Genetically encoded light-up sensors

Abstract

Disclosed herein is a dual aptamer system for detection of a target analyte, the system comprising: a signal input aptamer, wherein said signal input aptamer can change conformation upon interaction with a target analyte; and a signal output aptamer, wherein said signal output aptamer changes conformation upon binding of a target analyte to the signal input aptamer, wherein said change of conformation of the signal output aptamer allows for output of a detectable signal; wherein both the signal input aptamer and the signal output aptamer comprise deoxyribonucleic acid (DNA), and further wherein both the signal input aptamer and the signal output aptamer are linked to each other. Also disclosed are methods of making this dual aptamer system and methods of using this dual aptamer system.

Description

GENETICALLY ENCODED LIGHT-UP SENSORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 667,359, filed July 3, 2024, which is incorporated by reference herein in its entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. R35 GM141931 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The sequence listing submitted on July 3, 2025, as an .XML file entitled “10046- 624W01_ST26.xml” created on June 30, 2025, and having a file size of 56,809 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUND

[0004] Genetically encoded sensors that link fluorescent proteins

[0001] or fluorogenic RNAs [2], [3], [4], [5] to proteins [6], [7] or RNA molecules [8], [9],

[0010] ,

[0011] , respectively, have significantly advanced understanding of proteins and RNAs living cells. Despite the progress, their use in detecting small molecules like metabolites in cells is restricted, as directly linking the fluorescent proteins or fluorogenic RNAs to metabolites is impractical. Instead, a metabolite-specific component that can either regulate the synthesis of fluorescence proteins

[0023] ,

[0024] ,

[0025] or fluorogenic RNA

[0026] , or induce conformational changes that influence fluorescent signals of fluorescence proteins

[0012] ,

[0013] ,

[0014] ,

[0015] ,

[0016] ,

[0017] ,

[0018] ,

[0019] ,

[0020] ,

[0021] ,

[0022] or fluorogenic RNA

[0027] ,

[0028] ,

[0029] ,

[0030] is needed. However, most of these designs are customized to specific targets and thus lack generalizability. Since numerous metabolites are present in cells, it is desirable to develop a method that can be generally applied to many metabolites.SUMMARY

[0005] In some aspects, disclosed herein is a dual aptamer system for the detection of a target analyte, the system comprising: a signal input aptamer, wherein said signal input aptamer can change conformation upon interaction with a target analyte; and a signal output aptamer,wherein said signal output aptamer changes conformation upon binding of a target analyte to the signal input aptamer, wherein said change of conformation of the signal output aptamer allows for output of a detectable signal; wherein both the signal input aptamer and the signal output aptamer comprise deoxyribonucleic acid (DNA), and further wherein both the signal input aptamer and the signal output aptamer are linked to each other. Also disclosed are vectors and cells comprising the system.

[0006] Also disclosed is a method of screening to determine that a potential signal input aptamer can be used to detect a target analyte, the method comprises: providing a potential signal input aptamer, to determine if said signal input aptamer can change conformation upon interaction with a target analyte; and providing a signal output aptamer, wherein said signal output aptamer changes conformation upon binding of a target analyte to the signal input aptamer, wherein said change of conformation of the signal output aptamer allows for output of a detectable signal; determining whether the potential signal input aptamer can detect a target analyte by determining if a detectable signal is detected; wherein both the potential signal input aptamer and the signal output aptamer comprise deoxyribonucleic acid (DNA), and further wherein both the potential signal input aptamer and the signal output aptamer are linked to each other.

[0007] Also disclosed is a method of screening to determine that a potential signal output aptamer can be used to detect a target analyte, the method comprising: providing a signal input aptamer, wherein said signal input aptamer can change conformation upon interaction with a target analyte; and providing a potential signal output aptamer, and determining whether said signal output aptamer changes conformation upon binding of a target analyte to the signal input aptamer, wherein said change of conformation of the signal output aptamer allows for output of a detectable signal; determining whether the potential signal output aptamer can detect a target analyte by determining if a detectable signal is detected; wherein both the potential signal input aptamer and the signal output aptamer comprise deoxyribonucleic acid (DNA), and further wherein both the potential signal input aptamer and the signal output aptamer are linked to each other.

[0008] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0009] FIGURES 1A-1B depict a comparison of Lettuce aptamer and DIRFA in PBS buffer. FIG. 1A shows that the Lettuce aptamer displayed a significantly higher fluorescence signal compared to the point- mutated control in Tris and HEPES buffers; however, this difference was not observed in the PBS buffer. FIG. IB shows that DIRFA showed enhanced fluorescence intensity in PBS buffer when paired with DIR.

[0010] FIGURES 2A-2F depict the design and characterization of a DIRFA-based split Aptamer (DASA) sensor. FIG. 2A is a scheme that shows the designs of the truncational study for the dimethylindole red (DIR) specific Fluorogenic DNA Aptamer (SEQ ID NO: 5, DIRFA). DIRFA- 1 to DIRFA-5 correspond to 1- to 5- base-pair truncation from the bottom of stem DI, respectively. Compared to the canonical DIRFA, the Mutated DIRFA lacks a T-G base pair (highlighted in the box) but retains the stem D 1. FIG. 2B shows the spectrum of the truncated DIRFA showed a lower fluorescence intensity with a shorter stem length. FIG. 2C shows the fluorescence intensity of truncated DIRFAs (SEQ ID NO: 24) measured at 650 nm. FIG. 2D shows the scheme of the DASA sensor. A split ATP aptamer was used to replace the stem D 1. Without ATP (left), the stem structure is disrupted, and thus cannot interact with DIR. In the presence of ATP (right), the split ATP aptamer restores the stem structure with the structure switching upon binding to ATP. With the restored stem structure, DASA interacts with DIR and light up. FIG. 2E shows that the fluorometer recorded the spectrum of the DASA sensor with and without ATP incubations and its mutated controls with 5 mM ATP incubations. FIG. 2F shows that in the presence of 5 mM ATP, the DASA sensor showed a 244% increase in fluorescence intensity at 650 nm when compared to the group without ATP. However, point mutations in either split ATP aptamer, DIRFA, or both aptamers resulted in a constraint on the fluorescence increase. All the experiments were performed at room temperature upon excitation at 618 nm. ***, p < 0.001. Comparison was performed between DASA and groups carrying different mutations in DASA, or without ATP addition.

[0011] FIGURES 3A-3H depict the effect of stem DI length variations on connecting split ATP aptamer core and DIRFA. FIGS. 3A-3D show schemes of the design of the DASA sensors with different lengths of the DI stem region between the cores of the ATP aptamer and the DIRFA. FIG. 3A shows SEQ ID NO: 23. FIG. 3B shows SEQ ID NO: 24. FIG. 3C shows SEQ ID NO: 25. FIG. 3D shows SEQ ID NO: 26. FIGS. 3E-3H show fluorescence spectra obtained with fluorometer. Comparisons of the fluorescence signal between groups with and without 5 mM ATP were presented and correlated with the designs shown in FIGS. 3A-3D,respectively. The percentage showed increases in fluorescence intensity upon adding 5 mM ATP. The detection was performed at 22°C.

[0012] FIGURES 4A-4F depict the linear range and selectivity analysis of DASA-2, DASA-3, and DASA-4. FIGS. 4A-4C show the detection range of DASA-2, DASA-3, and DASA-4. FIGS. 4D-4F depict the selectivity of the DASA-2, DASA-3, and DASA-4 over different rNTPs. Fluorescence intensities were normalized using the equation (F-Fo) / Fo where F represents the fluorescence intensity at 650 nm for each group, and Fo represents the fluorescence intensity at 650 nm in the control group without the addition of any analogs. All the experiments were performed at 22 °C.

[0013] FIGURES 5A-5J depict a temperature comparison of DASA sensors at 22°C and 37 °C. FIGS. 5A-5B show a comparison of fluorescence intensities between DIRFA and its mutated control sequence in the presence of DIR. FIGS. 5C-5J show a comparison of fluorescence signals before and after the addition of 5 mM ATP at 22°C and 37°C, with percentages indicating fluorescence intensity increases upon ATP addition.

[0014] FIGURES 6A-6G depict the design and characterization of dimeric DASA (dDAS A) sensors. FIG. 6A is a scheme that shows the design for the dimerization of the DASA sensor (SEQ ID NO: 36). FIG. 6B shows the spectral analysis of dDASA 2-C and its mutant, which contains mutations in the binding site of the ATP aptamer, in the presence and absence of 5 mM ATP. The experiment was performed in the PBS buffer at room temperature (22°C). FIG. 6C shows the spectral analysis of dDASA 2-C and its mutant in PBS buffer at 37°C in the presence and absence of 5 mM ATP. FIG. 6D shows the detection range of dDASA 2-C under RT. FIG. 6E shows the detection range of dDASA 2-C under 37°C. FIG. 6F shows the selectivity of dDASA 2-C over different rNTPs at RT. FIG. 6G shows the selectivity of dDASA 2-C over different rNTPs under 37°C. The fluorescence intensity was normalized to the group without the addition of rNTPs with the equation (F-Fo) / Fo, where F represents the fluorescence intensity at 650 nm in each group, and Fo represents the fluorescence intensity at 650 nm without the addition of any of these rNTPs.

[0015] FIGURES 7A-7H a temperature comparison of DASA Dimers at 22°C and 37°C. The percentages showed increases in fluorescence intensities when adding 5 mM ATP into the sensing systems under 22°C or 37 °C. All the dimers used an 8 nt linker to link two monomers.

[0016] FIGURES 8A-8H depict a comparison of dDASA-2 dimers with variable linker lengths at different temperatures. In the dDASA-2 variants, different linker lengths were used to connect the two monomers: FIGS. 8A-8B show dDASA 2- A utilizing a 4-nt linker, FIGS. 8C-8D show dDASA 2-B utilizing an 8-nt linker with a different sequence than dDASA 2,FIGS. 8E-8F show dDASA 2-C utilizing a 10-nt linker, and FIGS. 8G-8H show dDASA 2-D utilizing a 13 -nt linker. The percentages reflect the increases in fluorescence intensity for each variant.

[0017] FIGURES 9A-9D depict monitoring ATP levels in live HeLa cells using dDASA. FIG. 9A shows cellular imaging of dDASA 2-C under varying ATP conditions demonstrated a decrease in fluorescence intensity following treatment with 10 pM oligomycin (ATP inhibition) and an increase in fluorescence intensity with 5 mM CaCh (ATP stimulation). In contrast, point mutations in the ATP binding pocket of dDASA 2-C resulted in a mutated variant with reduced fluorescence and an inability to distinguish between oligomycin and CaCh treatments. Scale bar = 20 pm. FIGS. 9B-9D show statistical analysis quantified the average fluorescence intensity in cells, with Student’s t-tests comparing the groups (*, p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; ns, p > 0.05). The results indicated that dDASA 2-C effectively detected reduced fluorescence under oligomycin treatment and increased fluorescence with CaCh treatment in FIG. 9B. In normal cells (DMSO control), the dDASA 2-C showed a lower baseline fluorescence signal in FIG. 9C. Notably, Mutated dDASA 2-C displayed no significant fluorescence changes after oligomycin or CaCh treatments, indicating its inability to discriminate between different ATP levels in FIG. 9D.

[0018] FIGURES 10A-10D depict a responsiveness evaluation of DASA sensor dimer 2- C. Fluorescence intensity at 650 nm was measured using a fluorometer immediately before and after the addition of ATP (FIGS. 10A-10B) or DIR (FIGS. 10C-10D) to the dimer 2-C sensing system at 22°C or 37°C, respectively. Measurements were repeated over several minutes. Fluorescence intensity reached a stable plateau within 7 seconds — the minimum interval achievable between compound addition and recording — and remained constant throughout the observation period. Error bars represent data from three independent experiments and measurements.

[0019] FIGURES 11A-11C depict the assessment of cell membrane permeability of DIR in HeLa cells using DIRFA. FIG. 11A shows that the DIRFA was delivered into HeLa cells without DIR. After the transfection and wash steps, DIR was then added to the cell culture media. The fluorescence of cells was recorded with a spinning disk confocal microscope at each time point during the DIR incubation. FIG. 11B shows images of the cells that were cotransfected with the same amount of DIRFA and DIR as used in FIG. 11A. The scale bars represent 50 pm. FIG. 11C shows the quantification of the average fluorescence intensity in cells. The blue line indicates the mean fluorescence intensity of the cells when DIR was delivered together with the aptamer (right- most bar). The stars represent the results fromstudent's t tests to compare the fluorescence intensity between different time points and the group without DIR treatments, ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0020] FIGURES 12A-12F depict real-time ATP tracking in HeLa cells using the dDASA sensor. FIG. 12A shows cellular images which demonstrated changes in ATP levels in response to varying glucose concentrations in the cell culture media. For the “-glucose” condition, cells were incubated in glucose-free DMEM for 1 h. In the “+glucose” condition, cells were cultured in DMEM containing 25 mM glucose for 1 h. Increased fluorescence signals were observed when treating the cells with glucose, while decreased fluorescence signals were observed when incubating the cells with glucose-free media. FIG. 12B shows cellular images which revealed changes in ATP levels in response to treatments with 10 pM oligomycin and 5 mM CaCh. Fluorescence images were collected using a 640 / 15 nm excitation light and a 680 / 25 nm emission light immediately before the cells were transitioned to a new condition. Scale bars represent 20 pm. FIGS. 12C-12D show a statistical quantification of the average fluorescence intensity in cells for FIGS. 12A-12B, respectively. The ATP levels were tracked in the same cell population.

