Modular functional RNA constructs and related compositions, systems, and methods

Abstract

Disclosed are RNA biosensor constructs and related, nucleic acids, vectors, cells, systems, and methods useful for detecting ligands of interest. Also disclosed are computer implemented methods for designing biosensors and constructs produced thereby. Exemplary embodiments include diverse RNA biosensors incorporating synthetic aptamers for the detection of ligands of interest.

Description

[0001] MODULAR FUNCTIONAL RNA CONSTRUCTS AND RELATED COMPOSITIONS, SYSTEMS, AND METHODS

[0002] BACKGROUND

[0003] RNA biomolecules have been proposed for use as molecular switches for sensing applications. For example, the development and production of high-value chemicals and biomolecules such as enzymes, biofuels, biomaterials, biochemicals, and pharmaceuticals constitute a multi-billion-dollar industry. RNA biomolecular switches can theoretically be employed as biosensors to improve the development and production of these chemicals and biomolecules.

[0004] Engineered RNA biomolecular switches have heretofore implicitly relied on the production of signals resulting from complex equilibria between a plethora of transient, ill-defined structurestates. This makes reliable and rational production of sensitive biomolecular sensors at the scale needed for the vast diversity of potential target ligands elusive. There has been a lack of significant success broadly in the field of computational design of functional RNA biomolecular switches. Furthermore, optimizing the sensitivity of existing RNA sensors to detect the low concentrations of metabolites available inside a cell has proven challenging.

[0005] Despite the advances in the art of biomolecular sensors, there remains a need for a systematic design pipeline to robustly produce sensitive RNA biomolecular switches. The present disclosure addresses these and related needs.

[0006] SUMMARY

[0007] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0008] In embodiments, the disclosure provides a biosensor construct. The biosensor construct includes a sensor domain that specifically binds to a molecule of interest. An output domain configured to modulate a detectable output signal when folded into a first conformation. The output domain is at an equilibrium between first and second conformations. Binding the molecule of interest by the sensor domain alters the equilibrium between the first and second conformations of the output domain. The alteration of the equilibrium modulates the detectable output signal.

[0009] In embodiments, the sensor domain has a binding element having a binding element subsequence located between a stem sequence and a stem target sequence. The sensor domain may also have an overhang sequence and / or a linker sequence. In embodiments, the overhang sequence is 5’ of the binding element and / or the linker sequence is 3’ of the binding element. In other embodiments, the overhang sequence is 3’ of the binding element and / or the linker sequence is 5’ of the binding element. The linker sequence is between the binding element and the output domain.

[0010] In embodiments, the output domain comprises a signaling element subsequence and a stem target sequence. In some embodiments, the signaling element subsequence is 3’ of the stem target sequence. In some embodiments, the signaling element subsequence is 5’ of the stem target sequence. The sensor domain comprises an overhang sequence and the output domain comprises an overhang target sequence located between a stem target sequence and a signaling element subsequence.

[0011] In embodiments, the overhang sequence of the sensor domain is the reverse complement of at least a portion of the overhang target sequence of the output domain. The stem sequence of the sensor domain is the reverse complement of at least a portion of the stem target sequence of the sensor domain and is the reverse complement of at least a portion of the stem target sequence of the output domain; and / or the linker sequence of the sensor domain is the reverse complement of at least a portion of linker target sequence of the sensor domain; in any combination.

[0012] In embodiments, the linker target sequence is the reverse complement to a portion of the binding element subsequence, optionally wherein the portion of the binding element subsequence is a discontinuous portion.

[0013] When the sensor domain is bound to the molecule of interest the stem sequence of the sensor domain is hybridized to the stem target sequence of the sensor domain thereby permitting folding of the output domain into one of the first or second conformations.

[0014] When the sensor domain is not bound to the molecule of interest the overhang sequence of the sensor domain is hybridized to the portion of the overhang target sequence of the output domain, the stem sequence of the sensor domain is hybridized to the portion of the stem target sequence of the output domain, and / or the linker target sequence of the sensor domain is hybridized to the portion of the linker sequence in the sensor domain, thereby permitting folding of the output domain into one of the first or second conformations.

[0015] In embodiments, at least two of the overhang sequence of the sensor domain, the stem sequence of the sensor domain, and the linker target sequence of the sensor domain form a continuous helix stem structure when hybridized to at least two of a portion of the overhang target sequence of the output domain, a portion of the stem target sequence of the output domain, and a portion the linker sequence of the sensor domain, respectively, thereby permitting the signaling element of the output domain to fold into one of the first or second conformations.

[0016] The overhang sequence is between 0 and about 15 nucleotides in length, the stem sequence is between 0 and about 15 nucleotides in length; and / or the linker sequence is between 0 and about 15 nucleotides in length. In embodiments, the overhang sequence is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15 nucleotides in length. In embodiments, the stem sequence is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15 nucleotides in length. In embodiments, the linker sequence is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, or 15 nucleotides in length. In embodiments, the various combinations of different lengths of stem, linker, and overhang are individually contemplated.In some embodiments, the biosensor includes an intermediate sequence disposed between the binding element and the linker sequence of the sensor domain, wherein the intermediate sequence has a length up to about 150 nucleotides in length.

[0017] The binding element of the sensor domain may be any of multiple types of sequences. In some embodiments, binding element of the sensor domain is a synthetic aptamer sequence. In some embodiments, the binding element of the sensor domain is a RNA sequence that exists in nature. In some of these embodiments, the RNA sequence is a sequence without a known ligand, wherein the ligand is a chemical, a metabolite, a protein, a peptide, a small molecule, optionally a drug molecule or drug precursor molecule, and the like.

[0018] In some embodiments, the biosensor construct includes a signaling molecule that produces a detectable output to indicate a specific conformation. In some embodiments, the output domain is configured to enhance the detectable output signal when folded into the first conformation. In some embodiments, the output domain is configured to enhance the detectable output signal when folded into the second conformation. In some embodiments, the output is fluorescence. In some embodiments, the signaling molecule is covalently linked to the biosensor construct. The signaling molecule may be covalently linked to an oligonucleotide sequence annealed to a complementary sequence appended to the biosensor construct. The signaling molecule may be covalently linked to a solid support. The signaling molecule may display enhanced fluorescence when the sensor domain is bound by a molecule of interest. The signaling molecule may display enhanced fluorescence when the sensor domain is not bound by a molecule of interest.

[0019] In some embodiments, the biosensor construct is a DNA biosensor construct. In some other embodiments, the biosensor construct is an RNA biosensor construct. In some embodiments, the biosensor construct is circular. In some embodiments, additional domains are appended to either end of the construct. In some of these embodiments, the additional domain is an aptamer appended to the 5’ end. In some of these embodiments, the additional domain is an aptamer appended to the 3’ end.

[0020] In some embodiments, when the output domain is folded into one of the first or second conformations, the output domain is or comprises a functional ribozyme, a functional nuclease guide RNA (gRNA), a functional ribosome binding site, or functional RNA aptamer.

[0021] In some embodiments, the molecule of interest is a chemical, a metabolite, a protein, a peptide, a small molecule, optionally a drug molecule or drug precursor molecule, and the like.

[0022] In some embodiments, the biosensor is immobilized on a solid support. The biosensor is attached to the solid support through a polynucleotide tail annealed to a capture oligonucleotide attached to the solid support. The biosensor is attached to the solid support through a covalently linked biotin. The solid support is magnetic or non-magnetic beads or the like.

[0023] In some embodiments, a polynucleotide molecule includes a sequence encoding the biosensor construct. In some embodiments the polynucleotide molecule encoding the biosensor construct is contained within a cell. In some embodiments, the cell is prokaryotic. In some embodiments, the cell is eukaryotic. In some embodiments, the cell is engineered or treated to modify expression or production of the molecule of interest.

[0024] The disclosure further provides a method of detecting a molecule of interest. The method includes introducing the nucleic acid biosensor construct into an environment that may contain the molecule of interest. A measured output signal indicates binding of the molecule of interest to the nucleic acid biosensor construct. The nucleic acid construct is a DNA construct or an RNA construct. The environment into which the nucleic acid biosensor construct is introduced is a cell- free synthesis environment. In some embodiments, the environment comprises a cell lysate. In some embodiments, the environment is in a cell. In some embodiments, the cell is engineered to modify production of a molecule of interest in the cell. In some embodiments, the cell is subjected to experimental conditions suspected to modify production of the molecule of interest in the cell. In some embodiments, the molecule of interest is a compound contacted to the cell or a metabolite thereof.

[0025] Designing a molecular construct can be implemented in several ways. Using computer software enables the construct to be designed and tested virtually. The disclosure describes a computer-implemented method for designing a biosensor construct. The method includes determining, by a computing device, one or more candidate biosensor constructs. For each of the one or more candidate biosensor constructs: Predicting, by the computing device, one or more folded structures that the biosensor construct will adopt. Determining, by the computing device, one or more metrics for the biosensor constructs based on the predicted one or more folded structures. Choosing, by the computing device, one or more of the one or more candidate biosensor constructs to be provided for synthesis based on the metrics. Determining the one or more metrics for the biosensor constructs based on the predicted one or more folded structures includes at least one of: determining an energy of a predicted folded structure for the biosensor construct. Comparing an energy of a predicted folded structure for the biosensor construct to energies of other predicted folded structures for the biosensor construct. Determining a barrier energy for converting a predicted folded structure for the biosensor construct to a target folded structure. In some embodiments, predicting one or more folded structures that the biosensor construct will form includes conducting a constraint folding analysis.

[0026] This disclosure further describes a computer-implemented method for designing a biosensor construct, the method comprising: determining, by a computing device, one or more candidate functional nucleic acid sequences; for each of the one or more candidate functional nucleic acid sequences: predicting, by the computing device, one or more folded structures that the functional nucleic acid sequence forms over time; and determining, by the computing device, one or more metrics for the functional nucleic acid sequence based on the predicted one or more folded structures; and choosing, by the computing device, one or more of the one or more candidate functional nucleic acid sequences to be provided for synthesis based on the metrics. In some embodiments of the computer-implemented method, the nucleic acid is DNA or RNA. In some embodiments of the computer-implemented method, predicting one or more folded structures that the functional nucleic acid sequence forms over time includes conducting a constraint folding analysis. In these embodiments, conducting a constraint folding analysis includes specifying a predetermined folded structure for a portion of the functional DNA or RNA sequence; and predicting an overall folded structure for the functional DNA or RNA sequence given the predetermined folded structure for the portion of the functional DNA or RNA sequence. In some embodiments of the computer-implemented method, the functional DNA or RNA is or comprises a biosensor molecule. In some of these embodiments, a non-transitory computer-readable medium having computer-executable instructions stored thereon that, in response to execution by one or more processors of a computing device, cause the computing device to perform actions of a method for predicting the overall folded structure of a portion of the biosensor construct. These computations can be performed on any computing device. DESCRIPTION OF THE DRAWINGS

[0027] The foregoing aspects and many of the attendant advantages described in this disclosure will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

[0028] Figure 1 is a schematic illustration of an embodiment of the RNA biosensor construct disclosed herein. The RNA biosensor construct has a Sensor domain, which itself comprises a binding element for binding a target ligand, and an Output domain, comprising a signaling element, that results in a measurable signal depending on the ligand-binding status of the target ligand to the binding element of the Sensor domain. The length and sequence of the closing stems of the binding element and the signaling element can generally be varied, so long as Watson-Crick base-pairing is maintained. The internal regions that possess more complex structures and activities are generally invariable in order to maintain function. "Ex. 1" and "Ex. 2" illustrate exemplary biosensors possessing the same input elements, but different lengths of Overhang, Stem, and Linker. Each of "Ex. 1" and "Ex. 2" are illustrated in ON (first conformation for modulation of detectable output signal) or OFF (second conformation for lack of modulation of detectable output signal). The Overhang, Stem, and Linker sequences are both shaded and labeled as such, while the unlabeled regions of the same shade are the reverse-complement of that region. The Linker regions is identified by a dashed box with alternating short and long dashes. The stem region is identified by a dashed box with uniform size dashes. The overhang region is identified by a box with dots and dashes.

[0029] Figure 2 is a stylized depiction of the specificity of a biosensor. The biosensor includes a fluorescing moiety, when the target molecule attaches to the biosensor, the fluorescing moiety lights up. In contrast, when a contaminant attaches to the biosensor, the fluorescing moiety does not light up.

[0030] Figure 3 is a stylized depiction of the use of biosensors in vitro showing the biosensor in use. The biosensor is placed in multiple wells. Engineered strains in each well produce target molecules that do not have a visible signature. The target molecule then binds the aptamer of the riboswitch and the functional RNA domain folds causing the fluorescing moiety to fluoresce. The fluorescence of the biosensor is read, and the fluorescence measures the target molecule titer.

