 0 to 100 C. Journal of Research of the National Bureau of Standards, 56(1 ), p.1 . Available at: http://nvlpubs.nist.gov/nistpubs/jres/56/jresv56n1 p1_A1 b.pdf.

Miller, S.L., Schopf, J.W. & Lazcano, A., 1997. Oparin's ^"Origin of Life": Sixty Years Later. Journal of Molecular Evolution, 44(4), pp.351-353.

Perry, S.L. & Sing, C.E., 2015. PRISM-Based Theory of Complex Coacervation: Excluded Volume versus Chain Correlation. Macromolecules, 48(14), pp.5040-5053. Phillips, R. et al., 2010. Physical Biology of the Cell. American Journal of Physics, 78(1 1 ), p.1230. Qin, J. et al., 2014. Interfacial tension of polyelectrolyte complex coacervate phases. ACS Macro Letters, 3(6), pp.565-568. Qin, J. & De Pablo, J.J., 2016. Criticality and connectivity in macromolecular charge complexation. Macromolecules, 49(22), pp.8789-8800.

Veis, A., 201 1 . A review of the early development of the thermodynamics of the complex coacervation phase separation. Advances in Colloid and Interface Science, 167(1-2), pp.2-1 1 .

Definitions

"Sequence identity" is herein defined as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, Wl. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps). Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; Ile to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

A "nucleic acid molecule" or "polynucleotide" (the terms are used interchangeably herein) is represented by a nucleotide sequence. A "polypeptide" is represented by an amino acid sequence. A "nucleic acid construct" is defined as a nucleic acid molecule which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids which are combined or juxtaposed in a manner which would not otherwise exist in nature. A nucleic acid molecule is represented by a nucleotide sequence. Optionally, a nucleotide sequence present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production or expression of said peptide or polypeptide in a cell or in a subject.

"Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleotide sequence coding for the polypeptide of the invention such that the control sequence directs the production/expression of the peptide or polypeptide of the invention in a cell and/or in a subject. "Operably linked" may also be used for defining a configuration in which a sequence is appropriately placed at a position relative to another sequence coding for a functional domain such that a chimeric polypeptide is encoded in a cell and/or in a subject.

"Expression" is construed as to include any step involved in the production of the peptide or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

A "control sequence" is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals. Optionally, a promoter represented by a nucleotide sequence present in a nucleic acid construct is operably linked to another nucleotide sequence encoding a peptide or polypeptide as identified herein.

The term "transformation" refers to a permanent or transient genetic change induced in a cell following the incorporation of new DNA (i.e. DNA exogenous to the cell). When the cell is a bacterial cell, as is intended in the present invention, the term usually refers to an extrachromosomal, self- replicating vector which harbors a selectable antibiotic resistance.

An "expression vector" may be any vector which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of a nucleotide sequence encoding a polypeptide of the invention in a cell and/or in a subject. As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes or nucleic acids, located upstream with respect to the direction of transcription of the transcription initiation site of the gene. It is related to the binding site identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Within the context of the invention, a promoter preferably ends at nucleotide -1 of the transcription start site (TSS).

A "polypeptide" as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non- limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a product or a composition or a nucleic acid molecule or a peptide or polypeptide of a nucleic acid construct or vector or cell as defined herein may comprise additional component(s) than the ones specifically identified; said additional components) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Example: Detection of a compound of interest (an RNA with specific sequence) by a method according to the invention.

1. Introduction to coacervate-based detection method using Cas13a

In the following we briefly recapitulate some introductory remarks to the method. The background of the invention lies in polymer physics and biochemistry. On the biochemistry side, the invention utilizes properties of genetic target recognition mechanisms as present in certain enzymes related to the prokaryotic immune system CRISPR/Cas. These mechanisms have been shown to be highly sensitive in recognizing specific sequences of DNA (Cas9, Cpfl) and RNA (C2c2/Cas13a). More specifically, such mechanisms include RNA (crRNA) and Cas proteins (CRISPR associated proteins) which together recognize sequences complementary to crRNA. Upon sequence recognition, the Cas proteins cut the nucleotide chain at the recognized site. Recently, it was demonstrated that the protein Cas13a, upon recognition of its target, undergoes a conformational change into a state of collateral cleavage, in which it cleaves any RNA sequence it encounters (1- 3). It hereby becomes an unspecific RNase, rather than a specific one. The loss of specificity upon target recognition of nucleases like Cas13a serves as one possible mechanism to implement our invention.