[0021] FIGURES 13A-13D depict evaluating and optimizing the aptamers for sensing in bacteria cells. FIG. 13A shows ATP detection in bacterial cells using a transformed dDASA sensor. Following transformation, DH10B cells were incubated with DIR for 30 minutes and analyzed using flow cytometry. Only a minimal increase in signal was observed upon incubation with 1 mM ATP. FIG. 13B shows the sequence and secondary structure prediction for the dimer of DIR aptamer (dDIRFA, SEQ ID NO: 39). FIG. 13C shows a fluorometer scan of a spectrum from 640 nm to 750 nm with the excitation light at 618 nm to compare the fluorescence intensity for the dDIRFA and the original monomer, DIRFA, mutated DIRFA, and groups in the absence of the aptamers or DIR. FIG. 13D shows that the fluorometer records the spectrum of DIRFA in the presence or absence of single- stranded DNA binding proteins (SSB and RecA). The presence of the single- stranded DNA binding proteins did not interfere with the interaction between DIRFA and DIR.

[0022] FIGURES 14A-14F depict genetically encoded Anorogenic DNA aptamer (GEFDA) sensor for monitoring ATP levels in E. coli. FIG. 14A is a scheme showing the major elements of the plasmid that generate GEFDA sensor in bacterial cells. The reverse transcriptase (RT) will be synthesized and bind to the tRNA binding site of the mRNA to initiate the reverse transcription of the GEFDA. With the help of protein and the tandem repeat structure of the sensor, the plasmid has a higher yield for producing GEFDA in cells. FIG. 14B shows a dot plot displaying the fluorescence intensity and size of individual DH10Bcells as detected by flow cytometry analysis. More than 40% of the bacterial cells with the plasmid that codes dDIRFA showed higher fluorescence intensity than the cells without the plasmid. FIG. 14C is a histogram from flow cytometry which shows the increase of fluorescence intensity in a population of the bacteria that expressed dDIRFA. FIG. 14D is a histogram from flow cytometry which reflects that the genetically encoded dDASA monitored ATP levels in bacterial cells. The DH10B cells were transformed with the plasmids that express dDASA, and then incubated with IPTG and DIR. After the transfection and induction, the cells were divided into three groups, which were maintained in the cell culture media, or treated with 50 pM ATP or 10 pM Venturicidin A (VentA), respectively. Comparison was also made with the cells that were not induced by IPTG (No IPTG) or not incubated with DIR (No DIR) after the transformation. FIG. 14E is a histogram from flow cytometry which reflects that the plasmid that expresses the Mutated dDASA was not able to monitor ATP levels. FIG. 14F shows statistical analysis for experiments in FIGS. 14D-14E. The cells that showed a fluorescence intensity higher than 103 were considered fluorescent cells. The data were obtained from 7 independent experiments, ns, p > 0.05; *, p < 0.05; **, p < 0.01 ; ***, p < 0.001.

[0023] FIGURES 15A-15B depict the genetically encoded light-up aptamer for monitoring ATP levels in HeLa cells. FIG. ISA shows that Hela cells were co-transfected with the plasmids that express dDIRFA and eGFP respectively. The eGFP plasmid was used as a control for transfection efficiency. No differences in transfection efficiency were observed between the group with or without adding DIR. No DIR means no DIR was added after the transfection. No plasmids meant incubating the cells with DIR, but no plasmids were transfected. Scale bar: 50 pm. FIG. 15B shows statistical analysis for pictures obtained in experiment FIG. 15A. The fluorescence intensity from 10 cells in each picture and 5 pictures per treatment were calculated with image J. Student t-tests were performed. ***, p < 0.001.

[0024] FIGURE 16 depicts a plasmid map for the gblock ordered from Thermo Fisher Scientific.

[0025] FIGURES 17A-17H depict split DIR-ATP aptamer pairs. The ATP aptamer was inserted into the stem- loop on the left or right side of the DIRFA. In the presence of ATP, only a small fluorescence enhancement was observed. FIG. 17A shows a representation of the insertion of the ATP -binding aptamer (SEQ ID NO: 40, left) into the left loop of the DIRFA scaffold (SEQ ID NO: 5, right). FIG. 17B shows fluorescence emission spectra of Design A with and without 5 mM ATP. Excitation was performed at 618 nm. FIG. 17C shows the ATP- binding aptamer was inserted into the left loop of DIRFA (SEQ ID NO: 5, left) with anextended stem structure (SEQ ID NO: 42, right). FIG. 17D shows fluorescence emission spectra of Design C in response to 5 mM ATP (excitation at 618 nm) FIGS. 17E shows that the ATP-binding aptamer (SEQ ID NO: 40, right) was inserted into the right loop of the DIRFA scaffold (SEQ ID NO: 5, left). FIG. 17F shows fluorescence emission spectra of Design E with and without 5 mM ATP (excitation at 618 nm). FIG. 17G shows the ATP-binding aptamer was inserted into the right loop of DIRFA (SEQ ID NO: 5, left) with an extended stem (SEQ ID NO: 42, right). FIG. 17H shows fluorescence emission spectra of Design G in response to 5 mM ATP (excitation at 618 nm). All experiments were performed in PBS buffer.

[0026] FIGURES 18A-18F depict titrating the insertion point for the conjugation of Split DIR- ATP aptamer. FIG. 18A shows a scheme of different insertion points of the ATP aptamer (Rl-5) into the right stem of the DIRFA (SEQ ID NO: 5). FIGS. 18B-18F shows that the fluorescence spectrum is recorded by fluorometer to test the performance of Split DIR-ATP aptamer pairs, which inserted the ATP aptamer at R1-R5 of the DIRFA, respectively, when sensing 5 mM ATP in PBS buffer.

[0027] FIGURES 19A-19F depict a comparison of the performance between aptamer pairs with different stem lengths between the Adenosine / ATP aptamer and DIR Anorogenic aptamer. The lengths were adjusted by using longer (L) or shorter versions of the Adenosine / ATP aptamers (S) on the insertion point R1 and R4. The fluorescence intensities were recorded with fluorometer in the presence or absence of 5 mM ATP in PBS buffer.

[0028] FIGURES 20A-20B depict a structure switching based design for Dir-ATP aptamer pair. FIG. 20A shows a scheme of the design of structure switching based aptamer pair (SEQ ID NO: 41). The adenosine / ATP aptamer was used to block the structure formation of the DIRFA. In the presence of ATP, the structure switches, and restores the structure of DIRFA. FIG. 20B shows that the aptamer pair was incubated with or without ATP, and a fluorescence spectrum was collected with a fluorometer. Only a minor increase in fluorescence intensity was observed in the presence of ATP.

[0029] FIGURES 21A-21B depict a stability comparison of DNA vs. RNA aptamers in cell lysates. FIG. 21A shows the signal-to-noise ratio of the Broccoli aptamer declined rapidly within 40 minutes, whereas DIRFA maintained stability over a 4-hour recording period. Fluorescence signals were continuously monitored using a fluorometer. For DIRFA, fluorescence was measured with an excitation wavelength of 618 nm and an emission wavelength of 650 nm. For Broccoli, an excitation wavelength of 488 nm and an emission wavelength of 520 nm were used. FIG. 21B shows the slope of the linear regression from the data in FIG. 21A indicates a significant difference in stability between the DIRFA and Broccoliaptamers. Specifically, the Broccoli aptamer exhibited noticeable degradation within 40 minutes, whereas the DIRFA signal showed minimal decrease over the same period, suggesting higher stability for DIRFA.

[0030] FIGURES 22A-22D depict the stability of dDIRFA and DIRFA in cell lysates. FIG. 22A shows that the sequence and predicted secondary structure of dDIRFA (SEQ ID NO: 39) were modeled using UNAfold8 software. FIG. 22B shows that a fluorometer scan measured fluorescence intensity across a spectrum from 640 nm to 750 nm, with excitation at 618 nm. The analysis compared dDIRFA, DIRFA, mutated DIRFA, and control groups lacking either the aptamer or DIR in a PBS buffer. Fluorescence intensity was collected with a fluorometer. FIG. 22C shows signal-to-noise ratio analysis which indicated that the fluorescence of both DIRFA and dDIRFA DNA aptamers, collected over time in a plate reader, are relatively stable within 300 min in a PBS buffer supplemented with 20% cell lysate. FIG. 22D shows that, when 20% cell lysate was added to the PBS buffer, dDIRFA exhibited a 35% increase in fluorescence intensity compared to monomeric DIRFA. Emission was recorded at 650 nm, with excitation at 618 nm. The reaction system used concentrations of 1 pM for aptamers and DIR.

[0031] FIGURES 23A-23F depict a stability comparison of DNA and RNA aptamers in live cells. Cellular images show cells transfected with Broccoli and its chromophore DFHBI- 1T (FIG. 23A), cells transfected with DIRFA and its chromophore DIR (FIG. 23B), and cells transfected with dDIRFA (FIG. 23C). Control images include cells incubated with DFHBI-1T (FIG. 23D) and cells incubated with DIR (FIG. 23E), both without transfection. FIG. 23F shows the quantification of FIGS. 23A-23E. The signal-to-noise ratio analysis shows that the fluorescence signal of the Broccoli RNA aptamer declined sharply and became nearly undetectable within 7 hours. In contrast, the signal-to-noise ratio of DIRFA gradually decreased but remained detectable for up to 21 hours. Dimerization of DIRFA further enhanced the stability of the DNA aptamer; although the overall fluorescence intensity of dDIRFA diminished, this decrease is likely due to photobleaching or fluorophore (DIR) degradation, as the sensor's signal-to-noise ratio remained steady. Scale bar: 20 pm.

[0032] FIGURES 24A-24D depict the impact of Mg2+on sensor performance and signal- to-noise ratio. FIGS. 24A-24B show that fluorescence intensities increased with the addition of 2.5 mM Mg2+but decreased at higher concentrations. FIGS. 24C-24D show that the impact on the signal-to-noise ratio was minimal under physiologically relevant Mg2+concentrations. Data were collected using a plate reader.

[0033] FIGURES 25A-25B depict fluorescence intensity of dDASA 2-C and mutant with / without 5 mM ATP. The experiment was performed in the PBS buffer at room temperature (22°C, FIG. 25A) or at 37°C (FIG. 25B), respectively. The fluorescence intensities were recorded with a fluorometer with excitation at 618 nm and an emission at 650 nm. The mutant contains mutations in the binding site of the ATP aptamer, ns, p > 0.05; *, p < 0.05; ***, p < 0.001.

[0034] FIGURES 26A-26B depict ATP-induced fluorescence enhancement in dDASA. FIG. 26A shows representative fluorescence intensity images showing the effect of ATP on DASA and dDASA. The upper row demonstrates fluorescence intensities for DASA and dDASA without 5 mM ATP, while the lower row shows the corresponding samples after ATP addition. The sensors were evaluated using an excitation wavelength of 635 nm, with emission measured at 685 / 40 nm. The color scale represents intensity values in photon counts (p.c.s.) per pixel, ranging from 0 to 225 p.c.s. FIG. 26B is a bar graph quantifying fluorescence intensity for DASA and dDASA in the presence or absence of 5 mM ATP. dDASA showed a greater increase (155%) in intensity upon ATP addition compared to DASA (43%). ***, p < 0.001.

[0035] FIGURE 27 depicts modulation of cellular ATP levels. Cells were incubated in opti-MEM containing 5 mM CaCh, 10 uM oligomycin, or DMEM supplemented with either 25 mM glucose or no glucose to modulate intracellular ATP levels. After a 1 h incubation, cells were lysed using cell lysis buffer, and ATP levels were measured using the Sigma adenosine 5'-triphosphate bioluminescent somatic cell assay kit. Bioluminescent intensities were normalized to cell counts to ensure accuracy.