[0031] Figure 4 is a stylized depiction of the biosensor in use in culture media. Variants of microbial strains produce a molecule of interest. That target molecule binds to the biosensor. The biosensor is switched on, causing the biosensor to fluoresce. The intensity of the fluorescence identifies the amount of the target molecule produced by each strain. Thus, strains that produce high amounts of the target molecule will appear brighter.

[0032] Figures 5A-5J are the chemical compositions of ten molecular targets. 5A is theophylline, 5B is flavin mononucleotide (FMN), 5C is p-aminophenylalanine (pAF), 5D is p-aminocinammic acid (pACA), 5E is tetracycline, 5F is guanosine monophosphate (GMP), 5G is tobramycin, 5H is tryptophan, 51 is lysozyme, and 5J is His-6; and four conditional fluorophores which have been used with the biosensor, 5K is malachite green, 5L is DFHBI, 5M is thiazole orange 1, and 5N is patent blue v.

[0033] Figure 6 are the graphs of sensor responses for the ten molecular targets showing the identification of those molecules.

[0034] Figures 7A and 7B are graphs showing the selectivity of the nucleic acid biosensors. Theophylline and Caffeine differ from one another by a single functional group. The biosensor is selective enough to distinguish between the theophylline and the Caffeine. Figure 7A is the graph for theophylline at various concentrations. The concentrations over 0 pM show florescence. The theophylline was tested at concentrations of 100 pM, 1 mM, and 10 mM. The caffeine was tested at the same concentrations. Caffeine shows a background response or essentially no response to the biosensor.

[0035] Figure 8 is a graph showing traces of Theophylline-responsive malachite green sensor (Theo_MG_4), showing sensor response to 4 concentrations of theophylline over the course of 60 minutes. The concentrations are 0 pM, 200 pM, 2 mM, and 20 mM. The biosensor is responsive even in non-ideal conditions. Yeast Broth includes many contaminants and various molecules that hinder or degrade the signal.

[0036] Figure 9A is a graph showing Kinetic traces of Tetracycline -responsive CP Broccoli / DFHBI sensor (WBI_0121), showing sensor response to 5 concentrations of tetracycline over the course of 60 minutes.

[0037] Figure 9B is a graph showing Titration of Tetracycline -responsive CP Broccoli / DFHBI sensor (WBI_0121), showing normalized sensor response at 60 minutes to concentrations ranging from 200 nM to 200 uM; EC50 of this sensor is 82 + 5 uM.

[0038] Figure 10A is a graph showing Kinetic traces of p-AF responsive malachite green sensor (WBI_0075), showing sensor response to 5 concentrations of p-AF over the course of 60 minutes. Figure 10B is graph showing titration of p-AF responsive malachite green sensor (WBI_0075), showing normalized sensor response at 60 minutes to concentrations ranging from 20 uM to 20 mM; EC50 of this sensor is 1.5 + 0.1 mM.

[0039] Figure 11 is a graph showing Kinetic traces of FMN-responsive malachite green sensor (FMN_MG_3), showing sensor response to 6 concentrations of FMN over the course of 60 minutes.

[0040] Figure 12A is a graph showing Kinetic traces of lysozyme-responsive malachite green sensor (WBI_0093), showing sensor response to 4 concentrations of lysozyme over the course of 60 minutes.

[0041] Figure 12B is a graph showing titration of lysozyme-responsive malachite green sensor (WBI_0093), showing normalized sensor response at 60 minutes to concentrations ranging from 2 uM to 10 mM; EC50 of this sensor is 970 + 170 nM.

[0042] Figure 13A is a graph showing Kinetic traces of His -Tag-responsive malachite green sensor (WBI_0047), showing sensor response to 6 concentrations of His-Mif over the course of 60 minutes.

[0043] Figure 13B is a graph showing Titration of His-Tag-responsive malachite green sensor (WBI_0047), showing normalized sensor response at 60 minutes to concentrations ranging from 100 nM to 4 uM; EC50 of this sensor is 3.6 + 0.8 uM.

[0044] Figures 14A and 14B are graphs depicting tetracycline responsive malachite green sensors. Figure 14A is a graph depicting Kinetic traces of Tetracycline -responsive malachite green sensor (WBI_0125), showing sensor response to 5 concentrations of tetracycline over the course of 60 minutes. Figure 14B is a graph depicting Titration of Tetracycline -responsive malachite green sensor (WBI_0125), showing normalized sensor response at 60 minutes to concentrations ranging from 100 nM to 100 uM; EC50 of this sensor is 1.0 + 0.2 uM.

[0045] Figure 15 is a graph showing Integrated brightness of beads vs bead area calculated from fluorescence microscopy images of theophylline- responsive malachite green sensors on agarose beads, where the beads have been incubated with four different concentrations of Theophylline, demonstrating a strong correlation between size and integrated brightness at each given concentration of theophylline. Thus, enabling quantification of the concentration of a sample through any single bead by mapping its size and fluorescence to this two-dimensional standard curve.

[0046] Figure 16A. In the absence of theophylline and FMN, beads with immobilized FMN- responsive sensors, and beads with immobilized theophylline-responsive sensors, generate low basal levels of fluorescence. 16B. In the presence of FMN, but absence of theophylline, only the FMN sensor beads generate increased fluorescence. 16C. In the presence of theophylline, but absence of FMN, only the theophylline sensor beads generate increased fluorescence. 16D. In the presence of both theophylline and FMN, both sets of beads generate increased fluorescence.

[0047] Figure 17 is a stylized depiction of sensor association with beads through covalently coupled ligands attached to beads. The sensors and switches can selectively associate to beads displaying the molecule that binds to the output module of the switches in the presence of the molecule that binds to the input module of the switches.

[0048] Figure 18 is a graphic representation showing covalently linked light up sensors (CLIPs). In cis-acting CLIPs (cCLIPs). Functional sensors may have a light-up fluorophore directly coupled to the sensors’ nucleic acid strand. Separately, functional trans-acting CLIPs (tCLIPs) have been demonstrated in which the sensor nucleic acid can detect target small molecules after being annealed to a separate DNA oligo with a covalently linked conditional fluorophore.

[0049] Figure 19 is a graph depicting quantification of theophylline-MG switch binding to beads coated with malachite green analogs. The sensors and switches can selectively associate to beads displaying the molecule that binds to the output module of our switches in the presence of the molecule that binds to the input module of the switches. This graph demonstrates quantification of the relative amounts of Theo-MG sensor RNA bound to beads coated with tetramethylrosamine, a malachite green analog using PAGE. Binding of the sensor to the tetramethylrosamine-coated beads is higher in the presence of theophylline compared to in the absence of theophylline.

[0050] Figures 20A is a graph showing traces of phenylalanine-responsive DAP DNA light-up sensor, showing sensor response to 4 concentrations of phenylalanine over the course of 60 minutes.

[0051] Figure 20B is a graph showing Titration of phenylalanine -responsive DAP DNA light-up sensor, showing normalized sensor response at 60 minutes to concentrations ranging from 50 uM to 50 mM; EC50 of this sensor is 1.6 + 0.2 mM.

[0052] Figure 21 is a depiction of tetracycline -responsive fluorescent sensors with diverse output ligands. All three sensors show increased fluorescence in the presence of tetracycline and no reaction or possible background reaction without tetracycline.

[0053] Figure 22A is a graph depicting Kinetic traces of FMN riboswitch-malachite green sensor (WBI_0366), showing sensor response to 4 concentrations of FMN over the course of 60 minutes. Figure 22B is a graph depicting Titration of FMN riboswitch-malachite green sensor

[0054] (WBI_0366), showing normalized sensor response at 60 minutes to concentrations ranging from 1 uM to 1 mM; EC50 of this sensor is 3.8 + 1.2 uM.

[0055] Figure 23 is a flow chart for a method of designing nucleic acid biosensor molecules.

[0056] Figure 24 is a flow chart for predicting one or more folded structures that a nucleic acid sequence forms over time.

[0057] DETAILED DESCRIPTION

[0058] The present disclosure is based on the development of a design platform for modular, tunable biosensors that can be implemented to produce a vast array of unique biosensors useful across molecular sensing applications.

[0059] Nucleic acid biosensors are disclosed herein. As used herein, the term refers to nucleic acid molecules that can sense (i.e., indicate binding to) a target ligand. Nucleic acid biosensors can be RNA or DNA biosensors. The biosensors will function in a similar manner whether they are RNA or DNA biosensors. Throughout this description, the biosensors may be referred to as nucleic acid biosensors, DNA biosensors, or RNA biosensors. The functional aspects of the biosensors are essentially the same whether the biosensor is DNA or RNA. The functional characteristics of the nucleic acid biosensor are conferred by the three-dimensional folding conformations. The conformations, and by extension the functionalities conferred thereby, are influenced by the presence or absence of the target ligand. The nucleic acid biosensors are useful because they allow modular coupling of diverse sensing (e.g. a natural or synthetic aptamer, or bindable natural sequence) and output domains resulting in the ability to generate signal in response to the desired concentration of an innumerable number of potential target molecules. Thus, the sensitivity of nucleic acid biosensors can be quantitatively tuned by manipulation of equilibria between the presence of a ligand and the absence of a ligand.

[0060] RNA biosensors are disclosed herein. As used herein, the term refers to RNA molecules that can sense (i.e., indicate binding to) a target ligand. The functional characteristics of the RNA biosensor are conferred by the three-dimensional folding conformations. The conformations, and by extension the functionalities conferred thereby, are influenced by the presence or absence of the target ligand. The RNA biosensors are useful because they allow modular coupling of diverse sensing (e.g. a natural or synthetic aptamer, or bindable natural sequence) and output domains resulting in the ability to generate signal in response to the desired concentration of an innumerable number of potential target molecules. Thus, the sensitivity of RNA biosensors can be quantitatively tuned by manipulation of equilibria between the presence of a ligand and the absence of a ligand. To implement reliable and reproducible designs of RNA biosensors, convergent expertise in in vitro RNA analysis, in silica prediction of RNA folding trajectories, and experimental analysis of biomolecular switch function were required. As described in more detail below, a novel molecular architecture has been developed that allowed the disclosed biomolecular switches to take advantage of RNA folding. This architecture, combined with the use of sequence complementarity to critical regions of the input domains, resulted in an efficient and elegant strategy to create in silica libraries of candidate switches with a probability of being functional much higher than through random sequence search. An algorithm for predicting folding in a way that allowed access to the underlying quantitative parameters, permitting the modular, tunable design has also been developed.

[0061] Figure 1 schematically illustrates exemplary embodiments for modulation of the RNA biosensor design and resulting architecture for and equilibrium between first ("ON") and second ("OFF") conformations or switch states depending on the presence of a ligand. To confer liganddependent activity, two different structures must be encoded: an "ON" conformation / conformation / state where both the sensor domain (including a binding element) and output domain are correctly folded and able to function as they would in isolation, and an "OFF" conformation / conformation / state where neither domain is correctly folded and, therefore, are nonfunctional. The base-pairing interactions that provide stabilizing energy to the "OFF" conformation / conformation / state are encoded within the sensor domain, which comprises the overhang, stem, and linker sequences adjacent to the invariable binding element sequence. The overhang, stem, and linker sequences can have variable lengths (e.g., from 0 to about 15 bases). Embodiments of the biosensor with different lengths for the overhang, stem, and linker sequences are illustrated in "Ex. 1" and Ex. 2". Their sequences allow hybridization with subregions within the binding element sequence, and subsequences of the output domain, such that they form one continuous helix within the OFF conformation / state. For example, the sequences for the named regions (overhang, stem, linker) can be the reverse complement or nearly reverse complement to the sequence in the corresponding subregions within the binding element sequence, and subsequences of the output domain. For example, the overhang and stem sequences can be the reverse complement to sequences within the 5' end of the output domain such that together they will bind to the 5'-most n bases of the output domain, where n is the sum of the lengths of the overhang and stem domains. The linker sequence can be the reverse complement to the 5' end of the invariable region of the binding element (which is also referred to herein as the linker target sequence). While the discussion presented herein is generally presented in the context of modulation of the equilibrium between the binary conformations / states of "ON" and "OFF" resulting from the folding in the presence and absence of a ligand, respectively, it will be understood that the configuration can also be inverted or can be further modulated. The features and elements described in more detail below can be modulated so that the elements are configured to result in "ON" and "OFF" conformations / states based on the shifting of the equilibrium between the conformations / states in the absence and presence of the ligand, respectively, or swapped, allowing for configurations of "0N" / "0N", "OFF" / "OFF", "ON" / "OFF", and "OFFV'ON" dependent on ligand binding or absence of ligand binding. Such inverted embodiments are also encompassed by the present disclosure.

[0062] Generally, the binary conformations / states of “ON / OFF” are depicted by fluorescence. That is either the ON conformation / conformation / state or the OFF conformation / conformation / state is operably linked to a signaling molecule that will fluoresce when a particular conformation occurs. Often the ON conformation / conformation / state is designed so that when the ligand is bound to the nucleic acid biosensor the signaling molecule will fold in such a way as to cause fluorescence. However, the conformations / states can be designed in the inverse so that the signaling molecule will fluoresce when the nucleic acid biosensor folds into the configuration without the ligand. This way the detection can be designed to test for either the presence or absence of the ligand.