The second ingredient of the invention relates to a method to convert target recognition (of highly sensitive nucleases like Cas13a for example) into a signal that is observable by the naked eye. Within polymer physics, it is known that under specific circumstances, different polymer species may aggregate into polymer-rich regions (coacervates) based on attractive interactions between different species of polymers. Such attractive interactions include, but are not limited to electrostatic interactions (4) and cation-π interactions (5). The presence of coacervates in a solution is visible to the naked eye, as these solutions are significantly more turbid (milky), because coacervates absorb/scatter visible light more than clear solutions. Whether coacervation occurs in aqueous solutions depends on many physical parameters (e.g. pH, temperature, salinity, solvent) (6), but for our purposes most interestingly, it highly depends on polymer chain lengths (7,8). Generally, the longer the polymers, the more readily coacervation occurs. We inferred that the combination of molecules, such as guided nucleases that can enter a state of collateral cleavage (like Cas13a), and coacervate formation provides a method to make the presence of a specific nucleotide sequence visible to the naked eye (Fig. 1). The conditions under which nucleotide chains form coacervates are currently an area of research receiving increasing attention (9, 10). Further, a set of other macroscopic and microscopic properties of the solution are affected by coacervate formation. These include rheological properties (assessed by mechanical manipulation) as well as other properties of liquids (viscosity, contact angle at interfaces), as well as the microscopic observation of liquid-liquid phase separation in droplets.

2. Materials and methods

Two mixes, depicted the "Active mix" and the "Inactive mix", of 220 μΙ_ were prepared containing the ingredients described in the table her above. Active mix Inactive mix

Cas13a reaction buffer Cas13a reaction buffer

0.1 wt% PolyU 0.1 wt% PolyU

0.3 ng/pL TcR target RNA

0.3 ng/pL crRNA 3

Nuclease free water Nuclease free water

After gentle mixing, both mixes were divided over 5 tubes (44 μΙ_ per tube), and 1 μΙ_ (from 0.05 wt% stock) of the protein Cas13a was added. All tubes were left at 37 °C, and at several time-points 5 pl_ of 10 wt% spermine stock was added to a final concentration of 1 .0 wt%. Preliminary experiments have given indication that Cas13a is not active in the presence of 1 .0 wt% spermine, so the spermine is added after Cas13a has been given the opportunity to cleave polyU. As shown in Fig. 5, after 0 minutes of incubation, both solutions showed coacervation indicating that the PolyU RNA was still intact regardless of whether Cas13a was activated or not (Fig. 5, left row of tubes). After 60 minutes however, no coacervation was observed when the spermine was added in the tube containing active Cas13a, proving that the coacervate based detection method can be used to detect the presence of a target nucleotide sequence with the naked eye (Fig. 6, right row of tubes).

3. Coacervate-based detection method using Cas12a

Loading Cas12a with crRNA was performed at 37 degrees C for at least 15 minutes before detection assays were performed. The incubation buffer was 1 X Cas12 reaction buffer at 100mM or 66.5mM KCI with 8.6 uM Cas12a and 10 uM crRNA.

Materials and Methods

Material Components Supplier (Product no.)