[0036] FIGURES 28A-28F depict the evaluation of the effect of single-stranded DNA (ssDNA) binding proteins. FIG. 28A shows a modified single-plasmid system for generating ssDNA in bacterial cells, using a 72-nt DNA sequence as a proof of concept. FIG. 28B shows ssDNA detected by denaturing polyacrylamide gel electrophoresis. M: standard ssDNA synthesized by IDT as a size marker. Lane 1 : 72-nt ssDNA produced by the three-plasmid system.9 Lane 2: no ssDNA control. Lane 3: 72-nt ssDNA produced by the modified singleplasmid system (pINV5-O72 from FIG. 28A). Lane 4: 72-nt ssDNA produced from the plasmid co-expressing protein (pINV5-O72-0, FIG. 28B). FIG. 28C shows an optimized plasmid co-expressing the 0 protein to enhance ssDNA stability. FIG. 28D shows a high-copy plasmid for ssDNA expression. FIG. 28E shows a comparison of ssDNA produced by the lower-copy plasmid (low, FIG. 28B) and the high-copy plasmid (high, FIG. 28D). FIG. 28F shows that the fluorometer records the spectrum of dDIRFA in the presence or absence ofssDNA binding proteins (SSB and RecA). The presence of the single- stranded DNA binding proteins did not interfere with the interaction between dDIRFA and DIR.

[0037] FIGURES 29A-29C depict expression of the genetically encoded light-up aptamer in bacteria. FIGS. 29A-29B are dot plots displaying the fluorescence intensity and size of individual DH10B cells as detected by flow cytometry analysis. FIG. 29A shows the dot plot for the bacteria without plasmid transfection, and FIG. 29B shows the bacteria with DIRFA plasmid transformation. Around 20% of the bacterial cells with the plasmid that codes DIRFA showed higher fluorescence intensity than the cells without the plasmid. FIG. 29C is a histogram from flow cytometry showing a small increase of fluorescence intensity in a population of the bacteria that expressed DIRFA.

[0038] FIGURES 30A-30B depict expression of the genetically encoded light-up aptamer in mammalian cells. FIG. 30A shows that HeLa cells were transfected with the plasmids that express dDIRFA without |3 protein. No DIR means no DIR was added after the transfection. No plasmids meant incubating the cells with DIR, but no plasmids were trans-fected. Scale bar: 50 pm. FIG. 30B shows statistical analysis for pictures obtained in FIG. 30A. ***, p < 0.001.

[0039] FIGURES 31A-31B depict a comparison of plasmids with and without the P protein coding gene. FIG. 31A shows the fluorescence intensity of cells transfected with plasmids containing the P protein compared to that of cells transfected with plasmids lacking the P protein. Scale bar: 50 pM. FIG. 31B shows statistical analysis of FIG. 31A. *, p<0.05; ***, p<0.001.

[0040] FIGURES 32A-32C depict monitoring ATP levels in HeLa cells via a dDASA- expressing plasmid. FIG. 32A shows cellular images for sensing ATP levels with the dDASA sensor expressed with a pBK-CMV plasmid. When decreasing the intracellular ATP levels with 10 pM oligomycin incubation, a 9% decrease in fluorescence intensity was observed. When increasing the intracellular ATP levels with 5 mM CaCh incubation, an 18% increase in fluorescence intensity was observed. This increase can no longer be observed with the mutated dDASA plasmid, in which a point mutation was introduced to the ATP binding site. Scale bar: 20 pm. FIGS. 32B-32C show statistical analysis of the cells in FIG. 32A. Unpaired student t- tests were used, ns, p >0.05; *, p <0.05.

[0041] FIGURES 33A-33D depict a GEFDA sensor for monitoring ATP Levels in HeLa cells. FIG. 33A shows that cellular imaging was performed to sense ATP levels using the dDASA sensor expressed via a pBK-CMV plasmid. Single fields of cell populations were monitored while modulating ATP levels. Adding 25 mM glucose to cell culture media results in a gradual increase of fluorescence intensity, indicating the elevation of ATP levels. FIG.33B shows single-field monitoring of ATP level changes when incubating cells with 10 .M oligomycin for 1.5 h followed. A slight initial increase in fluorescence intensity was observed, followed by a notable decline. Subsequent washing with PBS and the addition of cell culture media containing 5 mM CaCh led to a gradual increase in fluorescence intensity. Scale bars: 40 pm. FIG. 33C shows statistical analysis of the cells in FIG. 33A. FIG. 33D shows statistical analysis for the cells in FIG. 33B.DETAILED DESCRIPTION

[0042] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0043] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0044] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0045] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0046] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of eachof the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0047] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y ’ as well as the range greater than x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’ , and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0048] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0. 1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0049] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factorsknown to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0050] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors.

[0051] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0052] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0053] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment.Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0054] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0055] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0056] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0057] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0058] Reference is made herein to nucleic acid and nucleic acid sequences. The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be singlestranded or double- stranded and may represent the sense or the antisense strand).

[0059] Variants comprising deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of areference polypeptide or a 5 '-terminal or 3 '-terminal truncation or both of a reference polynucleotide).

[0060] Variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide / amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and / or the C-terminal region of a polypeptide or the 5 '-terminal region and / or the 3' terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.

[0061] Variants comprising insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.

[0062] Fusion polynucleotides also are contemplated herein. A “fusion polynucleotide” refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3' end of a first polynucleotide to a 5’ end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).

[0063] “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polypeptide sequences or polynucleotide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein.

[0064] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).

[0065] Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0066] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0067] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, atleast 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0068] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0069] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0070] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 3, Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0071] “Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0072] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.

[0073] The term “mismatched” or “mismatched target sequence” refers to an off-target sequence that is not perfectly complementary to the first DNA sequence or the second DNA sequence of the chimeric deoxyribonucleic acid described herein. The dual retargeted DNA may have at least one mismatch, but can also have 2, 3, 4, 5, 6 or 7 or more mismatched nucleotides to the off-target sequence.

[0074] As used herein, the term “detecting” used in context of detecting a signal from a detectable label to indicate the presence of a target nucleic acid (such as a signaling nucleic acid) in the sample does not require the method to provide 100% sensitivity and / or 100% specificity. As is well known, “sensitivity” is the probability that a test is positive, given that the sample has a target nucleic acid sequence, while “specificity” is the probability that a test is negative, given that the sample does not have the target nucleic acid sequence. A sensitivity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred. A specificity of at least 50% is preferred, although sensitivities of at least 60%, at least 70%, at least 80%, at least 90% and at least 99% are clearly more preferred. Detecting also encompasses assays with false positives and false negatives. False negative rates may be 1%, 5%, 10%, 15%, 20% or even higher. False positive rates may be 1%, 5%, 10%, 15%, 20% or even higher. The term “detecting” is also used in the context of detecting the amplified target nucleic acid by its melting temperature using melting curve analysis, as is known in the art.

[0075] As used herein, “labels” are chemical or biochemical moieties useful for labeling a nucleic acid (including a single nucleotide), amino acid, or antibody. “Labels” include fluorescent agents, chemiluminescent agents, chromogenic agents, quenching agents, radionuclides, enzymes, substrates, cofactors, inhibitors, magnetic particles, quantum dots, and other moieties known in the art. “Labels” are capable of generating a measurable signal, or can be used to capture nucleic acids, and may be covalently or noncovalently joined to an oligonucleotide or nucleotide (e.g., a non-natural nucleotide).

[0076] As used herein, the term “aptamer” refers to a non-naturally occurring nucleic acid that has a desirable action on a target molecule. A desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way thatmodifies or alters the target or the functional activity of the target, covalently attaching to the target, and facilitating the reaction between the target and another molecule. In one embodiment, the action is specific binding affinity for a target molecule, such target molecule being a three-dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which is independent of Watson / Crick base pairing or triple helix formation, wherein the aptamer is not a nucleic acid having the known physiological function of being bound by the target molecule. Aptamers to a given target include nucleic acids that are identified from a candidate mixture of nucleic acids, where the aptamer is a ligand of the target, by a method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to other nucleic acids in the candidate mixture can be partitioned from the remainder of the candidate mixture; (b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to yield a ligand- enriched mixture of nucleic acids, whereby aptamers of the target molecule are identified. It is recognized that affinity interactions are a matter of degree; however, in this context, the “specific binding affinity” of an aptamer for its target means that the aptamer binds to its target with a much higher degree of affinity than it binds to other, non-target, components in a mixture or sample. An aptamer can include any suitable number of nucleotides. “Aptamers” refer to more than one such set of molecules. Different aptamers can have either the same or different numbers of nucleotides. Aptamers may be DNA or RNA and may be single stranded, double stranded, or contain double stranded or triple stranded regions. Aptamers may be designed with any combination of the base modified nucleotides desired.

[0077] A “dual aptamer” is a system which comprises at least two different aptamers. Each aptamer can affect a different reaction. In one example, one of the aptamers can be a “signal input” aptamer, which is capable of functioning as an aptamer upon interaction with a target analyte. A dual aptamer system can also comprise a second aptamer, which can be a signal output aptamer, for example. The signal output aptamer can, upon receiving the proper input signal, cause an output signal to be released. This output signal can be, for example, a detectable signal. In one specific embodiment, the signal input aptamer can detect an analyte, which affects a change in the system which then provides input to the signal output aptamer. The signal output aptamer can then, in turn, provide a detectable signal.DUAL APTAMER SYSTEMS

[0078] Disclosed herein is a dual aptamer system for detection of a target analyte, the system comprising: a signal input aptamer, wherein said signal input aptamer can change aphysical feature upon interaction with a target analyte; and a signal output aptamer, wherein said signal output aptamer changes a physical feature upon binding of a target analyte to the signal input aptamer, wherein said change of physical feature of the signal output aptamer allows for output of a detectable signal; wherein both the signal input aptamer and the signal output aptamer comprise nucleic acid, and further wherein both the signal input aptamer and the signal output aptamer are linked to each other.

[0079] An example of the dual aptamer system can be seen in FIG. 2D. In the dual aptamer system described herein, both aptamers can be comprised of nucleic acid. This can be DNA,RNA, and / or modified nucleic acids, or any combination of these. It can be double stranded, single stranded, or a combination of double and single-stranded. They can be separated by a linker, or they can be directly fused to one another.

[0080] The dual aptamer, as the name suggests, can comprise at least two aptamers. One of these aptamers can be a “signal input aptamer.” This aptamer can interact with a target analyte, which can result in a physical change, such as a conformational change of the system, for example. In one example, the conformational change can be a change in secondary or tertiary structure of the nucleic acid of the aptamer. The conformational change and the target analyte are described in more detail below. The conformational change caused by the signal input aptamer can result in a signal being relayed to a second aptamer of the system, referred to herein as the “signal output aptamer.” The signal output aptamer, upon receiving a signal from the signal input aptamer, then provides an output signal, such as a detectable signal. Detectable signals are described in more detail below.

[0081] Regarding both the signal input aptamer and the signal output aptamer, the nucleic acid comprising each of these can be at least about 20 nucleotides (e.g., at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 85 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at least about 100 nucleotides in length.

[0082] In some aspects, when a target analyte binds the signal input aptamer, one or more physical characteristics, features, or properties in the dual aptamer system may change. In some such aspects, the one or more properties of the signal input aptamer can include a conformational change (such as a change in secondary or tertiary structure of a nucleic acid), a difference in melting temperature, and / or a variation in sensitivity to pH or anotherenvironmental condition. In some aspects, the changes to one or more properties of the signal input aptamer can cause an effect upon the signal output aptamer. In some aspects, this effect upon the signal output aptamer can cause the signal output aptamer to undergo a change. This can include, but is not limited to, a conformational change, a difference in melting temperature, and / or a variation in sensitivity to pH or another environmental condition. This change to the signal output aptamer can then result in a detectable signal, which is described in more detail below.

[0083] Changes in conformation of the nucleic acid can include changes in single-stranded and double- stranded binding areas, bulges, internal loops and hairpin loops, junctions, and helices. Stem-loop is formed when nucleic acid chains fold back on themselves to form a double helical tract called the 'stem', the unpaired nucleotides forms single stranded region called the 'loop'. A tetraloop is a four-base pairs hairpin structure. The change in conformation can occur when different bases pair or unpair with each other, causing new stems and / or loops to form. This change in conformation can occur with both the signal input and signal output aptamers.

[0084] The target analyte can be any molecule which is capable of interacting with, either directly or indirectly, the nucleic acid of the signal input aptamer. Examples include, but are not limited to, a small molecule, nucleic acid, virus, protein, or peptide. Specific examples include, but are not limited to, a metal ion, drug, or an environmental pollutant. Other examples include a metabolite. ATP is one example thereof.

[0085] The target analyte can interact with the signal input aptamer. This interaction can include binding to the aptamer. In some embodiments, there may be an increase or decrease in target analyte interacting with the signal input aptamer which can be detected. This can allow for a quantifiable assay, for example. More target analyte can result in an increased signal, whereas less target analyte can result in a decreased signal as compared to a control. The control can be a different time point, or can be compared to the lack of presence of any target analyte. For example, the amount of target analyte can be increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to a control. This can result in an increase in detectable signal of 1%, 2%, 3%, 4%, 5%, 6%, 7%,18%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or more.

[0086] When the signal input aptamer has a change in conformation, for example, this can result in a change in the signal output aptamer. Like the signal input aptamer, this change can be a change to the secondary or tertiary structure of the nucleic acid of the system. This change in conformation can then result in a detectable signal being produced. For example, the change to the signal output aptamer can result in the ability of a detection agent to interact with the signal output aptamer, or to cease interacting with a signal output aptamer. This change in interaction between a detection agent and the signal output aptamer can result in a detectable signal.