[0063] This detection occurs very rapidly. This is due in part to the conformation / state at which the detection reaction is prepared. The detection reaction is kept in a state of equilibrium. When the ligand is be added or removed the fluorescence will change along with the binding or unbinding of the ligand.

[0064] In order to ensure that the designed biosensor is able to respond to the presence of a target molecule on a relevant timescale, it is important that the kinetics of interconversion between the OFF and ON conformation / conformation states are rapid. In order to ensure this is the case, the predicted barrier height between the OFF and ON conformation / conformation states is at a minimum when screening for promising biosensor candidates. Sensors that possess very low values are selected for very low values.

[0065] Other approaches have been studied and described, including those that utilize co- transcriptional pathways. Such approaches may be differentiated from the biosensors described herein as the co-transcriptional pathways are not in a state of equilibrium as intended and described in this disclosure. Referring to Figure 1 and the disclosure of Example 1, below, the disclosed modular molecular architecture allows the equilibrium among variety of output domains (ribozyme, ribosome binding site, CRISPR gRNA, etc.) to be controlled by the binding conformation / state of a binding element using the same rules and quantitative screening metrics. The overhang, stem, and linker sequences are targeted to specific parts of the sensor and output domains. Their sequences can be the reverse-complement of sequences in the binding element and signaling element, providing thermodynamic incentive for the two domains to cooperatively misfold in the absence of stabilization by the target molecule (ligand). Their lengths can be systematically varied to thermodynamically couple the structures of the two domains, while still allowing the energy from the target ligand to ensure the correct structure upon binding. This enables a very efficient computational search for switch candidates that satisfy the thermodynamic requirements of switching.

[0066] Some thermodynamic biosensors rely on rapid interconversion between two conformations / states. First the binding element folds and allows the target ligand to associate. In one illustrative embodiment, if the target ligand does not associate, the RNA molecule structurally rearranges to a second conformation where neither the binding element, nor output domain, are functional. This is the "OFF / OFF" configuration indicated above. If, however, the target ligand is bound, it thermodynamically stabilizes the conformation containing the correctly folded binding element, allowing the output domain to fold into its functional conformation. This is the "ON / ON" configuration indicated above. In an alternative embodiment, if no ligand binds to the sensor domain, the output domain will fold resulting in a first or functional (i.e., "ON") output domain. This is the "OFF / ON" configuration indicated above. If a ligand does bind to the sensor domain, there is a thermodynamic incentive to (mis)fold into a configuration that results in a non-functional output domain. This is the "ON / OFF" configuration indicated above.

[0067] The described work provides a computational and experimental pipeline that takes a structured ligand binding element as an input, and outputs a genetic RNA biosensor that can be used to detect the concentration of molecules of interest and or the binding of molecules of interest to the structured ligand binding element. First, combinatorial variation of three variable regions in the molecular architecture (e.g., overhang, stem, and linker sequences) creates a diverse in silica pool of candidate sequences that can be screened using RNA folding simulations. Second, one can perform thermodynamic simulations, utilizing constraint folding, to ensure the proper switch conformations / states exist with the desired energies. In accordance with the foregoing, in one aspect the disclosure provides an RNA biosensor construct. Described from 5' to 3', the biosensor construct comprises: a sensor domain that specifically binds to a ligand of interest and an output domain configured to modulate a detectable output signal when folded into a first conformation. The output domain folds into either a functional ("ON") or non-functional "OFF") configuration depending on whether the sensor domain is bound to the ligand of interest. In some embodiments, the output domain folds into the first conformation when the sensor domain is bound to the ligand of interest ("ON / ON" configuration). In contrast, the output domain folds into a second conformation when the sensor domain is not bound to the ligand of interest ("OFF / OFF" configuration). In other embodiments, the configurations are inverted where the output domain folds into the second conformation when the sensor domain is bound to the ligand of interest ("ON / OFF" configuration) or, alternatively, the output domain folds into a first conformation when the sensor domain is not bound to the ligand of interest ("OFF / ON" configuration).

[0068] In some embodiments, the sensor domain comprises, from 5' to 3', at least two of: an overhang sequence, a binding element, and a linker sequence. The binding element itself comprises a stem sequence, a linker target, a binding element subsequence, and a stem target sequence. The output domain comprises, from 5' to 3': a stem target sequence, an overhang target sequence, and a signaling element subsequence.

[0069] In some embodiments, the overhang sequence of the sensor domain is the reverse complement of at least a portion of the overhang target sequence of the output domain. In some embodiments, the overhang sequence of the sensor domain is the reverse complement of at least a substantial portion (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%) of the overhang target sequence of the output domain. In additional or alternative embodiments, the stem sequence of the sensor domain is the reverse complement of at least a portion of the stem target sequence of the sensor domain and is the reverse complement of at least a portion of the stem target sequence of the output domain. In some embodiments, the stem sequence of the sensor domain is the reverse complement of at least a substantial portion (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%) of the stem target sequence of the sensor domain and is the reverse complement of at least a substantial portion (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%) of the stem target sequence of the output domain. In additional or alternative embodiments, the linker target sequence is the reverse complement of at least a portion of the binding element subsequence and is also the reverse complement of at least a portion of the linker sequence. In some embodiments, the linker target sequence is the reverse complement of at least a substantial portion (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or all) of the binding element subsequence and is also the reverse complement of at least a substantial portion (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or all) of the linker sequence. These embodiments can be present in any combination, including a further embodiment where the overhang sequence of the sensor domain is the reverse complement of at least a portion (e.g., a substantial portion such as at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or all) of the overhang target sequence; the stem sequence of the sensor domain is the reverse complement of at least a portion (e.g., a substantial portion such as at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or all) of the stem target sequence of the sensor domain and is the reverse complement of at least a portion of the stem target sequence of the output domain; and the linker target sequence is the reverse complement of at least a portion (e.g., a substantial portion such as at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or all) of the binding element subsequence.

[0070] In some embodiments, the portion of the binding element subsequence that is the reverse complement of at least a portion of the linker target sequence is a discontinuous portion of the binding element subsequence. When in the "ON" or first folding conformation, e.g., due to the binding of the ligand of interest, the discontinuous portion of the binding element subsequence can comprise two sequences with reverse complementarity to different portions of the linker target sequence, with an intervening sequence that forms a secondary structure, such as a hairpin domain that contributes to the ligand binding functionality within the binding element subsequence.

[0071] In some embodiments, when the sensor domain is bound to the ligand of interest, the stem sequence of the sensor domain is hybridized to the stem target sequence of the sensor domain, thereby permitting folding of the output domain into the first conformation. Alternatively, or in addition, in some embodiments the linker target sequence is hybridized to the portion of the binding element subsequence, thereby permitting folding of the output domain into the first conformation.

[0072] In some embodiments, the hybridization events indicated above are facilitated by the conformation of the sensor domain that is stabilized when a ligand binds to the sensor domain as it is transcribed. This results in a first or "ON" conformation that results in the correct folding of the output domain. In absence of a ligand, the elongating transcript will adopt a different conformation, allowing components of the sensor domain to hybridize with the output domain, resulting in a second or "OFF" conformation. For example, when the sensor domain is not bound to the ligand of interest, in some embodiments the overhang sequence of the sensor domain hybridizes to the overhang target sequence of the output domain, thereby permitting folding of the output domain into the second conformation. Additionally, or alternatively, when the sensor domain is not bound to the ligand of interest, the stem sequence of the sensor domain is hybridized to the stem target sequence of the output domain, thereby permitting folding of the output domain into the second conformation. Additionally, or alternatively, when the sensor domain is not bound to the ligand of interest the linker target sequence of the sensor domain is hybridized to the linker sequence of the sensor domain, thereby permitting folding of the output domain into the second conformation. In some embodiments, when the sensor domain is not bound to the ligand of interest, the overhang sequence of the sensor domain hybridizes to the overhang target sequence of the output domain, the stem sequence of the sensor domain is hybridized to the stem target sequence of the output domain, and the linker target sequence of the sensor domain is hybridized to the linker sequence of the sensor domain, thereby permitting folding of the output domain into the second conformation. While this disclosure is generally presented to illustrate the "ON / ON" or "OFF / OFF" configurations, it will be understood that the relative configurations can be adjusted to invert the biosensor to result in an "ON / OFF" or "OFF / ON" configuration. In this inverted embodiment, the sensor domain is stabilized by the binding on the ligand during its transcription and initial folding. This binding stabilizes the conformation in a manner that results in a non-functional output domain upon its transcription and resultant folding. Alternatively, when no ligand is present, the senor domain will assume a different conformation that provides thermodynamic incentive for the output domain to fold into a functional conformation upon its transcription.

[0073] In some embodiments, at least two of the overhang sequence of the sensor domain, the stem sequence of the sensor domain, and the linker target sequence form a continuous helix stem structure when hybridized to at least a portion of the overhang target sequence of the output domain, a portion of the stem target sequence of the output domain, and a portion of the linker sequence of the sensor domain, respectively, thereby permitting the signaling element subsequence to fold into the second conformation.

[0074] In some embodiments, the overhang sequence of the sensor domain is between 0 and about 15 nucleotides in length. In some embodiments, the stem sequence of the sensor domain is between 0 and about 15 nucleotides in length. In some embodiments, the linker sequence of the sensor domain is between 0 and about 15 nucleotides in length. In some embodiments, each of the overhang sequence of the sensor domain, the stem sequence of the sensor domain, and the linker sequence of the sensor domain are all between 1 and about 15 nucleotides in length, e.g., each is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. The biosensor can further comprise an intermediate sequence disposed between the binding element and the linker sequence of the sensor domain. The intermediate sequence can result in additional time between the transcription of the two domains or provide other beneficial functions orthogonal to the biosensor’s switching mechanism. In some illustrative embodiments, the intermediate sequence domain has a length up to about 150 nucleotides in length (e.g., about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, and 150 nucleotides in length). For example, the intermediate sequence can have a length of between 1 and about 50 nucleotides, between about 25 and 75 nucleotides, between about 50 and 100 nucleotides, between about 75 and 125 nucleotides, and between about 100 and 150 nucleotides. In some embodiments, the intermediate sequence is configured to fold into a secondary structure. For example, the intermediate sequence can be configured to form a stem-and- loop structure with each end of the intermediate sequence (i.e., the regions at the 5' end and the 3' end) forming part of a stem of the stem-and-loop structure. The loop structure is comprised of a middle section of the intermediate sequence.

[0075] The output domain can be any RNA-based construct that can be configured to modulate, directly or indirectly, a detectable signal when folded properly. The modulation can be the induction or increase of a detectable signal. In alternative embodiments, the modulation can be the reduction of a detectable second signal when the output domain is folded into the first conformation. In either approach, the change is detected and used to inform the presence and / or concentration of the ligand of interest.

[0076] To illustrate, in some embodiments, when the output domain is folded into a first conformation the output domain is or comprises a functional ribozyme, a functional nuclease guide RNA (gRNA), or RNA aptamer. In an exemplary embodiment, when the output domain is folded into an first conformation the output domain is or comprises a functional gRNA, wherein the functional gRNA is between about 40 and about 400 nucleotides in length.

[0077] In some embodiments, the functional gRNA associates with a nuclease, such as a nuclease selected from Cas9, Casl2a, Casl3, derivatives thereof, and the like. In this regard, the gRNA can integrate into a CRISPR-based reporting system. In some embodiments, the nuclease has ablated nuclease function. In some embodiments, the nuclease confers CRISPR activation (CRISPRa) function (see, e.g., Example 4). In some embodiments, the nuclease confers CRISPR inhibition (CRISPRi) function. In some embodiments, the nuclease has nuclease function.

[0078] In some embodiments, the biosensor sequences, the sequence architecture, are contained within larger sequences. In some embodiments, the biosensor is attached to the end of a sequence. This can be when the biosensor is attached to the end of a coding sequence. Additionally, an additional aptamer can be attached to the 5’ or 3’ end of the biosensor. In some embodiments, the additional structures are also being regulated. The biosensor may also be contained within sequences.

[0079] The biosensor is often in solution, and free to move within that solution. In alternative uses, the biosensor is immobilized on substrates such as beads. For RNA biosensors, a biotinylated DNA oligonucleotide is coupled to the beads, the biosensor anneals to the DNA oligonucleotide using a complementary tail sequence. For DNA biosensors, the DNA biosensor is directly biotinylated and coupled to the bead. The biosensor can be attached via either the 3’ or 5’ end.