Cas12a reaction 20 mM Tris-HCI Sigma

buffer 66.6 mM KCI

5 mM MgCI2

1 mM DTT

5% (v/v) glycerol

50

heparin

pH 7.5 (using NaOH)

Poly(dT) Polythymidylic acid sodium salt dissolved in Ella Biotech

nuclease free water. DNaseAlert IDT

Target DNA CGA GTA ACA GAC ATG GAC CAT CAG SEQ ID NO: 3

sequence ATC TAC AAC AGT AGA AAT TCT ATA GTG

(5'->3') AGT CGT ATT ACT T

Cas12a crRNA UAAUUUCUACUCUUGUAGAUcugauggucca SEQ ID NO: 4

sequence ugucuguuacuc

(5'->3')

DNaseAlert fluorescence assay

DNase Alert (IDT) is a fluorophore-quencher pair linked by ssDNA, and the fluorophore becomes fluorescent upon cleavage of the ssDNA linkage. This allows measuring a fluorescent signal as soon as the IDT is cleaved.

To test for collateral cleavage activity of Cas12a, two reaction mixes where prepared. The final Cas12a concentration was 86 nM.

Mix 1 Mix 2

Cas12a reaction buffer Cas12a reaction buffer

1 .25 pmol DNAseAlert 1 .25 pmol DNAseAlert

0.5 uM target DNA

86 nM Cas12a (preincubated with crRNA)

Nuclease free water Nuclease free water

After gentle mixing, Cas12a was added to both mixes after it had been loaded with crRNA for a minimum of 15 minutes, and subsequently measured. Fluorescence measurements were done (A_ex = 535 nm, A_em = 585 nm) in the Tecan Infinite M200 PRO plate-reader. Reactions were performed at 37°C. As shown in Fig. 7, Cas12a only cleaves IDT (thereby showing collateral activity) in the presence of the target DNA (black line), whereas no cleavage is observed when the target DNA is absent (grey line).

Turbidity assay

To analyse coarcevation by naked eye, a turbity test was performed. Again two mixes were prepared using as Cas12a reaction buffer 1 .1 x C12RB:

Mix 1 Mix 2

Cas12a reaction buffer Cas12a reaction buffer

22μΜ Poly(dT) poly(dT) 20μΜ

220nm target DNA

187nM Cas12a (preincubated with crRNA)

Nuclease free water Nuclease free water After gentle mixing, pre-loaded Cas12a was added to both mixes, after a 60 min digestion at 37C poly-L-Lysine (5mg/ml_, hydrobromide, 15-30 kDa) was added to induce turbidity. As can be seen in Figure 9, the solution is clear (see-through) indicating that no coarcevates are formed in the presence of the target DNA. In the absence of target DNA, coarcevates are formed and the solution is turbid.

References

1 . Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science (80- ) [Internet]. 2017;356(6336):438-42. Available from: http://www.sciencemag.Org/lookup/doi/10.1 126/science.aam9321

2. Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DBT, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 2016;353(6299):aaf5573. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27256883

3. Liu L, Li X, Ma J, Li Z, You L, Wang J, et al. The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a. Cell [Internet]. Elsevier Inc.; 2017;170(4):714-726. e10. Available from: http://dx.doi.Org/10.1016/j.cell.2017.06.050 4. Veis A. A review of the early development of the thermodynamics of the complex coacervation phase separation. Adv Colloid Interface Sci. Elsevier B.V.; 201 1 ;167(1-2) :2-1 1 .

5. Kim S, Huang J, Lee Y, Dutta S, Yoo HY, Jung YM, et al. Complexation and coacervation of like- charged polyelectrolytes inspired by mussels. Proc Natl Acad Sci. 2016;1 13(7):E847-53.

6. Blocher WC, Perry SL. Complex coacervate-based materials for biomedicine. Wiley Interdiscip Rev Nanomedicine Nanobiotechnology. 2016;76-8.

7. Qin J, Priftis D, Farina R, Perry SL, Leon L, Whitmer J, et al. Interfacial tension of polyelectrolyte complex coacervate phases. ACS Macro Lett. 2014;3(6):565-8.