[0087] The detection agent can be part of the dual aptamer system, or can be separately provided. When part of the system, the detection agent can be encoded by nucleic acid and then expressed within a cell, or can be provided separately as a protein or compound. The detection agent can be a fluorescent molecule. For example, the fluorescent molecule can comprises dimethylindole red (DIR), magnesium green (MG), or 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). The detection agent can be any agent which can interact differentially with the signal output aptamer depending upon the conformation of the signal output aptamer. For example, the detection agent can interact with the signal output aptamer at a rate which is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% greater or lesser when the conformation of the signal output aptamer has been triggered by the signal input aptamer. In other words, upon triggering by the signal input aptamer, the signal output aptamer interacts differently with a detection agent, and this change can be monitored or detected.

[0088] Both the signal input aptamer and the signal output aptamer of the dual aptamer system described herein can be from naturally occurring nucleic acids. Typically, these nucleicacids of the input and output aptamers do not naturally occur together, or not in the same proximity that they do within the dual aptamer system described herein. For example, they can be from naturally occurring sources, but can be truncated or otherwise engineered for detection optimization. Examples of truncated DIR can be seen in FIGS. 2A-2F for example. The aptamers described herein can be optimized for detectability by mutating them or by using non- naturally occurring nucleic acids.

[0089] The two aptamers of the dual aptamer system can be separated by a linker. This linker can comprise nucleic acid or amino acids. It can be varied in length, depending upon the intended use and aptamers being used. It can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids or nucleic acids in length, or longer.

[0090] The dual aptamer system disclosed herein can also comprise additional aptamers. For example, a multiplex system can be used wherein two or more different signal input aptamers are used, two or more different signal output aptamers are used, or both multiple signal input and signal output aptamers are used within the same system. They can be on contiguous nucleic acids or on separate nucleic acids. Also disclosed are multiple dual aptamer systems which are used in conjunction with each other to detect multiple target analytes and / or provide multiple different output signals, such as multiple different detectable signals such as that arising from a detection agent.

[0091] The dual aptamer system described herein can be reversible upon removal of the target analyte. In other words, the output signal (detectable signal) can be reduced or eliminated upon the removal or reduction of the target analyte. This reduction can be, for example, a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% reduction in target analyte, which can in turn result in a reduction of detectable signal by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% or more.

[0092] The dual aptamer system can further include one or more additional components for expression (e.g., two or more, three or more, four or more, five or more, or all six) selected from a reverse transcriptase, a sequence that codes a dimer form of one or both aptamers, an RNA polymerase terminator, a hairpin structure, a tRNA binding sequence, and a singlestranded DNA binding protein.

[0093] In some aspects, the reverse transcriptase can be IPTG-inducible. In some aspects, the reverse transcriptase can be used for reverse transcription of one or both aptamers. Reverse transcriptases (RTs) are enzymes that catalyze the synthesis of complementary DNA (cDNA) from an RNA template. They are primarily found in retroviruses but also occur in various cellular and viral systems, including but not limited to viral reverse transcriptases, telomerase reverse transcriptase, retrotransposon-encoded reverse transcriptases, bacterial reverse transcriptases, and engineered reverse transcriptases. In some aspects, the reverse transcriptase can be derived from Moloney mouse leukemia virus (MoMuLV), avian myeloblastosis virus (AMV), or HIV-1. In some aspects, the reverse transcriptase can be a thermostable reverse transcriptase (e.g., ThermoScript, SuperScript variants).

[0094] In some aspects, the RNA polymerase terminator can be an rrnB T1 terminator, a T7 terminator (e.g., T7Te), a TO terminator, a trp terminator, a T5 terminator, or a synthetic polythymidine sequence. In some aspects, the RNA polymerase terminator can be selected for its ability to promote efficient transcriptional termination and prevent read-through into downstream sequences.

[0095] In some aspects, the dual aptamer system can include both the reverse transcriptase and the hairpin structure, and the hairpin structure can terminate reverse transcription of said reverse transcriptase.

[0096] In some aspects, the tRNA binding sequence can be a val-tRNA binding sequence for initiation of reverse transcription.

[0097] In some aspects, the single-stranded DNA binding protein can be IPTG-inducible. In some aspects, the single-stranded DNA binding protein can be betA (i.e., RedP), ET SSB, RecA T4 Gene 32 Protein, SSB, RecT, Orf48, Sak, EF2132, any other phage- or bacteria-derived single-stranded annealing proteins (SSAPs) capable of promoting homologous recombination or strand annealing, or any homologs or functional fragments thereof.

[0098] The system disclosed herein can be encoded by a vector, for example, and expressed within a cell. This cell can be prokaryotic or eukaryotic, such as a bacteria or a yeast cell. Therefore, the system can be used in vivo to detect a target analyte. This vector can be provided to a cell and expressed therein.

[0099] The system can also be provided directly to a cell via microinjection, such as within a nanoparticle or other delivery system. Also disclosed are other components or compositions for the delivery of the dual aptamer system described herein. The system can also be provided in an in vitro setting, in cellulo, or ex vivo.METHODS OF USING DUAL APTAMER SYSTEMS

[0100] Also disclosed herein is a method of detecting a target analyte, the method comprising: providing the dual aptamer system disclosed herein in the presence of a target analyte, and detecting a signal via the signal output aptamer. This detection method can be carried out in real time, so results are available immediately. It can also be reversible, and can quantify the presence of a target analyte.

[0101] Also disclosed are methods of providing the dual aptamer system to a cell in the form of a vector, for example, or directly as nucleic acid. The detection agent can be provided before, after, or during the period of time when a target analyte is present.

[0102] Also disclosed herein are screening methods. These methods can determine whether the combination of a given target analyte and signal input aptamer can be used together, whether the combination of a given signal input aptamer and signal output aptamer can be used together, and whether the combination of a given signal output aptamer and detection agent can be used together. Any of these components can be varied and tested with each other to ensure that the dual aptamer system is capable of detecting a target analyte and producing a detectable signal.

[0103] Specifically, disclosed herein is a method of screening to determine that a potential signal input aptamer can be used to detect a target analyte, the method comprising: providing a potential signal input aptamer, to determine if said signal input aptamer can change conformation upon interaction with a target analyte; and providing a signal output aptamer, wherein said signal output aptamer changes conformation upon binding of a target analyte to the signal input aptamer, wherein said change of conformation of the signal output aptamer allows for output of a detectable signal; and determining whether the potential signal input aptamer can detect a target analyte by determining if a detectable signal is detected; whereinboth the potential signal input aptamer and the signal output aptamer comprise deoxyribonucleic acid (DNA), and further wherein both the potential signal input aptamer and the signal output aptamer are linked to each other.

[0104] Also specifically disclosed is a method of screening to determine that a potential signal output aptamer can be used to detect a target analyte, the method comprising: providing a signal input aptamer, wherein said signal input aptamer can change conformation upon interaction with a target analyte; and providing a potential signal output aptamer, and determining whether said signal output aptamer changes conformation upon binding of a target analyte to the signal input aptamer, wherein said change of conformation of the signal output aptamer allows for output of a detectable signal; and determining whether the potential signal output aptamer can detect a target analyte by determining if a detectable signal is detected; wherein both the potential signal input aptamer and the signal output aptamer comprise deoxyribonucleic acid (DNA), and further wherein both the potential signal input aptamer and the signal output aptamer are linked to each other.EXAMPLESExample 1: Genetically-Encoded DNA Light-Up Sensors

[0105] These DNA light-up sensors are regular DNA with specific sequences. They were developed based on a combination of two domains of specific DNA sequences, one of the domains can recognize specific small molecule or metal ion with high selectivity, while the other can show fluorescence in the presence of a small molecule dye that does not have background fluorescence without this specific DNA sequence in a correct structure. This DNA sequence connects two regions with rational design, which blocks the structure and cannot be accessed to the small molecule dye without binding to the target and conducting structural change. Thus, it has low background fluorescence without the target but shows fluorescence in the presence of the correlated light-up dye and upon binding to its target. This sensor can be used for in-solution, in cellulo, and in vivo sensing for the small molecule target. They can also be expressed in both bacteria and mammalian cells with a plasmid.

[0106] This type of DNA light-up sensor was engineered by optimizing a combination of a DNA aptamer that targets small metabolite and a DNA aptamer that can serve as signal output and show light-up in the presence of its organic small molecule target. The structure of the aptamer changes upon binding to its target and shows tunable detecting sensitivity upon binding to their targets. The fluorescence can be characterized with a microscope, fluorometer, or any instrument that can excite the sensor and collect fluorescence signaling at the expected wavelength. This sensor is generalizable and can be applied to different small molecules, metalions, or related targets with different light properties. The dimerization of the sensor can help stabilize the sensor inside of the cellular environment. Clone the sensor into a plasmid that contains a reverse transcriptase, a terminator, a sequence that coded the dimer of the aptamers, a tRNA binding sequence, and a single-stranded DNA binding protein, can express the DNA aptamer sensor in both bacteria and mammalian cells, and real-time monitor the concentrations of the small metabolites.

[0107] This type of DNA light-up sensor is cheap to produce and can be used for temporally controlled sensing. More importantly, the method for expressing the sensor in cells marks a significant advancement in genetically encoded sensors, transitioning from conventional fluorescent proteins and RNAs to fluorescent DNA-based sensors for small molecule detection. This sensing platform offers researchers a powerful tool for real-time monitoring of the dynamics of metabolites and other small molecules within biological systems.

[0108] These DNA light-up sensors were developed based on a rational design that engineered a DNA light-up aptamer that can serve as signal output and a DNA aptamer that can recognize small molecule targets specifically for label-free sensing of the small metabolites. It has low background fluorescence without the target but shows fluorescence in the presence of the correlated light-up dye and upon binding to its target. This sensor can be used for in-solution, in cellulo, and in vivo sensing for small molecule targets. To achieve better sensing in living cells, the dimer version of the single-stranded light-up aptamer are expressed with a plasmid that contains a reverse transcriptase, a terminator, a sequence that coded the dimer of the aptamers, a tRNA binding sequence, and a single- stranded DNA binding protein, BetaA. The sensing system is able to achieve a reversible and controllable detection of molecular targets in both mammalian cells and bacteria cells.

[0109] Small molecule sensors are developed for metal ions and metabolites, but they need case-by-case development and may encounter issues such as selectivity, relying on organic solvent, cannot being genetically encoded. For genetically encoded sensors, RNA and protein sensors are used for sensing protein and RNA in cells, but with limited success of detecting metabolites. In comparison, the DNA aptamer sensors can detect metabolites with high selectivity. More importantly, it is more stable than RNA and protein. Additionally, it is much easier to obtain DNA aptamers through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) than RNA riboswitches for new targets or new signal outputs, is more generalizable than the protein or organic dye sensors for sensing small molecule or metaltargets. These advantages made the DNA-based light-up sensor an effective alternative for developing new light-up sensors.

[0110] Despite the targets varying, they can be recognized by specific DNA aptamers. On the contrary, the light-up property of the sensor is fixed and can be easily detected under standard instruments, such as microscopy or fluorometer. This uniformed light property for different sensors is realized by conjugating the target recognition aptamer to a DNA light-up aptamer. To expand the application and broaden the options of the light property of the sensor, one can obtain new light-up aptamers via SELEX. Therefore, there’s a potential that different DNA light-up aptamers with different fluorescence wavelengths can be chosen for different targets, similar to choosing a secondary antibody in the immunochemistry staining. With a combination of different light-up aptamers and different targeting aptamers, the DNA light-up sensors could achieve multichannel sensing for tracing different targets simultaneously.

[0111] The DNA light-up sensor can show fluorescence signals in the presence of its target and small molecule dye. The sensor is label-free and can be adapted to other targets or small molecule dyes by changing the sensing domain or the signaling output domain. This advantage allows the DNA light-up sensor to be synthesized as regular DNA, which is more stable than protein and cheaper than fluorophore and quencher-based DNA sensors. Additionally, the light-up dyes can easily penetrate into cells. Thus, by adding the dye at different time points, this type of light-up sensor can achieve temporal controlled sensing towards their targets. Moreover, the DNA light-up sensor can be chemically synthesized on a large scale and shipped under ambient temperature, which is a significant advantage of DNA sensors over RNA sensors.

[0112] Another important feature of this sensor is that the aptamer binds to its target reversibly. Thus, the sensor can be used to track the real-time changes of the small metabolites.

[0113] The sensor was further optimized to improve its stability in the cells by dimerization and synthesized it in cells as a genetically encoded sensor. A plasmid was used which contained a reverse transcriptase, a terminator, a sequence that coded the dimer of the aptamers, a tRNA binding sequence, and a single-stranded DNA binding protein, to express the DNA aptamer sensor in both bacteria and mammalian cells.