[0080] The ligand of interest can be any compound or moiety that is capable of binding to a structured RNA sequence. Non-limiting examples of ligands of interest include chemicals, metabolites, proteins, peptides, small molecules (e.g., drug molecules, drug precursor molecules, drug metabolites, etc.), cells, and the like. Persons of ordinary skill in the art can readily identify other ligands that are encompassed by this disclosure.

[0081] The nucleic acid biosensor construct can be designed using computational approaches that ensure that the different domains (e.g., the sensor domain and the output domain) are independently functional but can operate in a binary "switch" manner when fused into a single construct, this binary “switch” modulates between “on” and “off.” For example, in some embodiments, the computer-implemented design method described in more detail below in the context of guide nucleic acid design is configured and applied to the design of the overall nucleic acid biosensor construct. A person of ordinary skill in the art can make the requisite adjustments to the described method to ensure design and output of function nucleic acid biosensors constructs, as described above.

[0082] In another aspect, the disclosure provides a polynucleotide molecule comprising a sequence encoding the RNA biosensor construct described herein. The polynucleotide can comprise or consist of DNA or RNA. In some embodiments, the polynucleotide is a DNA molecule, which also comprises a promoter operatively linked to the encoding sequence. The term "promoter" refers to a regulatory nucleotide sequence that can activate transcription (expression) of encoding DNA. A promoter is typically located upstream of the encoding DNA but can be located at other regions proximal to the encoding DNA. The promoter typically contains binding sites for RNA polymerase and one or more transcription factors, which participate in the assembly of the transcriptional complex. As used herein, the term "operatively linked" indicates that the promoter and the encoding DNA are configured and positioned relative to each other in a manner such that the promoter can activate transcription of the encoding nucleic acid by the transcriptional machinery of the cell. The promoter can be constitutive or inducible. Constitutive promoters can be determined based on the character of the target cell or transcription environment and the particular transcription factors available therein. A person of ordinary skill in the art can select an appropriate promoter based on the intended purpose, as various promoters are known and commonly used in the art.

[0083] In another aspect, the disclosure provides a vector comprising the DNA molecule described herein. The vector can be any construct that facilitates the delivery of the nucleic acid to the target cell or transcription environment (e.g., acellular environment) and / or expression of the nucleic acid within the cell or environment. The vectors can be viral vectors, circular nucleic acid constructs (e.g., plasmids), or nanoparticles. Various viral vectors are known in the art and are encompassed by the present disclosure. See, e.g., Machida, C. A. (ed.), Viral Vectors for Gene Therapy: Methods and Protocols, Humana Press, Totowa, New Jersey (2003); Muzyczka, N., (ed.), Current Topics in Microbiology and Immunology. Viral Expression Vectors, Springer- Verlag, Berlin, Germany (2012), each incorporated herein by reference in its entirety. In some embodiments, the viral vector is an adeno associated virus (AAV) vector, an adenovirus vector, a retrovirus vector, or a lentivirus vector. A specific embodiment of an AAV vector includes the AAV2.5 serotype.

[0084] In another aspect, the disclosure provides a cell comprising the nucleic acid or the vector described above. The cell is capable of transcribing the RNA biosensor construct from the DNA molecule. For example, a promoter operatively linked to the encoding DNA can be appropriately configured to allow binding of the cell's RNA polymerase and one or more transcription factors to permit assembly of the transcriptional complex.

[0085] The disclosure encompasses any type of cell for this aspect. For example, the cell can be prokaryotic or eukaryotic, without limitation.

[0086] The cell can be engineered to further comprise an expression construct comprising a reporter gene operatively linked to a promoter, wherein the promoter is targeted by the output domain when folded into the first ("ON") conformation. For example, the promoter can be a synthetic promoter optimized to be bound by a functional gRNA. In some embodiments, the output domain of the RNA biosensor construct is or comprises a functional gRNA when folded into an first conformation, and the functional gRNA hybridizes to the synthetic promoter. In some embodiments, the synthetic promoter contains a Protospacer Adjacent Motif (PAM) positioned between about 40 and about 120 bases 5' of the transcription start site. The synthetic promoter design and its use in CRISPR activation (CRISPRa) is described in more detail in Fontana, J., et al., "Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements," Nature Communications (2020) 11:1618, which is incorporated herein by reference. In some embodiments, the cell is engineered to further express a nuclease with ablated nuclease functionality and a transcription factor, wherein the expressed nuclease and transcription factor associate with the functional gRNA and are configured to facilitate transcription of the reporter gene when the gRNA hybridizes with the synthetic promoter of the expression construct. See, e.g., Mali P, Esvelt KM, and Church GM. Cas9 as a versatile tool for engineering biology. Nat Methods. 2013 Oct;10(10):957-63, and Dominguez AA, Lim WA, and Qi LS. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol. 2016 Jan;17(l):5-15, each of which is incorporated herein by reference in its entirety.

[0087] In some embodiments, the cell is engineered or treated to modify expression or production of the ligand which the biosensor binds to. For example, the cell can be engineered to increase or decrease biosynthesis of a particular metabolite or bioproduct. The expression of the RNA biosensor construct can facilitate the identification of cells that biosynthesize the metabolite or bioproduct, e.g., to screen for the success of the genetic manipulations.

[0088] The disclosed nucleic acid biosensor is not limited to use in cells. Thus, in another aspect, the disclosure provides a biosensor system that comprises: a sensor DNA expression cassette comprising sequence encoding the RNA biosensor construct as described herein; and an RNA polymerase and NTPs sufficient to facilitate synthesis of the RNA biosensor construct.

[0089] The biosensor system can be configured to perform in vitro, cell-free transcription (IVT). In further embodiments, the biosensor is configured to perform cell-free synthesis (CFS) of protein from transcribed RNA templates. Accordingly, in such embodiments, the biosensor system further comprises protein translation elements selected from ribosomes, tRNAs, aminoacyl-tRNA synthetase, initiation factors, elongation factors, termination factors, amino acids, ATP, GTP, and / or translation co-factors, in any combination.

[0090] In some embodiments, the biosensor system further comprises an expression construct comprising a reporter gene operatively linked to a synthetic promoter, wherein the output domain of the RNA biosensor construct is or comprises a functional gRNA when folded into an first conformation, and the functional gRNA hybridizes to the synthetic promoter.

[0091] In some embodiments, the biosensor system further comprises a nuclease with ablated nuclease functionality, and a transcription factor. The nuclease and transcription factor associate with the functional gRNA and are configured to facilitate transcription of the reporter gene when the associated gRNA hybridizes with the synthetic promoter of the expression construct.

[0092] In another aspect, the disclosure provides a method of detecting a ligand of interest. The method comprises synthesizing the RNA biosensor construct, as described herein, in an environment that may contain the ligand of interest and detecting an output signal. A detected output signal or modulation of an output signal indicates binding of the ligand of interest to the sensor domain.

[0093] In some embodiments, the environment is an in vitro environment capable of facilitating transcription of the RNA biosensor construct. For example, the in vitro environment can comprise an RNA polymerase, NTPs, and a template DNA molecule as described herein to facilitate synthesis of the RNA biosensor construct. In further embodiments, the environment is a cell-free synthesis (CFS) environment. For example, the CFS environment can comprise protein translation elements selected from ribosomes, tRNAs, aminoacyl-tRNA synthetase, initiation factors, elongation factors, termination factors, amino acids, ATP, GTP, and / or translation co-factors, in any combination. In some embodiments, the environment comprises a cell lysate.

[0094] In other embodiments, the environment is in a cell. The cell can be engineered, such as in a manner described above. For example, the cell can be engineered such that the output domain of the RNA biosensor construct is or comprises a functional gRNA when folded into an first conformation, and the functional gRNA hybridizes to the synthetic promoter. In some embodiments, the cell is engineered to further express a nuclease with ablated nuclease functionality and a transcription factor, wherein the expressed nuclease and transcription factor associate with the functional gRNA and are configured to modulate transcription of the reporter gene when the gRNA hybridizes with the synthetic promoter of the expression construct. In some embodiments, the nuclease with ablated nuclease functionality confers CRISPR activation (CRISPRa) function, and wherein the reporter gene encodes a fluorescent protein, an antibiotic resistance protein, beta-galactosidase, and the like. In some embodiments, the nuclease with ablated nuclease functionality confers CRISPR inhibition (CRISPRi) function. For example, in some embodiments the nuclease with ablated nuclease functionality confers CRISPR inhibition (CRISPRi) function and the reporter gene encodes a fluorescent protein, an antibiotic resistance protein, beta-galactosidase, and the like, or an endogenous gene. A change is detectable as a result of CRISPRi activity, indicating the status of ligand binding to the biosensor construct.

[0095] In some embodiments, the output domain of the RNA biosensor construct is or comprises a functional gRNA when folded into an first conformation, and the functional gRNA hybridizes to a target sequence of interest, and wherein the cell is engineered to further express a nuclease that has nuclease function.

[0096] In some embodiments, the cell is engineered to modify production of a ligand of interest in the cell, as described above. In other embodiments, the method further comprises subjecting the cell to experimental conditions suspected to modify production of the ligand of interest in the cell. In other embodiments, the ligand of interest is a compound contacted to the cell or a metabolite thereof. In such embodiments, the method can be a method of detecting whether the compound is transported into or out of the cell or is metabolized by the cell to produce the metabolite.

[0097] In another aspect, the disclosure provides a computer-implemented method for designing functional RNA molecules. As used in this context, the term " functional RNA" refers to an RNA molecule that has a functional capacity conferred by the three-dimensional conformation that is assumed during first transcription. In some instances, the functional conformation is distinct from a later-assumed conformation of the same RNA molecule (e.g., after prolonged storage). In other embodiments, the functional RNA molecule is an RNA biosensor molecule. Each of these embodiments are described in more detail elsewhere herein.

[0098] The method of this aspect comprises: determining, by a computing device, one or more candidate functional RNA sequences; for each of the one or more candidate functional RNA sequences: predicting, by the computing device, one or more folded structures that the functional RNA sequence forms over time; determining, by the computing device, one or more metrics for the functional RNA sequence based on the predicted one or more folded structures; and choosing, by the computing device, one or more of the one or more candidate functional RNA sequences to be provided for synthesis based on the scores.

[0099] In some embodiments, the step of determining the one or metrics for the functional RNA sequence based on the predicted one or more folded structures includes at least one of: determining an energy of a predicted folded structure for the functional RNA sequence; comparing an energy of a predicted folded structure for the functional RNA sequence to energies of other predicted folded structures for the functional RNA sequence; and determining a barrier energy for converting a predicted folded structure for the functional RNA sequence to a target folded structure.

[0100] In some embodiments, predicting one or more folded structures that the functional RNA sequence forms over time includes conducting a constraint folding analysis. In some embodiments, conducting a constraint folding analysis includes: specifying a predetermined folded structure for a portion of the functional RNA sequence; and predicting an overall folded structure for the functional RNA sequence given the predetermined folded structure for the portion of the functional RNA sequence.

[0101] In another aspect, the disclosure provides a non-transitory computer-readable medium having computer-executable instructions stored thereon that, in response to execution by one or more processors of a computing device, cause the computing device to perform actions of the computer-implemented method as described above.

[0102] In another aspect, the disclosure provides a computing device configured to perform actions of the computer-implemented method as described above.

[0103] In other aspects, the disclosure provides computer-implemented methods, non-transitory computer-readable media, and computing devices, as described above, but implemented for the design and implementation of RNA sensor constructs, also as described above. The above elements of the computer-implemented methods and related media and devices can be modified and implemented to the design of the disclosed RNA biosensor construct to ensure that the different domains (e.g., the sensor domain and the output domain) are independently functional but can operate in a binary "switch" manner when fused into a single construct with the sensor domain being transcribed before the output domain. Accordingly, for brevity the above elements are encompassed by the present aspects of the disclosure and are not repeated.

[0104] General Definitions

[0105] Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present disclosure. Practitioners are particularly directed to Ausubel, F.M., et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (2010); Coligan, J.E., et al. (eds.), Current Protocols in Immunology, John Wiley & Sons, New York (2010); Mirzaei, H. and Carrasco, M. (eds.), Modern Proteomics - Sample Preparation, Analysis and Practical Applications in Advances in Experimental Medicine and Biology, Springer International Publishing, 2016; Mali P, Esvelt KM, and Church GM. Cas9 as a versatile tool for engineering biology. Nat Methods. 2013 Oct;10(10):957-63; and Dominguez AA, Lim WA, and Qi LS. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol. 2016 Jan;17(l):5-15, for definitions and terms of art. For convenience, definitions for certain terms employed in this disclosure are provided here. The definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims.

[0106] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or."

[0107] The words "a" and "an," when used in conjunction with the word "comprising" in the claims or specification, denotes one or more, unless specifically noted.

[0108] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, which is to indicate, in the sense of "including, but not limited to." Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words "herein," "above," and "below," and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application. The word "about" indicates a number within range of minor variation above or below the stated reference number. For example, in some embodiments, the term "about" refers to a number within a range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% above and / or below the indicated reference number.