8. Qin J, De Pablo JJ. Criticality and connectivity in macromolecular charge complexation. Macromolecules. 2016;49(22):8789-800. 9. Aumiller WM, Pir Cakmak F, Davis BW, Keating CD. RNA-Based Coacervates as a Model for Membraneless Organelles: Formation, Properties, and Interfacial Liposome Assembly. Langmuir. 2016;32(39):10042-53.

10. Frankel EA, Bevilacqua PC, Keating CD. Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg2+, Nucleotides, and RNA. Langmuir. 2016;32(8):2041-9.

Claims

1 . A method for coacervate-based detection of the presence of a compound of interest in a sample, comprising contacting the sample with a target recognition moiety and a coacervation agent, wherein upon recognition of the compound of interest by the target recognition moiety, coacervate phase separation is either resolved or induced, wherein the extent of phase separation is a measure for the presence of the compound of interest.

2. A method according to claim 1 , wherein the coacervation agent comprises or consists of the compound or interest.

3. A method according to claim 1 or 2, wherein the extent of phase separation is binary.

4. A method according to any of the preceding claims, wherein the compound of interest is a metabolite, preferably a primary or secondary metabolite, or is a polymer, preferably a biopolymer, more preferably a polynucleotide such as a DNA or an RNA, preferably a viral RNA, or a polypeptide.

5. A method according to any of the preceding claims, wherein the coacervation agent is biopolymer such as a polypeptide or a polynucleotide.

6. A method according to claim 5, wherein the coacervation agent is a polypeptide that coacervates in the presence of a polynucleotide, preferably wherein the polypeptide binds to and/or interacts with the polynucleotide.

7. A method according to claim 6, wherein ), the polypeptide comprises:

a) amino acids selected from the group consisting of: Ser, Thr, Asn, Glu, Gly, and/or b) comprises amino acids selected from the group consisting of: Pro, Phe, Tyr, and Trp, or c) comprises less than 20 % amino acids selected from the group consisting of: Pro, Phe, Tyr, and Trp, and comprises more than 10% amino acids selected from the group consisting of Arg, Lys, Asp, and Glu.

8. A method according to any of the preceding claims, wherein the target recognition moiety is a molecular machine, a protein, an enzyme, an enzymatic network or a genome editing enzyme complex.

9. The method according to claim 8, wherein the genome editing enzyme within the genome editing enzyme complex is Cas13a or wherein the genome editing enzyme complex is Cas12a.

10. The method according to any of the preceding claims, wherein the coacervation agent is a compound that forms coacervates by electrostatic interaction, by cation-pi interaction, by polar/hydrophobic interaction and/or by entropic interaction.

1 1 . The method according to any of the preceding claims, wherein the genome editing enzyme complex is Cas13a and the coacervation agent is a single-stranded RNA molecule, preferably with CUG and/or CAG repeats.

12. The method according to any of the preceding claims, wherein the genome editing enzyme complex is Cas12a and the coacervation agent is a single-stranded DNA molecule, preferably an

A/T-rich DNA molecule.

13. The method according to any of the preceding claims, wherein the sample has been extracted from a plant, bacterium, fungus, animal or a human.

14. The method according to any of the preceding claims, wherein the sample is, or is derived from, a tissue, a bodily fluid such as blood, plasma, saliva, exudate, excrement, urine, stool.

15. The method according to any of the preceding claims, wherein the detection method is by naked eye or by detection technology.

16. The method according to any of the preceding claims, wherein the detection method does not require fluorescence technology.

17. A method of diagnosing a condition in a subject, comprising:

a) obtaining a sample from the subject;

b) performing a method according to any of the preceding claims; and

18. The method according to claim 17, wherein the condition preferably is a genetic condition and/or preposition.

19. A kit of parts comprising:

b) a coacervation agent.

20. A device, preferably a mobile device, for performing a method according to any one of claims 1 - 18, comprising a reaction container and means for detecting coacervation-associated phase separation.

21 . Use of a coacervation agent as defined in any one of the previous claims for the detection of a compound of interest.