[0114] Small molecules and metal ions are important components in life science. They serve important roles in metabolisms, enzymatic function, and signaling transductions, which are critical for human health and disease. Sensing those small molecules and metal ions will advance biological research in understanding the biological functions of those small molecules under health and disease status. More interestingly, this type of DNA light-up sensor can betemporally controlled by choosing a time window of interest to add the related small molecule dye, which is cell penetrable and necessary for the light-up. Thus, the sensors can easily be adapted for monitoring certain biological processes at the desired time point by preloading the sensors and activating them by adding the correlated dye at a specific time point. With the combination of different targeting elements and different light-up DNA aptamers, multichannel sensing for different small molecules and metal ions can be easily achieved to study the correlation between those targets in a certain biological process.

[0115] Genome encoding of the sensors helps the sensor to last longer in the cells and minimizes the dilution of the sensors caused by cell division. While genetically encoded fluorescent proteins and RNAs have significantly enhanced the visualization of proteins and RNAs, their capability to image small molecules in biological systems, particularly metabolites, remains limited. This innovative approach marks a significant advancement in genetically encoded sensors, transitioning from conventional fluorescent proteins and RNAs to DNA Anorogenic aptamer-based sensors for small molecule detection.

[0116] Although there are protein and organic dye sensors for sensing some of the small molecule and metal ion targets, these sensors largely rely on the limited number of specific proteins or dyes that are developed to recognize these targets. As a powerful alternative, DNA aptamers and DNAzyme sensors are developed for sensing small molecules and metal ions. They are more generalizable and can be identified for specific targets from a large library of IO14-15DNA sequences with systematic evolution of ligands by exponential enrichment (SELEX), which is a process that can identify and enrich specific DNA sequences that recognize a specific target. Currently, most of the DNA aptamer and DNAzyme-based Auorescence sensors are developed by adding Auorophores and quenchers to DNAzymes and aptamers sequences. Those DNA aptamers or DNAzymes show structural switching or binding affinity changes and thus change the distance between the lluorophore and quenchers, thus showing Auorescence upon target binding. This Auorescence labeling is expensive and has to be chemically synthesized and delivered to the system. Taking advantage of the generalizability of the aptamers and DNAzymes for small molecule and metal targets, the DNA sensors can be made label-free. While keeping the generalizability and selectivity, this type of DNA light-up sensor is more cost-effective. In addition, this type of DNA light-up sensor is more stable than protein or RNA sensors, and more importantly, they can be chemically synthesized into large batches at low cost. Thus, this type of light-up DNA sensor can be easily developed into commercialized imaging tools for small molecules and metal ions, and ready to be used in biological research.

[0117] More importantly, the sensor can be engineered into single-strand DNA expression plasmids, which contains a reverse transcriptase, a terminator, a sequence that coded the dimer of the aptamers, a tRNA binding sequence, and a single-stranded DNA binding protein. With these elements, the plasmids were able to generate genetically encoded sensors for small metabolites in both bacteria and mammalian cells. This innovative approach marks a significant advancement in genetically encoded sensors, transitioning from conventional fluorescent proteins and RNAs to fluorescent DNA-based sensors for small molecule detection. This sensing platform offers researchers a powerful tool for real-time monitoring of the dynamics of metabolites and other small molecules within biological systems.

[0118] The plasmid that expresses the aptamer sensor can be engineered to generate other types of DNA sensors, such as DNAzymes. Also, by changing the target recognition element and the signaling output element of the sensor, the sensors can achieve multiplex sensing with different aptamer pairs. These sensors can be applied to track important metabolites in biological systems and study their functions.

[0119] Small molecules, such as metabolites, are important components in life science. They serve important roles in metabolisms, enzymatic function, and signaling transductions, which are critical for human health and disease. Sensing those small molecules will advance biological research in understanding the biological functions of those small molecules under health and disease status. Developing genetically encoded sensors presents a promising avenue for advancing cellular imaging and monitoring techniques. While genetically encoded fluorescent proteins and RNAs have significantly enhanced the visualization of proteins and RNAs, their capability to image small molecules in biological systems, particularly metabolites, remains limited. In contrast, aptamer sensors offer distinct advantages due to their high specificity and sensitivity towards small molecules. However, the challenge lies in ensuring the stability of these sensors within the cellular environment for effective long-term monitoring. To overcome these limitations, a class of Genetically Encoded DNA Aptamer Sensors has been developed in this study. These sensors are engineered by incorporating an ATP aptamer for target recognition, and a Anorogenic DNA aptamer that binds dimethylindole red (DIR) as the readout component to generate a detectable signal output. Following the optimization of interactions between these two aptamers in test tubes, a plasmid was constructed containing a reverse transcriptase, a terminator, a sequence that coded the dimer of the aptamers, a tRNA binding sequence, and a single-stranded DNA binding protein, to express the DNA aptamer sensor in both bacteria and mammalian cells. The study successfully demonstrated the sensor's capability to detect ATP in both types of cells. By leveraging theexisting DNA aptamers towards a diversity of targets, and the ability to obtain new aptamers with in vitro selection, the method developed in this work can be generally applied for detecting a wide range of other targets. This innovative approach marks a significant advancement in genetically encoded sensors, transitioning from conventional fluorescent proteins and RNAs to fluorescent DNA-based sensors for small molecule detection. This sensing platform offers researchers a powerful tool for real-time monitoring of the dynamics of metabolites and other small molecules within biological systems.Example 2: Genetically Encoded Fluorogenic DNA aptamers for Metabolite Imaging in Living Cells

[0120] Among existing methods for developing genetically encoded sensors, fluorogenic RNAs combined with riboswitches

[0039] ,

[0041] ,

[0042] ,

[0043] ,

[0044] ,

[0045] ,

[0046] ,

[0047] ,

[0048] have generalizability, as the riboswitches can recognize metabolites and cause conformational changes of the fluorogenic RNAs to produce an increase of fluorescent signal for visualization of various small metabolite targets

[0028] ,

[0049] ,

[0050] ,

[0051] ,

[0052] ,

[0053] . Despite the notable successes, it is desirable to expand the repertoire of riboswitches for many other metabolites, while addressing lower stability for some applications that require it. To achieve the goals, a study was conducted that developed genetically encoded DNA aptamers, as DNA aptamers for a wide variety of metabolites can be obtained more easily than riboswitches through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX)

[0042] ,

[0043] . DNA aptamers also have higher stability than proteins and riboswitches. These advantages position DNA aptamers as effective alternatives as sensors for metabolites. Indeed, many DNA aptamers have been applied for imaging metabolites. However, all these studies require labeling of the aptamers with a fluorophore outside the cells and then delivered into the cells. As a result, when the DNA is degraded within the cells or when the cells divide, the aptamer sensors’ signals decrease, making it difficult to quantify the metabolites. Converting such sensors into genetically encoded DNA fluorogenic aptamer sensors will address this issue, as the DNA can be continuously expressed in the cells, including the dividing cells.

[0121] Despite the promise, genetically encoded DNA fluorogenic aptamers have not been applied in imaging any target in living cells due to two major challenges. First, even though fluorogenic DNAs that bind various organic fluorophores like MG

[0054] , dapoxyl

[0055] , crystal violet

[0056] , Hoechst

[0057] , DFHBI

[0058] , Auramine O

[0059] , and dimethylindole red (DIR)

[0014] have been developed and some of them have been paired with DNA aptamers to detect molecules such as thrombin, ATP, and serotonin in controlled environments such as test tubes

[0055] ,

[0059] , most of the organic fluorophores do not meet all the requirements of applications inliving cells, such as low auto-fluorescence, high specificity to the target aptamer, and significant fluorescence increase upon binding to the DNA aptamer, under intracellular conditions. For instance, while Hoechst is widely used for cellular applications due to its high fluorescent intensity

[0061] ,

[0062] ,

[0063] , it lacks specificity toward any target. On the other hand, while crystal violet has shown selectivity, it displays only a modest fluorescent increase (around 30%) upon binding to its aptamer compared to other G-quadruplexes in cells

[0056] .

[0122] In addition to the lack of effective fluorogenic DNAs for signaling in cells, it has been difficult to genetically encode both the fluorogenic DNAs and DNA aptamers for their targets in cells. Unlike fluorogenic RNAs and riboswitches, most genetically encoded DNA molecules in cells are in double-stranded form, but the fluorogenic DNAs and DNA aptamers are required to be expressed in single-stranded forms in order to function. To meet both challenges of developing an effective fluorogenic DNA and to express the fluorogenic DNA and its associated DNA aptamer in cells, the study developed and optimized a DIR-specific fluorogenic DNA linked to a ATP aptamer

[0064] that meets all the requirements of applications in cells. After demonstrating reversible detection of ATP in mammalian cells under temporal control, the study converted them into genetically encoded sensors by constructing a plasmid, which contains a tRNA binding sequence, terminators, dimerized sensors, and sequences that encoding IPTG inducible reverse transcriptase and single-stranded DNA binding protein, to express the DNA aptamer sensor and sensing ATP in both bacteria and mammalian cells. With the selection markers on plasmids, this system allows continuous sensor generation, preventing degradation and dilution over time. This platform has the potential to be adapted to other DNA aptamers and is generalizable for imaging different metabolites in cells.Methods and Materials

[0123] Characterization of light-up aptamer sensors'. DNA sequences were ordered from IDT with standard desalting. Upon receiving, the DNA sensors were dissolved in Milli-Q water. For each fluorometer test, a 200 pL reaction system was used, which includes IxPBS buffer, 0.5 pM DIR, and 0.5 pM DNA sensor or control DNA. When sensing ATP or other analogous, a master mix was made with the sensor and DIR, and then different concentrations of analytes were spiked into the system before detection. ATP was dissolved in buffer and the pH was adjusted to 7.0 before spiking to the system. Serial dilutes were made when studying the detection range of the sensors. A FluoroMax-P fluorometer (HORIBA Jobin Yvon Inc., Edison, NJ, USA) and ISS ChronosDFD fluorometer (ISS Inc., Champaign, IL, USA) were used for the luminescent spectra measurements. Fluorescence was excited at 618nm and a spectrum from 630-750 nm was recorded. When plotting the sensing curves with a singleemission wavelength, the fluorescence intensity was presented at 650 nm. When testing the samples at 37°C, the mixtures were pre-warmed at 37°C with a heating block before transferring them into the fluorometer. Temperature control of the fluorometer was used during the detection.

[0124] Light-up aptamer sensor for imaging ATP levels in mammalian cells'. HeLa cells were brought from ATCC and cultured in DMEM. 4.2xl04cells per well were seeded into 8- well strips one day before the experiments. During the transfection, 150 pL transfection system that includes opti-MEM, 0.4 pM synthesized DNA sensor, control DNA, or 300 ng plasmid, and 1 pL TurboFect transfection reagent, was vortexed briefly and then stood at 37 °C for 15 minutes before adding to the cells. The cells were incubated in the transfection system for 4 hrs, and were washed twice with PBS, then changed into DMEM before imaging. To regulate cellular ATP levels, the cells were incubated with 5 mM CaCh or 10 pM oligomycin (contains l%c DMSO) in opti-MEM, 25 mM glucose in DMEM, or glucose-free media after transfection. 0.5 pM DIR was supplied during the transfection or before imaging as implicated in the main text. The cells were stained with 1 :1000 Hoechst 33342 for 12 minutes and washed twice with PBS before changing into opti-MEM for imaging.

[0125] To test the potential of temporally controlled sensing with DIR aptamer, the DIR aptamer was delivered into Hela cells with the Turbofect transfection reagent. After the transfection, the cells were washed three times with PBS buffer to remove transfection reagents and exceeded aptamers. The DIR was then added to the cell culture media, and the fluorescence signal of the cells was recorded immediately and every 5-10 mins with a microscope.

[0126] To test if the light-up sensor can monitor the ATP levels in real-time in living cells, the dDASA 2-C sensor and DIR were delivered to the cells as described above and regulated the intracellular ATP levels by treating the cells alternatively between 30 min incubation with 5 mM CaCh followed by 1 hr incubation with 10 pM oligomycin for two cycles. For evaluating the glucose induced ATP level changes, 25 mM glucose was added to complete cell culture media, and the cells were incubated in the high-glucose media for 1 hr. Afterward, the glucose was removed by washing out the high-glucose media and replacing it with glucose-free media. The cells were then incubated in the glucose-free media for 1 hr and changed back into the high-glucose media that contained 25 mM of added glucose again. This media switch was applied to the cells for two cycles. The cells were washed with PBS before changing the solutions.

[0127] Express DASA sensors in bacteria cells'. One plasmid system for generating singlestranded DNA in bacterial cells was produced through modification of the system by Chen et,al.