[0109] A nucleic acid is a polymer of monomer units or "residues". The monomer subunits, or residues, of the nucleic acids each contain a nitrogenous base (i.e., nucleobase) a five-carbon sugar, and a phosphate group. The identity of each residue is typically indicated herein with reference to the identity of the nucleobase (or nitrogenous base) structure of each residue. Canonical nucleobases include adenine (A), guanine (G), thymine (T), uracil (U) (in RNA instead of thymine (T) residues) and cytosine (C). However, the nucleic acids of the present disclosure can include any modified nucleobase, nucleobase analogs, and / or non-canonical nucleobase, as are well-known in the art. Modifications to the nucleic acid monomers, or residues, encompass any chemical change in the structure of the nucleic acid monomer, or residue, which results in a noncanonical subunit structure. Such chemical changes can result from, for example, epigenetic modifications (such as to genomic DNA or RNA), or damage resulting from radiation, chemical, or other means. Illustrative and nonlimiting examples of noncanonical subunits, which can result from a modification, include uracil (for DNA), 5 -methylcytosine, 5-hydroxymethylcytosine, 5- formethylcytosine, 5 -carboxy cytosine b-glucosyl-5-hydroxy-methylcytosine, 8-oxoguanine, 2- amino-adenosine, 2-amino-deoxyadenosine, 2-thiothymidine, pyrrolo-pyrimidine, 2-thiocytidine, or an abasic lesion. An abasic lesion is a location along the deoxyribose backbone but lacking a base. Known analogs of natural nucleotides hybridize to nucleic acids in a manner similar to naturally occurring nucleotides, such as peptide nucleic acids (PNAs) and phosphorothioate DNA.

[0110] The five-carbon sugar to which the nucleobases are attached can vary depending on the type of nucleic acid. For example, the sugar is deoxyribose in DNA and is ribose in RNA. In some instances, herein, the nucleic acid residues can also be referred with respect to the nucleoside structure, such as adenosine, guanosine, 5-methyluridine, uridine, and cytidine. Moreover, alternative nomenclature for the nucleoside also includes indicating a "ribo" or deoxyribo" prefix before the nucleobase to infer the type of five-carbon sugar. For example, "ribocytosine" as occasionally used herein is equivalent to a cytidine residue because it indicates the presence of a ribose sugar in the RNA molecule at that residue. A nucleic acid polymer can be or comprise a deoxyribonucleotide (DNA) polymer, a ribonucleotide (RNA) polymer. The nucleic acids can also be or comprise a PNA polymer, or a combination of any of the polymer types described herein (e.g., contain residues with different sugars)

[0111] As used herein, the term "polypeptide" or "protein" refers to a polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L- isomers being preferred. The term polypeptide or protein as used herein encompasses any amino acid sequences and includes modified sequences such as glycoproteins. The term polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

[0112] "Percent sequence identity" or grammatical equivalents means that a particular sequence has at least a certain percentage of nucleic acid or amino acid residues identical to those in a specified reference sequence using an alignment algorithm. An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) website.

[0113] Reverse complement refers to the sequences of corresponding sequences that can mutually hybridize according to Watson-Crick base pairing rules. The term refers to each of the corresponding sequence (i.e., the sense and the anti-sense) hybridizing in reverse orientations with respect to the 5' to 3' directionalities. For example, a sense strand will have a sequence from 5' to 3' that is the complement of a sequence in the corresponding anti-sense strand when the anti-sense strand is aligned in the 3' to 5'. As used herein, it is contemplated that sequences indicated as being the reverse complement of a reference sequence does not have to have perfect, i.e., 100% complementation, but can have some residues that do no complement so long as the corresponding sequences still mutually hybridize under normal operating conditions.

[0114] RNA biosensors are defined as RNA biomolecules that bind a target ligand and regulate the folding of an output RNA domain in response. As indicated above, the RNA molecule will fold into three-dimensional conformations, and which can confer discrete functionalities.

[0115] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. It is understood that, when combinations, subsets, interactions, groups, etc., of these materials are disclosed, each of various individual and collective combinations is specifically contemplated, even though specific reference to each and every single combination and permutation of these compounds may not be explicitly disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in the described methods. Thus, specific elements of any foregoing embodiments can be combined or substituted for elements in other embodiments. For example, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. Additionally, it is understood that the embodiments described herein can be implemented using any suitable material such as those described elsewhere herein or as known in the art.

[0116] Publications cited herein and the subject matter for which they are cited are hereby specifically incorporated by reference in their entireties.

[0117] The following examples are provided to illustrate certain features and / or embodiments of the disclosure. This example should not be construed to limit the invention to the particular features or embodiments described.

[0118] This example discloses the development of a molecular architecture and computational workflow for the in-silico engineering of representative modular, nucleic acid biosensors according to some embodiments of the disclosure. Through in vitro assays, a set of computational design parameters are identified that enable the robust in silica identification of functional RNA switches. High performance switches and tunable ligand sensitives are demonstrated. The biosensors can be used, e.g., for screening applications in industrial biotechnology, medical biotechnology, pharmaceutical development, diagnostics, etc. The described biosensors are highly sensitive. The biosensors are able to distinguish between the desired target molecule and molecules with similar composition. The sensitivity can be tuned by implementation of changes in equilibrium concentrations to lengthen or shorten the time available for ligand binding. The modular design allows implementation and optimization of biosensors for any ligand of interest using a variety of output signals. The RNA biosensors are highly specific. Even a contaminant that has a similar atomic composition will not turn the RNA biosensor to the “ON” position and cause fluorescence. (Figure 2).

[0119] The biosensors are having a real time response, that is they give near-immediate results as to whether the desired molecule is present or absent. Another advantage to the biosensors is that they come ready to use and can be easily stored.

[0120] The biosensors can be used in in vitro high throughput screening of biomolecules. The target molecule or ligand will be introduced into the solution containing the biosensor as can be seen in Figure 3. The biosensor may be used with plate readers or fluorescence-activated cell sorting (FACS). As has been previously described, the target molecule will be introduced, and the molecule will bind to the biosensor. The binding of the target molecule triggers folding in the biosensor which turns the biosensor on and causes it to fluoresce. By screening with this method 104-106strains can be screened per day.

[0121] The process can be carried further to use the biosensors to detect target molecules secreted by cells as depicted in Figure 3. Candidate cells are grown in multi-well plates, a specific biosensor is developed to detect a target molecule secreted by the cells. The biosensor is added to each well and the fluorescence of each well is measured to determine the amount of target molecule produced by the cells. The amount of fluorescence of each well can be used as a comparison to the other wells and determine a titer of the target molecule.

[0122] In addition to extracellular screening and in vitro screening the RNA biosensors can be used in in vivo screening as shown in Figure 4. Microbial strains may produce a target molecule naturally or may be engineered to produce a target molecule. Then the biosensor is introduced to the microbial strain. There are several ways for the RNA biosensor to be introduced. Once introduced into the microbial strain, the RNA biosensor will function as it does in the extracellular screens and turn on in the Prescence of the target molecule. The microbial strains will be able to be screened and the specific strains that produce the target molecule will be identified.

[0123] Identifying cells that produce a desired molecule is useful and can be carried a step further to isolate those cells expressing the desired molecule, such as that shown in Figure 4. There are myriad reasons to isolate the cells producing the desired molecules. In another aspect, the disclosure provides a computer-implemented method for designing functional RNA molecules. As used in this context, the term " functional RNA" refers to an RNA molecule that has a functional capacity conferred by the three-dimensional conformation that is assumed during first transcription. In some instances, the functional conformation is distinct from a later-assumed conformation of the same RNA molecule (e.g., after prolonged storage). In other embodiments, the functional RNA molecule is an RNA biosensor molecule. Each of these embodiments are described in more detail elsewhere herein.

[0124] The method of this aspect comprises: determining, by a computing device, one or more candidate functional RNA sequences; for each of the one or more candidate functional RNA sequences: predicting, by the computing device, one or more folded structures that the functional RNA sequence forms over time; determining, by the computing device, one or more metrics for the functional RNA sequence based on the predicted one or more folded structures; and choosing, by the computing device, one or more of the one or more candidate functional RNA sequences to be provided for synthesis based on the scores.

[0125] In some embodiments, the step of determining the one or metrics for the functional RNA sequence based on the predicted one or more folded structures includes at least one of: determining an energy of a predicted folded structure for the functional RNA sequence; comparing an energy of a predicted folded structure for the functional RNA sequence to energies of other predicted folded structures for the functional RNA sequence; and determining a barrier energy for converting a predicted folded structure for the functional

[0126] RNA sequence to a target folded structure.

[0127] In some embodiments, predicting one or more folded structures that the functional RNA sequence forms over time includes conducting a constraint folding analysis. In some embodiments, conducting a constraint folding analysis includes: specifying a predetermined folded structure for a portion of the functional RNA sequence; and predicting an overall folded structure for the functional RNA sequence given the predetermined folded structure for the portion of the functional RNA sequence.

[0128] In some embodiments, choosing the first structure or the second structure includes: determining a barrier energy for converting from the first structure to the second structure; determining a time for adding the subsequent set of one or more additional nucleotides of the functional RNA sequence to the second incomplete portion of the functional RNA sequence; choosing the first structure in response to determining that the barrier energy is too high for the second incomplete portion of the functional RNA sequence to transition from the first structure to the second structure during the time for adding the subsequent set of one or more additional nucleotides; and choosing the second structure in response to determining that the barrier energy is not too high for the second incomplete portion of the functional RNA sequence to transition from the first structure to the second structure during the time for adding the subsequent set of one or more additional nucleotides. Referring now to Figure 3 which is a stylized depiction of the use of biosensors in vitro showing the biosensor in use. The biosensor may be placed in multiple wells. Engineered strains in each well produce target molecules that do not have a visible signature. Then the target molecule may bind the aptamer of the riboswitch and the functional RNA domain may fold causing the fluorescing moiety to fluoresce. The fluorescence of the biosensor is read, and the fluorescence measures the target molecule titer. The brighter the fluorescence, the more target molecules are bound to the riboswitch, thus, the more target molecules are being produced by the engineered strains. The fluorescence may also enable the strains producing the target molecule to be chosen for replication, thus enabling the further replication of the target molecule and increasing the yield of the target molecule by increasing the strains producing the molecule.

[0129] Referring to Figure 4 which is a stylized depiction of the biosensor in use in culture media. Variants of microbial strains produce a molecule of interest. That target molecule binds to the biosensor. The biosensor is switched on, causing the biosensor to fluoresce. The intensity of the fluorescence identifies the amount of the target molecule produced by each strain. Thus, strains that produce high amounts of the target molecule will appear brighter. Those strains that are fluorescing brighter and thus producing more target molecules may be selected for replication and the further production of the target molecules.

[0130] Referring to Figures 5A-5J which are some exemplary chemical compositions used as molecular targets. 5A is theophylline, 5B is flavin mononucleotide (FMN), 5C is p- aminophenylalanine (pAF), 5D is p-aminocinammic acid (pACA), 5E is tetracycline, 5F is guanosine monophosphate (GMP), 5G is tobramycin, 5H is tryptophan, 51 is lysozyme, and 5J is His-6. Additionally, and four conditional fluorophores have been shown to be compatible with and used with the biosensor, 5K is malachite green, 5E is DFHBI, 5M is thiazole orange 1, and 5N is Patent Blue V.

[0131] Referring to Figure 6 which are graphs of sensor responses for the ten molecular targets showing the identification of those molecules. Each of these molecules is theophylline, flavin mononucleotide (FMN), p-aminophenylalanine (pAF), p-aminocinammic acid (pACA), tetracycline, guanosine monophosphate (GMP), tobramycin, is tryptophan, lysozyme, and His-6 may be bound to the ligand. When bound to the ligand or riboswitch, each target shows increased fluorescence as compared to the target molecule without the ligand.

[0132] Referring to Figures 7 A and 7B which are graphs showing the selectivity of the nucleic acid biosensors. Theophylline and Caffeine differ from one another by a single functional group. The biosensor is selective enough to distinguish between the theophylline and the Caffeine. Figure 7 A is the graph for theophylline at various concentrations. All concentrations over 0 pM show florescence. The theophylline was tested at concentrations of 100 pM, ImM, and 10 mM. The caffeine was tested at the same concentrations. Caffeine only shows a background response or essentially no response to the biosensor. Thus, the biosensor is able to distinguish between molecules with very small differences.

[0133] Referring to Figure 8 which is a graph showing traces of Theophylline-responsive malachite green sensor (Theo_MG_4), showing sensor response to 4 concentrations of theophylline over the course of 60 minutes. The concentrations are 0 pM, 200 pM, 2 mM, and 20 mM. The biosensor is responsive even in non-ideal conditions. Yeast Broth includes many contaminants and various molecules that hinder or degrade the signal. The contaminants in yeast broth include proteins and enzymes and in general molecules that could interfere with the biosensor. However, the selectivity of the biosensor enables functioning even through conditions that could interfere with binding the correct targe molecules.