[0082] ,

[0083] ,

[0084] ,

[0085] ,

[0086] . To increase the productivity of the single- stranded DNA, a betA protein was added to increase the stability of single-strand DNA. Additionally, the DNA was designed into a dimer to minimize the exposure of the end of DNA to nucleases. The dimer of the DIR aptamer was used for the initial demonstration, and the dimer of the DASA sensor was cloned to replace the dimer of the DIR fluorogenic aptamer DIRFA. Gblocks were synthesized with GeneArt Gene and Protein Synthesis Services from Thermo Fisher Scientific in a pMA- RQ (AmpR) plasmid (FIG. 16). Pad and Xhol were used to double digest the plasmids, and the fragments were ligated with T4 ligase after agarose gel-based product purifications.

[0128] DH10B competent cells were purchased from NEB. Sensors or plasmids that code the sensors or control DNA were transformed into competent cells following the standard protocol. Fresh clones were picked and inoculated into TB media containing 50 pg / ml of kanamycin and 10 mM of IPTG. Then the cells were grown in a shaking incubator at 37 °C overnight until the culture reached the early stationary phase. The cells were collected and detected as soon as possible. The ATP level of bacterial cells was regulated by incubating with 50 pM ATP or 10 pM Venturicidin A in the culture media. DIR was also added to the culture media at a concentration of 0.5 pM 20 min before detection.

[0129] Flow cytometry assay of bacteria cells: The fluorescence intensities of the cells were recorded with flow cytometry assay and compared between groups. The APC or PE-Cy5 channel with DB LSRFortessa Flow Cytometers was used for analysis. When processing the data from the flow cytometer, bacterial cells were gated based on the size using side scatter (SSC) and forward scatter (FSC) parameters. Subsequently, singlets were isolated from the bacterial cell population. Following this, cells exhibiting fluorescence intensity above 103 were classified as fluorescent cells, while those below 103 were categorized as non-fluorescent cells.

[0130] Express DASA sensors in mammalian cells: pBK-CMV plasmid was used as the backbone for the mammalian cell expression system. The ssDNA expression systems that contain betA, ssDNA sequences of interest, and reverse transcriptase were obtained by using Xbal and Avril to double digest the plasmids that were generated for the bacterial system, and by agarose gel purification to collect the correct DNA fragments. Next, pBK-CMV plasmid was linearized by PCR to generate two ends that each has 30 bps homologous sequence with the previously agarose gel purified DNA fragments. The primers used to linearize pBK-CMV plasmid are 5’-TTTCCAGTCGGGAAACCTGTCGTGCTAGCTTGCAGGCGCGCGAGCTCC-3’ (SEQ ID NO: 1) and 5’-ACGAGCCGATGATTAATTGTCAACACAGCCGGTCGACACTAGTGGATCCAAAGAATTCAAAAAGCTTCTC-3’ (SEQ ID NO: 2). NEBuilder HiFi DNA Assembly Master Mix was then used to assemble the linearized pBK-CMV backbone with the DNA fragments that contain the ssDNA expression system. NEB 5-ahpla competent E. coli cells were used for transformation and plasmids were purified using QIAprep Miniprep Kit (QIAGEN). 300 ng of the correctly constructed plasmids were transfected into HeLa cells with the same protocol described in the ATP level imaging in the mammalian cell sensing section. After 4 hrs of transfection, the cells were changed into DMEM and cultured overnight. Then the ATP levels were regulated with 5 mM CaCb or 10 M oligomycin incubation in opti-MEM. Afterward, the cells were incubated in 0.5 LI M DIR and 1 : 1000 Hoechst 33342 for 30 min and washed with PBS twice before imaging with confocal microscopy at 37°C in the opti-MEM media. 640 nm channel was used for imaging DIR aptamer-based sensors, and 408 nm channel was used for imaging the Hoechst staining.

[0131] Statistical analysis: All experiments were repeated at least three times independently. The standard error of the mean was used as the error bars. GraphPad was used to analyze and present data. To quantify the fluorescence intensity in the cellular images, the study used Fiji imageJ to measure the mean intensity of each cell. Five cells per picture and three to six pictures per group were quantified. Unpaired student t-test were used to quantify if the differences between two different groups were significant or not.

[0132] TABLE 1 provides the sequences used in this study.TABLE 1. DNA sequences used in the study. In SEQ ID NOs: 30-38: the underlined segment corresponds to the hairpin structure involved in dimer formation; the bold segment indicates the split ATP aptamer; and the unformatted sequence represents the DIRFA region.Results and Discussion

[0133] Development and Characterization of DIR-ATP Aptamer Sensors: The study initially screened the reported DNA Anorogenic aptamers to select a suitable candidate for intracellular applications. Among the fluorophores used in Anorogenic sensors, DFHBI and its derivatives are commonly used in mammalian cells due to their low Auorescence background and a significant increase in Auorescence upon binding to specific RNA aptamers like Spinach, Broccoli, Spinach2, and Baby Spinach

[0065] . Recently, a DNA aptamer called Lettuce that binds to DFHBI was identified

[0058] . Although Lettuce showed increased Auorescence when bound to DFHBI in HEPES and Tris buffers, it was found that its Auorescence enhancement in PBS buffer, which mimics cellular conditions, was minimal compared to a control sequence with a point mutation (FIG. 1A). In contrast, the DNA aptamer for dimethylindole red (DIR)

[0066] showed a 10-fold increase in Auorescence compared to its mutated control sequence, which removed the highlighted TG pair illustrated in FIG. 2A, when tested in PBS buffer. Removal of either the DIR Auorogenic aptamer (DIRFA) or DIR from the system resulted in a 100-fold decrease in Auorescence signal (FIG. IB). These results signify the potential of the DIR / DIRFA complex for cellular sensor applications. Therefore, the study selected the DIRFA as the signal output and carried out a systematic truncational analysis by removing one base pair at a time to investigate the functionality of its stem structure (FIGS. 2A-2B). When more than 3 base pairs were removed, the Auorescence intensity of the truncated DIRFA becamelower than the mutated control sequence (FIG. 2B). Based on this observation, it was hypothesized that the stem structure at the bottom of the DIR aptamer is very important for the light-up effect and replaced the stem structure with a target recognition aptamer to generate the light-up sensor.

[0134] To test the above hypothesis, the study chose the ATP aptamer as proof of concept, because ATP serves as a primary energy source in cells, and its aptamer has been extensively researched and applied in sensing applications

[0043] ,

[0067] , The stem structure of the DIRFA was replaced with a split ATP aptamer

[0068] ,

[0069] (FIG. 2C). In the absence of ATP, the stem structure was disrupted since the two strands of the split ATP aptamer were not perfectly base paired with each other. The binding of the ATP helped the two strands of the split ATP aptamer to hybridize and restore the stem structure of the DIRFA, which could potentially restore the light-up ability of the DIR aptamer. This design of the DIR aptamer-based split aptamer sensor (called DASA sensor hereafter) was tested using a fluorometer and showed a 215% fluorescence increase at 650 nm upon the addition of 5 mM ATP. Introducing point mutations in either the split ATP aptamer, DIR aptamer, or both aptamers reduced fluorescent increase (FIG. 2D). These results demonstrated that the DASA sensor is capable of sensing ATP in test tubes.

[0135] To optimize the performance of the DASA sensors for cellular applications, the length of the stem was systematically varied between the core of the split ATP aptamer and that of the DIR aptamer (FIGS. 3A-3D) and compared the fluorescent increase upon adding 5 mM ATP in PBS buffer (FIGS. 3E-3H). When the stem length is reduced from 7 to 3 base pairs, the fluorescence increases upon ATP binding increased from 23% to 334%. Further shortening the stem from 3 bases to 2 bases reduced the fluorescence increase to 176%. The study determined the linear ranges, limit of detection (LOD), and selectivity of DASA 2-4 sensors for ATP concentrations. The linear ranges showed a small difference between DASA- 2 (0.05-5 mM ATP), DASA-3 (0.05-25 mM ATP), and DASA-4 (0.05-2.5 mM ATP) (FIG. 2E and FIGS. 4A-4B). The LOD values for DASA-2, DASA-3, and DASA-4 were 0.7 mM, 1.0 mM, and 0.6 mM, respectively. The LOD values were calculated using the formula LOD = 3.3 x (Sy / S), where Sy is the standard deviation of the fluorescence signal response and S is the slope of the calibration curve

[0070] . Furthermore, the selectivity of the sensors was assessed by comparing the fluorescence signals when incubating the sensors with ATP, UTP, CTP, and GTP. A >550% higher fluorescence signal was observed at 650 nm when the sensor was incubated with ATP compared to the other rNTPs (FIG. 2F and FIGS. 4C-4D). The study further assessed the performance of DASA sensors at 37 °C for potential cellular applications.The DIR aptamer showed a 17-fold higher fluorescence increase, while all DASA sensors exhibited a lower fluorescence enhancement when targeting their ligands at 37 °C relative to room temperature (FIGS. 5A-5J). This reduction may be attributed to the compromised stability of the ATP aptamer structure at a higher temperature due to thermostability

[0071] . Notably, DASA-2 demonstrated minimal variation between room temperature and 37°C, therefore selected for further investigations.

[0136] Dimerization of the DASA Sensors: To improve the stability, binding affinity, and brightness of the DASA sensors for cellular applications, the DASA sensors were dimerized by adding a linker to connect two copies of the monomers

[0072] ,

[0073] ,

[0074] ,

[0075] ,

[0076] (FIG. 6A). The study evaluated the dimers of DASA-1 to -4 (called dDASA 1-4 hereafter) by comparing their fluorescence spectra with and without ATP at either RT or 37°C. In FIGS. 7A-7H, dDASA- 1 did not clearly distinguish between the presence and absence of ATP at room temperature (RT) and 37 °C. In contrast, dD ASA-2 effectively differentiated ATP presence from absence at both temperatures. dD ASA-3 exhibited a significant signal increase with ATP at RT but had a weaker signal and differentiation at 37°C. Additionally, while DASA-4 displayed strong fluorescence and differentiation as monomers, its sensing ability decreased as a dimer.

[0137] To evaluate if the length of the linker between monomers will influence the sensor performance, the study tested 4 nt, 8 nt, 10 nt, and 13 nt linkers, and found that the 10-nt linker (dDASA 2-C) exhibited strong fluorescence and substantial fluorescence enhancement when detecting ATP in PBS solution at RT and 37°C (FIGS. 6B-6C and FIGS. 8A-8H). Therefore, the study used dDASA 2-C for further experiments. When point mutations were introduced to the ATP aptamer to disrupt its binding affinity towards ATP, the mutated dDASA 2-C showed -50% lower fluorescence intensity. Interestingly, the mutant ATP aptamer still displays an increase in the fluorescence intensity upon the addition of ATP (FIGS. 6B-6C), even if the overall fluorescence intensity and increase are less than the original ATP aptamer. This result may indicate that the point mutation of the ATP aptamer made the binding affinity between the aptamer and ATP notably weaker but did not completely abolish the binding between them.

[0138] To evaluate the performance of the dDASA 2-C under RT and 37°C, the study measured its detection range and observed a linear range between 0.05-2.5 mM of ATP at RT and 0.05-5 mM ATP at 37°C, respectively (FIGS. 6D-6E). Moreover, the dDASA 2-C sensor showed good selectivity over other rNTPs under both RT and 37°C (FIGS. 6F-6G).

[0139] Temporally controlled sensing of ATP levels in living cells: After optimizing dDASA 2-C in test tubes, it was transfected into Hela cells using TurboFect transfectionreagent to assess its performance. The cells were then treated with 5 mM CaCh to increase intracellular ATP levels

[0043] ,

[0077] or 10 pM oligomycin, a commonly used inhibitor of ATP synthase

[0078] ,

[0079] to decrease ATP levels. Cells exposed to CaCF exhibited higher fluorescence intensity, while those treated with oligomycin showed reduced intensity (FIGS. 9A-9B). Introducing point mutations to disrupt the ATP binding pocket in dDASA 2-C (Mutated dDASA 2-C) resulted in displaying lower fluorescence intensity compared to the original dDASA 2-C (FIG. 9A, FIG. 9C). More importantly, Mutated dDASA 2-C displayed minimal changes with CaCh or oligomycin (FIG. 9D), indicating that its fluorescence intensity variations were linked to the interaction between ATP and ATP aptamer. Hence, dDASA 2-C effectively sensed ATP levels in living cells.

[0140] To evaluate the possibility of temporally controlled sensing, the study evaluated how fast the sensor can respond to ATP or DIR by recording the fluorescence change with a fluorometer before and immediately after adding ATP or DIR. The fluorescence signal reached a plateau in < 7s (FIGS. 10A-10D), the minimal handling time and instrument response time that could be achieved with the fluorometer. This fast response suggests that the design has a potential for temporally controlled sensing. More importantly, the fluorescence signal remained the same after more than 40 readings, suggesting that the sensor and DIR have good photostability. To further evaluate if DIR can penetrate the cell membrane without a delivery reagent at a desired time point to initiate ATP sensing, the study assessed how fast DIRFA can display observable fluorescent signals after incubating the DIR with cells. The DIRFA was delivered into Hela cells, and the fluorescence signal of the cells was recorded with a microscope (FIGS. 11A-11C). The cell showed a 52% increase in fluorescence intensity as fast as being incubated with DIR for 1 min. The fluorescence intensity increased over time and plateaued at 20 minutes, with no significant statistical differences observed beyond this point (FIG. 11A, FIG. 11C).

[0141] As a comparison, the same amount of DIRFA and DIR as used in the posttransfection incubation study was co-transfected, and a fluorescence intensity comparable to that observed at 4 minutes post-transfection of DIR was observed (p = 0.1136, FIGS. 11B- 11C). This intensity was significantly lower than the plateau achieved after post-transfection incubation (p=0.0028, when comparing the 30 min after post-transfection incubation with cotransfect the DIR), suggesting an efficient cell penetration by DIR for rapid sensing as fast as 1 minute while generating a stabilized signal after 20-30 min of DIR incubation.

[0142] Tracking ATP level in real-time in living cells: To test if the dDASA sensors can monitor the ATP levels in real-time in living cells, the study regulated intracellular ATP levelsby incubating CaCb and oligomycin alternatively after delivering the dDASA 2-C sensor into the cells. The study observed an increase in fluorescence signal with CaCh treatment and a decreased signal with oligomycin treatment (FIGS. 12A-12B, FIG. 12E), demonstrating reversible ATP monitoring with dDASA 2-C. To confirm this observation, the study used dDASA 2-C to monitor intracellular ATP levels while alternating the glucose concentrations in the cell media, since glucose is an important energy source for producing ATP

[0080] . The study observed a bright higher fluorescence signal with the high glucose media and a low fluorescence signal when treating the cells with glucose-free media (FIGS. 12C-12D, FIG. 12F), which is consistent with earlier reports

[0081] , and further supports the conclusion that the fluorescence increase of the dDASA 2-C sensor upon ATP detection is reversible and can reflect the dynamics of ATP levels inside the living cells.

[0143] Construction of genetically encoded sensors for sensing ATP levels in bacteria cells: To expand the application of the DASA sensor, the study tested the performance of the sensor in bacteria cells by delivering the dDASA 2-C into bacteria cells and incubating them with DIR and 1 mM ATP, the study observed only a small (3.9% and 4.8%, respectively) increase of fluorescence signal when comparing them with bacteria cells without ATP incubation or transformed with the mutated 2-3 that contains point mutations and weaken the ATP binding (FIG. 13A). This limited performance could be caused by the fast division of bacteria cells and subsequent dilution of the aptamer sensors. This observation is consistent with previous reports that multiple copies of the RNA aptamers are necessary for imaging its target in E. coli cells

[0010] .

[0144] To address the above issue by consistently generating the sensors in each bacteria cell, the study modified the single plasmid system established by Chen et al.

[0082] ,

[0083] ,

[0084] ,

[0085] ,

[0086] . The study used the original DIR fluorogenic aptamer, which does not contain the ATP sensing element, to optimize the system. The study designed dimers for the DIR fluorogenic aptamer (dDIRFA) and tested their performance with a fluorometer. dDIRFA, which maintained the secondary structure of DIRFA based on the UNAfold

[0087] prediction (FIG. 13B), showed significant fluorescence enhancement (226% increase with dDIRFA) when compared with negative controls that used mutated DIRFA sequence or in the absence of either the aptamer or DIR (FIG. 13C).

[0145] The study further hypothesized that single-stranded DNA (ssDNA) binding proteins can help stabilize the ssDNA products in bacteria cells and protect them from degradation since ssDNA binding proteins (SBP) are essential for stabilizing single-stranded genome regions during replication or transcription

[0088] ,

[0089] . To test this hypothesis, the study first tested ifSBP will interfere with the binding between the DIRFA and DIR. Two commercially available SBP, ET SSB and Rec A, were added to the DIRFA and their influences in the fluorescence spectrum were tested with a fluorometer. In the presence of either SBP, DIRFA showed similar fluorescence intensity as the DIRFA alone, which indicates no significant interference of SBP on the performance of the DIR aptamer (FIG. 13D). With the above observations, the plasmid for generating ssDNA in bacteria cells was constructed with the following elements: 1) An IPTG inducible reverse transcriptase (RT) from Moloney mouse leukemia virus (MoMuLV) for reverse-transcription of ssDNA; 2) A dimer form of the ssDNA of interest; 3) A rmB T1 terminator for termination of RNA polymerase; 4) A hairpin structure for termination of reverse transcription of RT; 5) A val-tRNA binding sequence for initiation of reverse transcription (val- tRNA primer) in E. coli; and 6) An IPTG inducible single-stranded DNA binding protein, betA protein, which is also known as Red[3

[0090] ,

[0091] ,

[0092] (FIGS. 14A-14B).

[0146] In evaluating this system, the expression of dDIRFA in DH10B bacteria resulted in over 40% fluorescence increase in transformed cells compared to non-transformed cells (FIGS. 14C-14D), indicating the successful generation of dDIRFA in these bacterial cells. Therefore, the sequence of dDIRFA was replaced with a dDAS A 2-C sensor to upgrade it into a genetically encoded sensor (dDIRFA 2-C plasmid). After transforming the DH10B cells with the plasmid, the cells were induced by IPTG and incubated with DIR. Then the bacteria were aliquoted into three identical groups and incubated with cell culture media only or the media containing 50 pM ATP, or 10 pM Venturicidin A (VentA), an inhibitor of bacterial ATP-synthase complexes

[0093] that depletes intracellular ATP level of bacteria cells. The fluorescence intensities of the cells were recorded with flow cytometry assay and compared between groups. The result showed that the incubation of DIR introduced a small increase in background fluorescence (FIG. 14E, No DIR vs No IPTG), while IPTG induction largely increased the fluorescence signal in the cells (FIG. 14E, No IPTG vs Medium). This observation indicates the detection of intrinsic ATP levels in the bacteria with the dDASA 2-C plasmid. A further increase of fluorescence signal was observed in a population of bacteria cells when incubating the bacteria cells with 50 pM ATP (FIG. 14E, Medium vs 50 pM ATP). In comparison, when incubating the DH10B cells with VentA, the fluorescence signal in bacteria cells decreased (FIG. 14E Medium vs 10 pM VentA). These results indicate successful detection of intracellular ATP with the genetically encoded sensor (FIG. 14E). To further confirm that the signal changes were from the expression of the plasmid of dDASA 2-C and represent its response to intracellular ATP levels, point mutations were introduced in the ATP binding site of the dDASA 2-C plasmid and the mutated dDASA 2-C plasmid, was expressed into the samesystem. While the addition of the DIR increased the fluorescence signal of mutated dDASA 2- C plasmid (FIG. 14F, No DIR vs No IPTG), adding IPTG or changing the ATP levels did not result in any changes (FIG. 14F). These results suggest that the changes in fluorescence intensity are associated with the ATP recognition and binding of the dDASA 2-C sensor. Therefore, the study successfully generated dDASA sensors for sensing ATP levels in bacteria cells.

[0147] Generation of genetically encoded sensors for sensing ATP levels in mammalian cells'. To further apply this genetically encoded sensor for detecting ATP levels in mammalian cells, the key elements were cloned into pBK-CMV under the CMV promoter to express the sensor in mammalian cells. The study used the dDIRFA to establish the system. After transfecting the plasmid expressing dDIRFA, the study observed a fluorescence increase in the cells that were incubated with DIR. In comparison, DIR did not increase the fluorescence intensity in cells without plasmid transfection. This observation indicates a successful expression of dDIRFA in mammalian cells (FIGS. 15A-15B).

[0148] To evaluate the performance of the genetically encoded sensor in Hela cells, the study replaced the sequence of dDIRFA with dDASA 2-C in the plasmid. The plasmid was cotransfected with a GFP coding plasmid to visualize the potential influence on transfection and protein synthesis efficiency under different intracellular ATP concentrations. The study observed elevated fluorescence in cells that upregulated ATP with CaCh incubation with dDASA 2-C plasmid but not mutated dDASA 2-3 plasmid or in cells that down-regulated ATP with oligomycin incubation. This observation indicates the successful detection of intracellular ATP levels with the genetically encoded sensor. To this end, the study demonstrated the potential of using genetically encoded dDASA sensors to trace ATP levels in both bacteria and mammalian cells.Conclusions

[0149] Genetically encoded sensors like fluorescence protein and RNA have been widely used for visualizing cellular events. Despite their advances, the application of genetically encoded sensors for imaging metabolites is limited. Taking advantage of the DNA aptamers, which can recognize metabolites specifically, the study developed a class of genetically encoded sensors for controlled real-time detection of metabolites in cells. The genetically encoded sensors include a dimeric aptamer DNA sensor, which conjugated a split target recognition DNA aptamer and a truncated Anorogenic DNA aptamer, termed dDASA. The dDASA were expressed with a plasmid that contains a tRNA binding sequence, terminators, dimerized sensors, and sequences that code IPTG inducible reverse transcriptase and single-stranded DNA binding protein to overcome the limitations of DNA aptamer sensors related to dilution and degradation. With the genetically encoded dDASA sensors, the study monitored the ATP levels in both bacteria and mammalian cells. Utilizing the DNA aptamers allowed the expansion of the aptamer library from both existing DNA aptamers and obtaining aptamers for new targets with SELEX. Therefore, this method signifies a major advancement in genetically encoded sensors by adding genetically encoded fluorogenic DNA aptamer as a new member, along with the fluorescent proteins and RNAs to detect small molecular metabolites as valuable tools for monitoring the real-time dynamics of metabolites and other small molecules in biological systems.Example 3: Additional Aptamers

[0150] Investigation and optimization of light-up aptamer system for detecting ATP'. Firstly, the adenosine / ATP aptamer was inserted into the left or right stem-loop of the DIR fluorogenic aptamer DIRFA to generate the DIR-ATP aptamer pair following the traditional light-up aptamer design

[0027] ,

[0044] . To evaluate the performance of these DIR-ATP aptamer pairs, a fluorometer was used to record their fluorescence spectrum in the presence or absence of 5 mM ATP. Both pairs showed only a small increase in fluorescence intensity when ATP was added to the system (FIGS. 17A-17D). Interestingly, when inserting the ATP aptamer into the right stem-loop (FIGS. 17A-17B), the DIR-ATP aptamer pair showed a higher absolute fluorescence intensity than the pairs that inserted the ATP aptamer into the left loop (FIGS. 17C-17D). Based on these results, the study focused on inserting the adenosine / ATP aptamer into the right stem-loop of the DIR aptamer to generate the DIR-ATP aptamer pair for further optimizations. To test the influence of the position where the DIR aptamer was split on the performance of the DIR-ATP aptamer pair, the study tested all five positions in the right loop of the DIR aptamer for inserting the adenosine / ATP aptamer (FIG. 18A, Rl-5). The study used the fluorometer to record and compare the changes in fluorescence intensity for each DIR-ATP aptamer pair before and after the addition of 5 mM ATP. The study observed the highest fluorescence intensity when inserting the ATP aptamer at the R1 and R2 positions. However, when the study compared the quantitative increase of fluorescence intensity at 650 nm, the peak of the emission spectrum, the largest increase was observed when the ATP aptamer was inserted at the R4 position (FIGS. 18A-18F). Despite the differences, all the designs showed <50% fluorescence increases upon the addition of 5 mM ATP.

[0151] To optimize the fluorescence increase upon binding ATP with the traditional design, that linked the ATP aptamer in the right loop of DIRFA, the length of the stem between the cores of the DIR and ATP aptamer was adjusted by using a shorter or longer version of theATP aptamer that was reported by Huizenga et al.

[0037] . The tests were focused on the positions R1 and R4, since they showed the highest absolute fluorescence intensity and the largest increase upon addition of ATP, respectively. When the length of the stem was a few bases shorter, the DIR-ATP aptamer pair showed a fluorescent increase in the presence of ATP (FIGS. 19A-19B). In contrast, when the length of the stem was a few bases longer, the sensor lost the ability to sense ATP (FIGS. 19D-19E). Interestingly, when the stem was further shortened, the study observed a decrease in fluorescence intensity when adding ATP to the system (FIG. 19C, FIG. 19F). This observation indicates that there is likely an optimal length of the stem for the best performance of the aptamer pair. This indication matched the earlier finding that the length of the stem between the light-up aptamer and the aptamer for analytes regulates the sensing performance of the aptamer pair

[0027] . However, none of these designs allowed the aptamer pairs to achieve > 50% increase after the addition of 5 mM ATP.