[0134] Referring now to Figure 9A which is a graph showing Kinetic traces of Tetracyclineresponsive CP Broccoli / DFHBI sensor. (WBI_0121). The graph shows sensor response to 4 concentrations of tetracycline over the course of 60 minutes. Increased concentration of tetracycline exhibit increased fluorescence.

[0135] Referring to Figure 9B which is a graph showing Titration of Tetracycline-responsive CP Broccoli / DFHBI sensor (WBI_0121). The graph shows normalized sensor response at 60 minutes to concentrations ranging from 200 nM to 200 uM; EC50 of this sensor is 82 + 5 uM. Increased concentration exhibits increased fluorescence.

[0136] Referring to Figure 10A which is a graph showing Kinetic traces of p-AF responsive malachite green sensor (WBI_0075), showing sensor response to 5 concentrations of p- AF over the course of 60 minutes. Increased concentration exhibits increased fluorescence.

[0137] Referring to Figure 10B which is graph showing titration of p-AF responsive malachite green sensor (WBI_0075), showing normalized sensor response at 60 minutes to concentrations ranging from 20 uM to 20 mM; EC50 of this sensor is 1.5 + 0.1 mM. Referring to Figure 11 which is a graph showing Kinetic traces of FMN-responsive malachite green sensor (FMN_MG_3), showing sensor response to 6 concentrations of FMN over the course of 60 minutes. Increased concentration exhibits increased fluorescence.

[0138] Referring to Figure 12A which is a graph showing Kinetic traces of lysozyme-responsive malachite green sensor (WBI_0093). The graph shows sensor response to 4 concentrations of lysozyme over the course of 60 minutes. The higher the concentration of lysozyme, the higher the intensity of fluorescence.

[0139] Referring to Figure 12B which is a graph showing titration of lysozyme-responsive malachite green sensor (WBI_0093). The graph shows normalized sensor response at 60 minutes to concentrations ranging from 100 uM to 10 mM; EC50 of this sensor is 970 + 170 nM.

[0140] Referring to Figure 13A which is a graph showing Kinetic traces of His-Tag-responsive malachite green sensor (WBI_0047). The graph shows sensor response to 6 concentrations of His- Mif over the course of 60 minutes. The higher the concentration of His-tag, the higher the intensity of fluorescence.

[0141] Referring to Figure 13B which is a graph showing Titration of His-Tag-responsive malachite green sensor (WBI_0047), showing normalized sensor response at 60 minutes to concentrations ranging from 100 nM to 4 uM; EC50 of this sensor is 3.6 + 0.8 uM.

[0142] Referring to Figures 14A and 14B which are graphs depicting tetracycline responsive malachite green sensors. Figure 14A is a graph depicting Kinetic traces of Tetracycline-responsive malachite green sensor (WBI_0125), showing sensor response to 5 concentrations of tetracycline over the course of 60 minutes. Figure 14B is a graph depicting Titration of Tetracycline-responsive malachite green sensor (WBI_0125), showing normalized sensor response at 60 minutes to concentrations ranging from 100 nM to 100 uM; EC50 of this sensor is 1.0 + 0.2 uM.

[0143] Referring to Figure 15 which is a graph showing Integrated brightness of beads vs bead area calculated from fluorescence microscopy images of theophylline- responsive malachite green sensors on agarose beads. The beads were incubated with four different concentrations of Theophylline, demonstrating a strong correlation between size and integrated brightness at each given concentration of theophylline. Quantification of concentration of a sample through any single bead is possible by mapping its size and fluorescence to this two-dimensional standard curve.

[0144] Referring to Figures 16A-16D which are photos of the surface of beads with various immobilized biosensors attached. Referring to Figure 16A in the absence of theophylline and FMN, beads with immobilized FMN-responsive sensors, and beads with immobilized theophyllineresponsive sensors, generate low basal levels of fluorescence. 16B. In the presence of FMN, but absence of theophylline, only the FMN sensor beads generate increased fluorescence. 16C. In the presence of theophylline, but absence of FMN, only the theophylline sensor beads generate increased fluorescence. 16D. In the presence of both theophylline and FMN, both sets of beads generate increased fluorescence.

[0145] Referring to Figure 17 which is a stylized depiction of sensor association with beads through covalently coupled ligands attached to beads. The sensors and switches can selectively associate to beads displaying the molecule that binds to the output module of the switches in the presence of the molecule that binds to the input module of the switches. The output molecule will not bind to the switch or biosensor unless the input small molecule is already bound to biosensor. This adds a level of control for binding to target molecules

[0146] Referring now to Figure 18 which is a graphic representation showing Covalently linked light up sensors (CLIPs). In cis-acting CLIPs (cCLIPs) functional sensors may have a light-up fluorophore directly coupled to the sensors’ nucleic acid strand. Separately, functional trans-acting CLIPs (tCLIPs) have been demonstrated in which the sensor nucleic acid can detect target small molecules after being annealed to a separate DNA oligo with a covalently linked conditional fluorophore.

[0147] To reduce the number of different components for the biosensors and to solve problems resulting from toxicity of malachite green in solutions, covalently linked light up sensors (CLIPs) were built, in which the malachite green output fluorophore is covalently linked to the sensors directly or to other nucleic acid elements that interact with the sensors.

[0148] Two ways to implement CLIPs: trans-acting and cis-acting (tCLIPs and cCLIPs)

[0149] In cis-acting CLIPs (cCLIPs), functional sensors have a light-up fluorophore directly coupled to the sensors’ nucleic acid strand. In an alternative embodiment, functional trans-acting CLIPs (tCLIPs) were developed, in which the sensor nucleic acid can detect target small molecules after being annealed to a separate DNA oligo with a covalently linked conditional fluorophore.

[0150] Referring to Figure 19 which is a graph depicting quantification of theophylline-MG switch binding to beads coated with malachite green analogs. The sensors and switches can selectively associate to beads displaying the molecule that binds to the output module of our switches in the presence of the molecule that binds to the input module of the switches. This graph demonstrates quantification of the relative amounts of Theo-MG sensor RNA bound to beads coated with tetramethylrosamine, a malachite green analog using PAGE. Binding of the sensor to the tetramethylrosamine-coated beads is higher in the presence of theophylline compared to in the absence of theophylline. Referring to Figure 20A which is a graph showing traces of phenylalanine-responsive DAP DNA light-up sensor. The graph shows sensor response to 4 concentrations of phenylalanine over the course of 60 minutes. Figure 20B is a graph showing Titration of phenylalanine -responsive DAP DNA light-up sensor, showing normalized sensor response at 60 minutes to concentrations ranging from 50 uM to 50 mM; EC50 of this sensor is 1.6 + 0.2 mM.

[0151] Referring to Figure 21 which is a graph of tetracycline -responsive fluorescent sensors with diverse output ligands. All three sensors show increased fluorescence in the presence of tetracycline and no reaction or possible background reaction without tetracycline. The Mango / TOl sensor exhibits increased response. However, the Mango / TOl also shows an increased level of response with no-tetracycline.

[0152] Referring to Figure 22A which is a graph depicting Kinetic traces of FMN riboswitchmalachite green sensor (WBI_0366). The graph shows sensor response to 4 concentrations of FMN over the course of 60 minutes. Increasing the concentration of FMN increase the fluorescent intensity. Figure 22B is a graph depicting Titration of FMN riboswitch-malachite green sensor (WBI_0366), showing normalized sensor response at 60 minutes to concentrations ranging from 1 uM to 1 mM; EC50 of this sensor is 3.8 + 1.2 uM.

[0153] Referring now to figure 23 which illustrates a non-limiting example embodiment of a method for designing functional nucleic acid biosensor molecules according to various aspects of the present disclosure.

[0154] Figure 23 is a flow chart for a method of designing nucleic acid biosensor molecules. From a start block, the method 2300 proceeds to block 2302, where a computing device determines one or more candidate \ functional nucleic acid sequences. The method 2800 then proceeds to a for- loop defined between a for-loop start block 2804 and a for-loop end block 2810, wherein each of the candidate functional nucleic acid sequences is processed to predict folded structures and determine metrics for the functional nucleic acid sequences based on the predicted structures.

[0155] From the for-loop start block 2304, the method 2300 proceeds to subroutine block 2306, where a subroutine is executed wherein the computing device predicts one or more folded structures that the functional nucleic acid sequence forms over time. Any suitable technique for predicting the folded structures may be used, including but not limited to the procedure 2400 described below.

[0156] At block 2308, the computing device determines one or more metrics for the functional nucleic acid sequence based on the predicted one or more folded structures. Any suitable metrics may be used, including but not limited to one or more of determining an energy of a predicted folded structure for the functional nucleic acid sequence, comparing an energy of a predicted folded structure for the functional nucleic acid sequence to energies of other predicted folded structures for the functional nucleic acid sequence, and determining a barrier energy for converting a predicted folded structure for the functional nucleic acid sequence to a target folded structure.

[0157] The method 2300 then proceeds to the for-loop end block 2310. If further candidate nucleic acid sequences remain to be processed, then the method 2300 returns to for-loop start block 2304 to process the next candidate functional nucleic acid sequence. Otherwise, the method 2300 proceeds to block 2312.

[0158] At block 2312, the computing device chooses one or more of the one or more candidate functional nucleic acid sequences to be provided for synthesis based on the metrics. The method 2800 then proceeds to an end block and terminates.

[0159] Figure 24 is a flow chart for predicting one or more folded structures that a nucleic acid sequence forms over time.

[0160] Figure 24 illustrates a non-limiting example embodiment of a procedure for predicting one or more folded structures that a functional nucleic acid sequence forms over time. In the procedure 2400, a constraint folding analysis is conducted. The procedure 2900 is a non-limiting example of a procedure suitable for use at subroutine block 2906 in figure 28.

[0161] From a start block, the procedure 2400 advances to block 2402, where a computing device specifies a predetermined folded structure for a portion of the functional nucleic acid sequence. At block 2404, the computing device predicts an overall folded structure for the functional nucleic acid sequence given the predetermined folded structure for the portion of the functional nucleic acid sequence. Further details of each of these actions are included above. The procedure 2400 then proceeds to an end block and terminates. Figure 24 illustrates a non-limiting example embodiment of a procedure for predicting one or more folded structures that a functional RNA sequence forms over time.

[0162] As indicated above, in some embodiments, the functional RNA of this aspect is a guide RNA (gRNA) molecule. In some embodiments, the functional RNA of this aspect is an RNA biosensor. In some specific embodiments, the functional RNA of this aspect is an RNA biosensor that comprises guide RNA (gRNA) molecule as an output domain. In other embodiments, the output domain does not comprise a gRNA but rather an alternative functional RNA domain.

[0163] In another aspect, the disclosure provides a non-transitory computer-readable medium having computer-executable instructions stored thereon that, in response to execution by one or more processors of a computing device, cause the computing device to perform actions of the computer-implemented method as described above.

[0164] In another aspect, the disclosure provides a computing device configured to perform actions of the computer-implemented method as described above.

[0165] In other aspects, the disclosure provides computer-implemented methods, non-transitory computer-readable media, and computing devices, as described above, but implemented for the design and implementation of RNA sensor constructs, also as described above. The above elements of the computer-implemented methods and related media and devices can be modified and implemented to the design of the disclosed RNA biosensor construct to ensure that the different domains (e.g., the sensor domain and the output domain) are independently functional but can operate in a binary "switch" manner when fused into a single construct with the sensor domain being transcribed before the output domain. Accordingly, for brevity the above elements are encompassed by the present aspects of the disclosure and are not repeated.

[0166] A first embodiment is a nucleic acid biosensor construct. The nucleic acid biosensor construct comprises a sensor domain that specifically binds to one or more molecules of interest; and an output domain configured to modulate a detectable output signal when folded into a first conformation. The output domain is at an equilibrium between first and second conformations. Binding the molecule of interest by the sensor domain alters the equilibrium between the first and second conformations of the output domain. The alteration of the equilibrium modulates the detectable output signal.

[0167] In a second embodiment, the sensor domain of the nucleic acid biosensor construct of embodiment 1 comprises a binding element having a binding element subsequence located between a stem sequence and a stem target sequence.

[0168] In a third embodiment, the sensor domain of the nucleic acid biosensor construct of embodiment 2 comprises an overhang sequence and / or a linker sequence.

[0169] In a fourth embodiment, the overhang sequence of embodiment 3 is 5’ of the binding element and / or the linker sequence is 3’ of the binding element.

[0170] In a fifth embodiment, the overhang sequence of embodiment 3 is 3’ of the binding element and / or the linker sequence is 5’ of the binding element.

[0171] In a sixth embodiment, the linker sequence of embodiment 3 is between the binding element and the output domain.

[0172] In a seventh embodiment, the output domain of embodiment 3 comprises a signaling element subsequence and a stem target sequence. In an eighth embodiment, the signaling element subsequence of embodiment 7 is 3’ of the stem target sequence.

[0173] In a ninth embodiment, the signaling element subsequence of embodiment 7 is 5’ of the stem target sequence.

[0174] In a tenth embodiment, the sensor domain of embodiment 7 comprises an overhang sequence and the output domain comprises an overhang target sequence located between a stem target sequence and a signaling element subsequence.

[0175] In an eleventh embodiment, the biosensor construct of embodiment 10 comprises an overhang sequence of the sensor domain that is the reverse complement of at least a portion of the overhang target sequence of the output domain. A stem sequence of the sensor domain that is the reverse complement of at least a portion of the stem target sequence of the sensor domain and is the reverse complement of at least a portion of the stem target sequence of the output domain. The linker sequence of the sensor domain is the reverse complement of at least a portion of linker target sequence of the sensor domain. These elements may be combined in any combination.

[0176] In a twelfth embodiment, the linker target sequence of embodiment 11 is the reverse complement to a portion of the binding element subsequence, optionally wherein the portion of the binding element subsequence is a discontinuous portion.

[0177] In a thirteenth embodiment, when the sensor domain of embodiment 11 is bound to the molecule of interest the stem sequence of the sensor domain is hybridized to the stem target sequence of the sensor domain thereby permitting folding of the output domain into one of the first or second conformations.

[0178] In a fourteenth embodiment, when the sensor domain of embodiment 11 is not bound to the molecule of interest the overhang sequence of the sensor domain is hybridized to the portion of the overhang target sequence of the output domain, the stem sequence of the sensor domain is hybridized to the portion of the stem target sequence of the output domain, and / or the linker target sequence of the sensor domain is hybridized to the portion of the linker sequence in the sensor domain, thereby permitting folding of the output domain into one of the first or second conformations.

[0179] In a fifteenth embodiment, at least two of the overhang sequence of the sensor domain, the stem sequence of the sensor domain, and the linker target sequence of the sensor domain of embodiment 11 form a continuous helix stem structure when hybridized to at least two of a portion of the overhang target sequence of the output domain, a portion of the stem target sequence of the output domain, and a portion the linker sequence of the sensor domain, respectively, thereby permitting the signaling element of the output domain to fold into one of the first or second conformations.

[0180] In a sixteenth embodiment, the overhang sequence of the biosensor construct of embodiment 3 is between 0 and about 15 nucleotides in length. The stem sequence of the biosensor construct of embodiment 3 is between 0 and about 15 nucleotides in length. The linker sequence of the biosensor construct of embodiment 3 is between 0 and about 15 nucleotides in length.

[0181] In a seventeenth embodiment, the biosensor construct of embodiment 3, further comprises an intermediate sequence disposed between the binding element and the linker sequence of the sensor domain, wherein the intermediate sequence has a length up to about 150 nucleotides in length.

[0182] In an eighteenth embodiment, the sensor domain of the biosensor construct of any of embodiments 1-17 is a synthetic aptamer sequence.

[0183] In a nineteenth embodiment, the sensor domain of the biosensor construct of any of embodiments 1-17 is a RNA sequence that exists in nature.

[0184] In a twentieth embodiment, the sensor domain of the biosensor construct of embodiment 19 is a natural RNA sequence without a known ligand, wherein the ligand is a chemical, a metabolite, a protein, a peptide, a small molecule, optionally a drug molecule or drug precursor molecule, and the like.

[0185] In a twenty-first embodiment, the biosensor construct of any of embodiments 1 to 20, further comprises a signaling molecule that produces a detectable output to indicate a specific conformation.

[0186] In a twenty-second embodiment, the output domain of the biosensor construct of embodiments 1-21 is configured to enhance the detectable output signal when folded into the first conformation.

[0187] In a twenty-third embodiment, the output domain of the biosensor construct of any of embodiments 1 to 21, is configured to enhance the detectable output signal when folded into the second conformation.

[0188] In a twenty-fourth embodiment, the output of the biosensor construct of embodiment 21 is fluorescence.

[0189] In a twenty-fifth embodiment, the signaling molecule of the biosensor construct of embodiment 21 is covalently linked to the biosensor construct. In a twenty-sixth embodiment, the signaling molecule of the biosensor construct of embodiment 21 is covalently linked to an oligonucleotide sequence annealed to a complementary sequence appended to the biosensor construct.

[0190] In a twenty-seventh embodiment, the signaling molecule of the biosensor construct of embodiment 21 is covalently linked to a solid support.

[0191] In a twenty-eighth embodiment, the signaling molecule of the biosensor construct of embodiment 24 displays enhanced fluorescence when the sensor domain is bound by a molecule of interest.

[0192] In a twenty-ninth embodiment, the signaling molecule of the biosensor construct of embodiment 24 displays enhanced fluorescence when the sensor domain is not bound by a molecule of interest.

[0193] In a thirtieth embodiment, the biosensor construct of any of embodiments 1-29 is a DNA biosensor construct or an RNA biosensor construct.

[0194] In a thirty-first embodiment, the biosensor construct of embodiment 30 is circular.

[0195] In a thirty-second embodiment, the biosensor construct of any of embodiments 1 to 29, further comprises additional domains appended to either end of the construct.

[0196] In a thirty-third embodiment, the additional domain of the biosensor construct of any of embodiments 1 to 29 is an aptamer appended to the 5’ end.

[0197] In a thirty-fourth embodiment, when the output domain of the biosensor construct of any of embodiments 1 to 33 is folded into one of the first or second conformations, the output domain is or comprises a functional ribozyme, a functional nuclease guide RNA (gRNA), a functional ribosome binding site, or functional RNA aptamer.

[0198] In a thirty-fifth embodiment, the molecule of interest of the biosensor construct of any of embodiments 1 to 34 is a chemical, a metabolite, a protein, a peptide, a small molecule, optionally a drug molecule or drug precursor molecule, and the like.

[0199] In a thirty-sixth embodiment, the biosensor of the biosensor construct of any of embodiments 1 to 35 is immobilized on a solid support.

[0200] In a thirty-seventh embodiment, the biosensor of the biosensor construct of embodiment 36 is attached to the solid support through a polynucleotide tail annealed to a capture oligonucleotide attached to the solid support.

[0201] In a thirty-eighth embodiment, the biosensor of the biosensor construct of embodiment 37 is attached to the solid support through a covalently-linked biotin. In a thirty-ninth embodiment, the solid support of the biosensor construct of any of embodiments 36 to 38 is magnetic or non-magnetic beads or the like.

[0202] A fortieth embodiment is a polynucleotide molecule comprising a sequence encoding the biosensor construct of any of embodiments 1 to 39.

[0203] A forty-first embodiment is a cell comprising the polynucleotide of embodiment 40.

[0204] In a forty-second embodiment, the cell of embodiment 41 is prokaryotic.

[0205] In a forty-third embodiment, the cell of embodiment 42 is eukaryotic.

[0206] In a forty-fourth embodiment, the cell of any of embodiments 41-43 is engineered or treated to modify expression or production of the molecule of interest.

[0207] A forty- fifth embodiment is a method of detecting a molecule of interest. The method comprises introducing the nucleic acid biosensor construct, as recited in any one of embodiment 1 to embodiment 34, into an environment that may contain the molecule of interest and measuring an output signal. A measured output signal indicates binding of the molecule of interest to the nucleic acid biosensor construct.

[0208] In a forty-sixth embodiment, the nucleic acid construct used in the method of detecting a molecule of interest of embodiment 45 is a DNA construct or an RNA construct.

[0209] In a forty-seventh embodiment, the environment of the method of any of embodiments 45 or 46 is a cell-free synthesis environment.

[0210] In a forty-eighth embodiment, the environment of the method of embodiments 45 or 46 comprises a cell lysate.

[0211] In a forty-ninth embodiment, the environment of the method of embodiments 45 or 46 is in a cell.

[0212] In a fiftieth embodiment, the cell of the method of embodiment 49 is engineered to modify production of a molecule of interest in the cell.

[0213] In a fifty-first embodiment, the method of any of embodiments 49 or 50, further comprising subjecting the cell to experimental conditions suspected to modify production of the molecule of interest in the cell.

[0214] In a fifty-second embodiment, the molecule of interest of the method of embodiments 49 to 51 is a compound contacted to the cell or a metabolite thereof.

[0215] A fifty-third embodiment is a computer-implemented method for designing a biosensor construct of any of embodiments 1 to 52. The method comprises determining, by a computing device, one or more candidate biosensor constructs according to any of embodiments 1 to 12. For each of the one or more candidate biosensor constructs: predicting, by the computing device, one or more folded structures that the biosensor construct will adopt; determining, by the computing device, one or more metrics for the biosensor constructs based on the predicted one or more folded structures; and choosing, by the computing device, one or more of the one or more candidate biosensor constructs to be provided for synthesis based on the metrics.

[0216] In a fifty-fourth embodiment, determining the one or more metrics for the biosensor constructs based on the predicted one or more folded structures of the computer-implemented method of embodiment 53, includes at least one of: determining an energy of a predicted folded structure for the biosensor construct; comparing an energy of a predicted folded structure for the biosensor construct to energies of other predicted folded structures for the biosensor construct; and determining a barrier energy for converting a predicted folded structure for the biosensor construct to a target folded structure.

[0217] In a fifty-fifth embodiment, predicting, with the computer-implemented method of embodiment 54, one or more folded structures that the biosensor construct will form includes conducting a constraint folding analysis.

[0218] Turing to Figures 7-22. RNA biosensors for 10 different targets have been engineered. These targets include p-amino cinnamic acid, p-amino phenylalanine, GMP, tetracycline, theophylline, flavin mononucleotide, His-tag, lysozyme, and tobramycin.

[0219] Example 1

[0220] Figures 7A and 7B are graphs showing the selectivity of the nucleic acid biosensors. Theophylline and Caffeine differ from one another by a single functional group. The biosensor is selective enough to distinguish between the theophylline and the Caffeine. Figure 7A is the graph for theophylline at various concentrations. All concentrations over 0 pM show florescence. The theophylline was tested at concentrations of 100 pM, ImM, and 10 mM. The caffeine was tested at the same concentrations. Caffeine only shows a background response or essentially no response to the biosensor.

[0221] Example 2

[0222] Figure 8 is a graph showing traces of Theophylline-responsive malachite green sensor (Theo_MG_4), showing sensor response to 4 concentrations of theophylline over the course of 60 minutes. The concentrations are 0 pM, 200 pM, 2 mM, and 20 mM. The biosensor is responsive even in non-ideal conditions. Yeast Broth includes many contaminants and various molecules that hinder or degrade the signal.

[0223] Example 3 Figure 9A is a graph showing Kinetic traces of Tetracycline -responsive CP Broccoli / DFHBI sensor (WBI_0121), showing sensor response to 5 concentrations of tetracycline over the course of 60 minutes.

[0224] Figure 9B is a graph showing Titration of Tetracycline -responsive CP Broccoli / DFHBI sensor (WBI_0121), showing normalized sensor response at 60 minutes to concentrations ranging from 200 nM to 200 uM; EC50 of this sensor is 82 + 5 uM.

[0225] Example 4

[0226] Figure 10A is a graph showing Kinetic traces of p-AF responsive malachite green sensor (WBI_0075), showing sensor response to 5 concentrations of p-AF over the course of 60 minutes.

[0227] Figure 10B is graph showing titration of p-AF responsive malachite green sensor (WBI_0075), showing normalized sensor response at 60 minutes to concentrations ranging from 20 uM to 20 mM; EC50 of this sensor is 1.5 + 0.1 mM.

[0228] Example 5

[0229] Figure 11 is a graph showing Kinetic traces of FMN-responsive malachite green sensor (FMN_MG_3), showing sensor response to 6 concentrations of FMN over the course of 60 minutes.

[0230] Example 6

[0231] Figure 12A is a graph showing Kinetic traces of lysozyme-responsive malachite green sensor (WBI_0093), showing sensor response to 4 concentrations of lysozyme over the course of 60 minutes.

[0232] Figure 12B is a graph showing titration of lysozyme-responsive malachite green sensor (WBI_0093), showing normalized sensor response at 60 minutes to concentrations ranging from 2 uM to 10 mM; EC50 of this sensor is 970 + 170 nM.

[0233] Example 7

[0234] Figure 13A is a graph showing Kinetic traces of His -Tag-responsive malachite green sensor (WBI_0047), showing sensor response to 6 concentrations of His-Mif over the course of 60 minutes.

[0235] Figure 13B is a graph showing Titration of His-Tag-responsive malachite green sensor (WBI_0047), showing normalized sensor response at 60 minutes to concentrations ranging from 100 nM to 4 uM; EC50 of this sensor is 3.6 + 0.8 uM.

[0236] Example 8 Figures 14A and 14B are graphs depicting tetracycline responsive malachite green sensors. Figure 14A is a graph depicting Kinetic traces of Tetracycline -responsive malachite green sensor (WBI_0125), showing sensor response to 5 concentrations of tetracycline over the course of 60 minutes. Figure 14B is a graph depicting Titration of Tetracycline -responsive malachite green sensor (WBI_0125), showing normalized sensor response at 60 minutes to concentrations ranging from 100 nM to 100 uM; EC50 of this sensor is 1.0 + 0.2 uM.

[0237] Example 9

[0238] Figure 15 is a graph showing Integrated brightness of beads vs bead area calculated from fluorescence microscopy images of theophylline- responsive malachite green sensors on agarose beads, where the beads have been incubated with four different concentrations of Theophylline, demonstrating a strong correlation between size and integrated brightness at each given concentration of theophylline. Allowing quantification of concentration of a sample through any single bead by mapping its size and fluorescence to this two-dimensional standard curve.

[0239] Example 10

[0240] Figure 16A. In the absence of theophylline and FMN, beads with immobilized FMN- responsive sensors, and beads with immobilized theophylline-responsive sensors, generate low basal levels of fluorescence. 16B. In the presence of FMN, but absence of theophylline, only the FMN sensor beads generate increased fluorescence. 16C. In the presence of theophylline, but absence of FMN, only the theophylline sensor beads generate increased fluorescence. 16D. In the presence of both theophylline and FMN, both sets of beads generate increased fluorescence. Integrated brightness of beads vs bead area calculated from fluorescence microscopy images of theophylline- responsive malachite green sensors on agarose beads, where the beads have been incubated with four different concentrations of Theophylline, demonstrating a strong correlation between size and integrated brightness at each given concentration of theophylline. This will allow us to quantify concentration of a sample through any single bead by mapping its size and fluorescence to this two-dimensional standard curve. Fluorescence microscopy images of theophylline and FMN- responsive malachite green sensors on beads, incubated in the combinatorial space of the presence and absence of theophylline and FMN, demonstrating spatial multiplexing where sensor beads only respond to the addition of the small molecule that they respond to.

[0241] Example 11

[0242] Figure 19 is a graph depicting quantification of theophylline-MG switch binding to beads coated with malachite green analogs. The sensors and switches can selectively associate to beads displaying the molecule that binds to the output module of our switches in the presence of the molecule that binds to the input module of the switches. This graph demonstrates quantification of the relative amounts of Theo-MG sensor RNA bound to beads coated with tetramethylrosamine, a malachite green analog using PAGE. Binding of the sensor to the tetramethylrosamine-coated beads is higher in the presence of theophylline compared to in the absence of theophylline.

[0243] Example 12

[0244] Figure 20A is a graph showing traces of phenylalanine -responsive DAP DNA light-up sensor, showing sensor response to 4 concentrations of phenylalanine over the course of 60 minutes.

[0245] Figure 20B is a graph showing Titration of phenylalanine -responsive DAP DNA light-up sensor, showing normalized sensor response at 60 minutes to concentrations ranging from 50 uM to 50 mM; EC50 of this sensor is 1.6 + 0.2 mM.

[0246] Example 13

[0247] Figure 21 is a depiction of tetracycline -responsive fluorescent sensors with diverse output ligands. All three sensors show increased fluorescence in the presence of tetracycline and no reaction or possible background reaction without tetracycline.

[0248] Example 14

[0249] Figure 22A is a graph depicting Kinetic traces of FMN riboswitch-malachite green sensor (WBI_0366), showing sensor response to 4 concentrations of FMN over the course of 60 minutes.

[0250] Figure 22B is a graph depicting Titration of FMN riboswitch-malachite green sensor (WBI_0366), showing normalized sensor response at 60 minutes to concentrations ranging from 1 uM to 1 mM; EC50 of this sensor is 3.8 + 1.2 uM.

[0251] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMSThe embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A nucleic acid biosensor construct, comprising: a sensor domain that specifically binds to one or more molecules of interest; and an output domain configured to modulate a detectable output signal when folded into a first conformation; wherein the output domain is at an equilibrium between first and second conformations; wherein binding the molecule of interest by the sensor domain alters the equilibrium between the first and second conformations of the output domain; and wherein the alteration of the equilibrium modulates the detectable output signal.

2. The nucleic acid biosensor of claim 1 , wherein the sensor domain comprises a binding element having a binding element subsequence located between a stem sequence and a stem target sequence.

3. The nucleic acid biosensor of claim 2, wherein the sensor domain comprises an overhang sequence and / or a linker sequence.

4. The nucleic acid biosensor of claim 3, wherein the overhang sequence is 5’ of the binding element and / or the linker sequence is 3’ of the binding element.

5. The nucleic acid biosensor of claim 3, wherein the overhang sequence is 3’ of the binding element and / or the linker sequence is 5’ of the binding element.

6. The nucleic acid biosensor of claim 3, wherein the linker sequence is between the binding element and the output domain.

7. The nucleic acid biosensor of claim 3, wherein the output domain comprises a signaling element subsequence and a stem target sequence.

8. The nucleic acid biosensor of claim 7, wherein the signaling element subsequence is 3’ of the stem target sequence.

9. The nucleic acid biosensor of claim 7, wherein the signaling element subsequence is 5’ of the stem target sequence.

10. The nucleic acid biosensor of claim 7, wherein the sensor domain comprises an overhang sequence and the output domain comprises an overhang target sequence located between a stem target sequence and a signaling element subsequence.

11. The biosensor construct of claim 10, wherein: an overhang sequence of the sensor domain is the reverse complement of at least a portion of the overhang target sequence of the output domain; a stem sequence of the sensor domain is the reverse complement of at least a portion of the stem target sequence of the sensor domain and is the reverse complement of at least a portion of the stem target sequence of the output domain; and / or the linker sequence of the sensor domain is the reverse complement of at least a portion of linker target sequence of the sensor domain; in any combination.

12. The biosensor construct of claim 11, wherein the linker target sequence is the reverse complement to a portion of the binding element subsequence, optionally wherein the portion of the binding element subsequence is a discontinuous portion.

13. The biosensor construct of claim 11, wherein when the sensor domain is bound to the molecule of interest the stem sequence of the sensor domain is hybridized to the stem target sequence of the sensor domain thereby permitting folding of the output domain into one of the first or second conformations.

14. The biosensor construct of claim 11, wherein when the sensor domain is not bound to the molecule of interest the overhang sequence of the sensor domain is hybridized to the portion of the overhang target sequence of the output domain, the stem sequence of the sensor domain is hybridized to the portion of the stem target sequence of the output domain, and / or the linker target sequence of the sensor domain is hybridized to the portion of the linker sequence in the sensordomain, thereby permitting folding of the output domain into one of the first or second conformations.

15. The biosensor construct of claim 11, wherein at least two of the overhang sequence of the sensor domain, the stem sequence of the sensor domain, and the linker target sequence of the sensor domain form a continuous helix stem structure when hybridized to at least two of a portion of the overhang target sequence of the output domain, a portion of the stem target sequence of the output domain, and a portion the linker sequence of the sensor domain, respectively, thereby permitting the signaling element of the output domain to fold into one of the first or second conformations.

16. The biosensor construct of claim 3, wherein: the overhang sequence is between 0 and about 15 nucleotides in length the stem sequence is between 0 and about 15 nucleotides in length; and / or the linker sequence is between 0 and about 15 nucleotides in length.

17. The biosensor construct of claim 3, further comprising an intermediate sequence disposed between the binding element and the linker sequence of the sensor domain, wherein the intermediate sequence has a length up to about 150 nucleotides in length.

18. The biosensor construct of any of claims 1 to 17, wherein the sensor domain is a synthetic aptamer sequence.

19. The biosensor construct of any of claims 1 to 17, wherein the sensor domain is a RNA sequence that exists in nature.

20. The biosensor construct of claim 19, wherein the sensor domain is a natural RNA sequence without a known ligand, wherein the ligand is a chemical, a metabolite, a protein, a peptide, a small molecule, optionally a drug molecule or drug precursor molecule, and the like.

21. The biosensor construct of any of claims 1 to 20, further comprising a signaling molecule that produces a detectable output to indicate a specific conformation.

22. The biosensor construct of any of claims 1 to 21, wherein the output domain is configured to enhance the detectable output signal when folded into the first conformation.

23. The biosensor construct of any of claims 1 to 21, wherein the output domain is configured to enhance the detectable output signal when folded into the second conformation.

24. The biosensor construct of claim 21, wherein the output is fluorescence.

25. The biosensor construct of claim 21, wherein the signaling molecule is covalently linked to the biosensor construct.

26. The biosensor construct of claim 21, wherein the signaling molecule is covalently linked to an oligonucleotide sequence annealed to a complementary sequence appended to the biosensor construct.

27. The biosensor construct of claim 21, wherein the signaling molecule is covalently linked to a solid support.

28. The biosensor construct of claim 24, wherein the signaling molecule displays enhanced fluorescence when the sensor domain is bound by a molecule of interest.

29. The biosensor construct of claim 24, wherein the signaling molecule displays enhanced fluorescence when the sensor domain is not bound by a molecule of interest.

30. The biosensor construct of any of claims 1 to 29, wherein the biosensor construct is a DNA biosensor construct or an RNA biosensor construct.

31. The biosensor construct of claim 30, wherein the biosensor construct is circular.

32. The biosensor construct of any of claims 1 to 29, further comprising additional domains appended to either end of the construct.

33. The biosensor construct of any of claims 1 to 29, wherein the additional domain is an aptamer appended to the 5’ end.

34. The biosensor construct of any of claims 1 to 33, when the output domain is folded into one of the first or second conformations, the output domain is or comprises a functional ribozyme, a functional nuclease guide RNA (gRNA), a functional ribosome binding site, or functional RNA aptamer.

35. The biosensor construct of any of claims 1 to 34, wherein the molecule of interest is a chemical, a metabolite, a protein, a peptide, a small molecule, optionally a drug molecule or drug precursor molecule, and the like.

36. The biosensor construct of any of claims 1 to 35, wherein the biosensor is immobilized on a solid support.

37. The biosensor construct of claim 36, wherein the biosensor is attached to the solid support through a polynucleotide tail annealed to a capture oligonucleotide attached to the solid support.

38. The biosensor construct of claim 37, wherein the biosensor is attached to the solid support through a covalently-linked biotin.

39. The biosensor construct of any of claims 36 to 38, wherein the solid support is magnetic or non-magnetic beads or the like.

40. A polynucleotide molecule comprising a sequence encoding the biosensor construct of any of claims 1 to 39.

41. A cell comprising the polynucleotide of claim 40.

42. The cell of claim 41, wherein the cell is prokaryotic.

43. The cell of claim 42, wherein the cell is eukaryotic.

44. The cell of one of claims 41 to 43, wherein the cell is engineered or treated to modify expression or production of the molecule of interest.

45. A method of detecting a molecule of interest, comprising: introducing the nucleic acid biosensor construct as recited in any one of claim 1 to claim 34 into an environment that may contain the molecule of interest, and measuring an output signal, wherein a measured output signal indicates binding of the molecule of interest to the nucleic acid biosensor construct.

46. The method of detecting a molecule of interest of claim 45, wherein the nucleic acid construct is a DNA construct or an RNA construct.

47. The method of any of claims 45 or 46, wherein the environment is a cell-free synthesis environment.

48. The method of claim 45 or 46, wherein the environment comprises a cell lysate.

49. The method of claim 45 or 46, wherein the environment is in a cell.

50. The method of claim 49, wherein the cell is engineered to modify production of a molecule of interest in the cell.

51. The method of any of claims 49 or 50, further comprising subjecting the cell to experimental conditions suspected to modify production of the molecule of interest in the cell.

52. The method of any of claims 49 to 51 , wherein the molecule of interest is a compound contacted to the cell or a metabolite thereof.

53. A computer-implemented method for designing a biosensor construct of any of claims 1 to 52, the method comprising: determining, by a computing device, one or more candidate biosensor constructs according to any of claims 1 to 12; for each of the one or more candidate biosensor constructs:predicting, by the computing device, one or more folded structures that the biosensor construct will adopt; and determining, by the computing device, one or more metrics for the biosensor constructs based on the predicted one or more folded structures; and choosing, by the computing device, one or more of the one or more candidate biosensor constructs to be provided for synthesis based on the metrics.

54. The computer-implemented method of claim 53, wherein determining the one or more metrics for the biosensor constructs based on the predicted one or more folded structures includes at least one of: determining an energy of a predicted folded structure for the biosensor construct; comparing an energy of a predicted folded structure for the biosensor construct to energies of other predicted folded structures for the biosensor construct; and determining a barrier energy for converting a predicted folded structure for the biosensor construct to a target folded structure.

55. The computer-implemented method of claim 54, wherein predicting one or more folded structures that the biosensor construct will form includes conducting a constraint folding analysis.

Images