[0152] As an alternative, the study constructed a design using the ATP aptamer to block the secondary structural formation of DIR aptamer first and then undergo a structural switching to release the blockage upon binding ATP and thus allow the binding of DIRFA to DIR dye that results in a fluorescent increase (FIG. 20A). However, this design resulted in only a 23% increase in fluorescence intensity when ATP was added (FIG. 20B). These results may be due to the differences between the binding affinities of the two aptamers, as the stronger binding affinity between DIR and DIRFA (Ka = 0.65+0.17pM)

[0042] may not be efficiently overcome by the structural switching of the ATP aptamer upon binding to ATP, because the binding affinity between ATP and its aptamer is one order of magnitude weaker (Kd ~ 6 pM)

[0037] ,

[0045] . As a result, the secondary structure of DIRFA may be restored in the presence of DIR regardless of the presence or absence of ATP.Example 4: Genetically Encoded Fluorogenic DNA aptamers for Imaging Metabolite in Living Cells

[0153] Genetically encoded sensors employing fluorescent proteins have revolutionized biochemical and biomedical imaging. These protein-based sensors fuse fluorescent protein with a target molecule or a target-specific binding module via a short peptide linker [1-3]. The recognition of target molecules induces conformational changes in the protein construct [4-10] or regulates the synthesis of the fluorescent protein [11-13], resulting in detectable fluorescence signal changes in living cells. Despite significant advancements in this field, developing protein-based sensors for diverse small-molecule analytes remains challenging due to the limited availability of proteins that bind a wide variety of small molecules and the timeconsuming linker optimization [14-16]. Since numerous metabolites are present in cells,developing a method that can be generally applied to almost all metabolites is desirable. Toward this goal, Anorogenic RNAs combined with riboswitch added the toolbox of genetically encoded sensors for imaging small metabolites [17-30]. These fluorogenic RNAs undergo conformational changes upon binding to their targets, leading to an increase in fluorescence. Despite their adaptability to various analytes (e.g., SAM, TPP) [28,31], these fluorogenic RNA sensors have half- lives ranging from 0.25 to 1.25 h in cells

[0032] . While circularized RNA sensors extend their half-lives by 5- to 10-fold (7-10 h)

[0033] , they still exhibit lower stability compared to protein sensors. Therefore, it is desirable to increase stability, while increasing the number of targets that genetically encoded sensors can detect in living cells.

[0154] Compared to riboswitches or RNA aptamers, DNA aptamers can be obtained more easily through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) [34,35], because the selection does not require reverse transcription. As a result, DNA aptamers that can bind a wide variety of targets, including small molecular metabolites, have been obtained and thus significantly expand the target molecules one can detect [36-39]. In addition, DNA aptamers also exhibit higher stability than proteins and RNAs [40,41], positioning them as effective alternatives for metabolite sensing. Due to these advantages, DNA aptamers have been widely used to image and detect small molecules for over a decade [42-57]. Despite the above advantages and decades of research in this area, most DNA aptamer sensors for intracellular applications reported to date still require prelabeling with a fluorophore and then delivery into the cells. As a result, delivery efficiency and locations can vary widely. More importantly, since the cells are dividing and replicating, the delivered prelabeled DNA aptamers are constantly diluted, making them ineffective for long-term monitoring beyond the first generation of cells. To overcome these limitations and to maximize the full potential of DNA aptamers in imaging metabolites in cells, a study was conducted to develop a Genetically Encoded Fluorogenic DNA Aptamers (GEFDA) sensor that enables continuous expression of the aptamers even after cell division. However, GEFDA has never been demonstrated before.

[0155] The development of GEFDA sensors has two major challenges. First, although fluorogenic DNA aptamers such as those binding MG [58,59], dapoxyl

[0060] crystal violet

[0061] Hoechst

[0062] , and Auramine O

[0063] have been developed for detecting small molecules in controlled environments [60,63] most of their chromophores exhibit high autofluorescence, low specificity, high cytotoxicity, and poor signal-to-noise ratio (around or below 30% increase in fluorescence upon binding to their aptamers). The recent development of the Lettuce aptamer64 employs chromophores with low cytotoxicity [65-68] and was expressed in bacterial cells with a retron system

[0069] . However, this fluorogenic DNA aptamer has not beenshown to work in mammalian cells, because of the differences in regulatory elements of gene expression between bacterial and mammalian cells, and thus limited its applications. More importantly, the Anorogenic aptamer needs to be coupled to a target recognition element to create GEFDA sensors for sensing metabolites

[0070] . It is challenging to express the coupled sensors that the binding of a target can transduce an increase of Auorescence signals inside the cells.

[0156] To meet these challenges, this study reports the first GEFDA sensor for imaging metabolites in live bacterial and mammalian cells. To achieve the goal, the study first evaluated the Anorogenic Lettuce aptamer and dimethylindole red (DIR) specific Fluorogenic Aptamer (termed DIRFA hereafter)

[0071] , and identified DIRFA as an excellent choice of Auorogenic aptamer in living cells, as it displays a > 10-fold increase in red Auorescence at 650 nm compared to its mutated control in a buffer mimicking the cellular environment. Encouraged by the result, the study coupled DIRFA with an ATP aptamer

[0072] to develop a DIRFA-based Split Aptamer (DASA), which turns bright red upon forming a DASA / DIR / ATP complex. To improve the signal-to-noise ratio and stability in living cells, the study constructed a dimeric DASA (dDASA) with dual chromophore and analyte bindings. This dimeric dDASA construct exhibited a 60% increase in brightness compared to the monomeric DASA sensor and displayed responsive detection of ATP levels in cells over 5 h. By integrating dDASA into plasmids that encode reverse transcriptase and a single- stranded DNA binding protein , the study created the GEFDA sensor to image ATP in live bacterial and mammalian cells. This platform has the potential to be adapted to express other DNA aptamers for imaging different metabolites in cells.Experimental Procedures

[0157] Characterization of DASA and dDASA fluorogenic aptamer sensors with fluorometer or plate reader. DNA sequences were ordered from IDT with standard desalting. Upon receiving, the DNA sensors were dissolved in Milli-Q water. For each Auorometer test, a 200 pL reaction system was used, which includes lx PBS buffer, 0.5 pM DIR, and 0.5 pM DNA sensor or control DNA. When sensing ATP or other analogous, a master mix was made with the sensor and DIR, and then different concentrations of analytes were spiked into the system before detection. ATP was dissolved in buffer and the pH was adjusted to 7.0 before spiking it to the system. Serial dilutes were made when studying the detection range of the sensors. A FluoroMax-P Auorometer (HORIBA Jobin Yvon Inc., Edison, NJ, USA), ISS ChronosDFD Auorometer (ISS Inc., Champaign, IL, USA), or BioTek Synergy Hl Plate Reader were used for the luminescent spectra measurements. Fluorescence was excited at 618nm and a spectrum from 630-750 nm was recorded. When plotting the sensing curves with a single emission wavelength, the fluorescence intensity was presented at 650 nm, the emission peak of all the DIRFA-based sensors. When testing the samples at 37°C, the mixtures were pre-warmed at 37°C with a heating block before transferring them into the fluorometer. Temperature control of the fluorometer was used during the detection.

[0158] Characterization of DASA and dDASA fluorogenic aptamer sensors with fluorescence microscopy: Solutions of DASA+DIR, DASA+DIR+ATP, dDASA+DIR, and dDAS A+DIR+ATP were mixed at the above concentration and placed in an 8-well Lab-Tek™ chambered cover glass (Thermo Fisher Scientific, Cat. No. 154534) for optical imaging. A 635 nm diode laser served as the excitation source, passing through a multi -band dichroic mirror to illuminate the samples. The emitted fluorescence was collected along the same optical path and detected using an avalanche photodiode (SPCM-AQR-15, Perkin Elmer) after filtering through a 685 / 40 nm band-pass filter (Semrock). Fluorescence images were acquired using a 60x, 1.2 NA water immersion objective (CFI Plan Apochromat Lambda 60x C objective lens (Nikon, Japan)). Each image (256x256 pixels) was scanned with a dwell time of 0.1 ms / pixel. Bar plots were generated from the fluorescence intensity values of each pixel (n=65,536) for comparative analysis.

[0159] Manipulation of ATP levels in mammalian cells: HeLa cells were obtained from ATCC and cultured in DMEM. Cells were seeded into 6-well plates and allowed to grow overnight before conducting experiments. To elevate ATP levels, cells were incubated with either DMEM supplemented with 25 mM glucose or Opti-MEM containing 5 mM CaCF. To decrease ATP levels, glucose-free DMEM or Opti-MEM containing 10 pM oligomycin was used. Cells were incubated in these conditions for 1 hour, followed by a wash with lx PBS. The cells were then incubated with 0.025% trypsin for 5 minutes. After trypsinization, an equal volume of DMEM was added to the cell suspension, and the cells were centrifuged at 3000 rpm for 3 minutes. The supernatant was removed, and the cells were resuspended in 200 pL of PBS. A 10 pL aliquot of cells was used for counting cell density, and the remaining cells were diluted to a concentration of approximately 2xl05cells / mL. The diluted cells were recounted, and ATP concentrations were measured using the Sigma adenosine 5 '-Triphosphate Bioluminescent Somatic Cell Assay Kit. Bioluminescence was recorded both before and after cell lysis, and the absolute bioluminescent intensity was normalized to cell number.

[0160] Utilizing dDASA sensors for imaging ATP levels in mammalian cells: HeLa cells were seeded into 8-well strips at the concentration of 4.2x104cells per well one day before the experiments. During the transfection, 150 pL transfection system that includes opti-MEM, 0.4pM synthesized DNA sensor, control DNA, or 300 ng plasmid, and 1 pL TurboFect transfection reagent, was vortexed briefly and then stood at 37 °C for 15 minutes before adding to the cells. The cells were incubated in the transfection system for 4 hrs, and were washed twice with PBS, then changed into DMEM before imaging. To regulate cellular ATP levels, the cells were incubated with 5 mM CaCh or 10 pM oligomycin (contains 0.1% DMSO) in opti-MEM, 25 mM glucose in DMEM, or glucose-free media after transfection. 1 hr after manipulating the ATP level, 1 pM DIR was added to the system for 30 minutes of incubation. The cells were stained with 1 :1000 Hoechst 33342 for 12 minutes and washed twice with PBS before changing into opti-MEM for imaging.

[0161] To test the cell membrane penetrability of DIR, DIRFA was directly delivered into HeLa cells with the Turbofect transfection reagent. After the transfection, the cells were washed three times with PBS to remove transfection reagents and excess aptamers. The study then added 1 pM DIR to the cell culture media and started recording the fluorescence emission of the cells every 5-10 mins under a ZEISS AXIO observer microscope.

[0162] To test the real-time sensing capability in live cells, dDASA sensor and DIR were co-delivered to the cells, and the intracellular ATP levels were regulated by incubating cells in DMEM with 25 mM additional glucose for 1 hr, followed by incubating in glucose-free DMEM for 1 hr, and repeated two cycles. Alternatively, the cells were treated with 10 pM oligomycin for 1 hr or 5 mM CaCh in opti-MEM for two cycles. The cells were washed twice with PBS in between the medium changes. The same cell populations were tracked during the treatments.

[0163] Transform and express dDASA sensors in bacterial cells: High-efficiency competent DH10B bacterial cells were brought from NEB (C3019H). The dDASA sensors were transformed into competent bacterial cells following the standard protocol. After transformation, the cells were resuspended into SOC buffer and recovered at 37 degrees for 1 hr. DIR was added to the culture media at a concentration of 0.5 pM 20 min before detection with a flow cytometer.

[0164] One plasmid system for generating single-stranded DNA (ssDNA) in bacterial cells was constructed through modification of the system by Chen et al. [118-122]. The study adopted the high-copy-number origin of replication from pUC19, the kanamycin resistance gene, and the lad gene and Plac promoter from the pFNK-101 plasmid (GenBank accession number AY952935), a synthesized gBlock DNA fragment containing the Moloney mouse leukemia virus (MoMuLV) reverse transcriptase (RT) gene, a terminator sequence (SL) for reverse transcription, the GEFDA sequence and val-tRNA binding sequence, and the transcription terminator Tl. These components were assembled to generate the plasmid pUC-RT-GEFDA through Gibson assembly. To increase the productivity and stability of the singlestranded GEFDA, the gene for protein from phage lambda was cloned from pKD46 (GenBank accession number MF287367) into pUC-RT-GEFDA, generating the plasmid pUC- RT-GEFDA- . Additionally, the DNA was designed into a dimer to minimize the exposure of the end of DNA to nucleases. The dimer of the DIRFA was used for the initial demonstration, and the dDASA sensor was cloned to replace the dDIRFA. Gblocks were synthesized with GeneArt Gene and Protein Synthesis Services from Thermo Fisher Scientific in a pMA-RQ (AmpR) plasmid. Pad and Xhol were used to double digest the plasmids, and the fragments were ligated with T4 ligase after agarose gel-based product purifications. Sanger sequencing was performed to confirm the insertion and sequence of the main compounds.

[0165] Plasmids that code the sensors or control DNA were transformed into DH10B competent cells following the standard protocol. Fresh clones were picked and inoculated into TB media containing 50 pg / ml of kanamycin and 10 mM of IPTG. Then the cells were grown in a shaking incubator at 37°C overnight until the culture reached the early stationary phase. The cells were collected and detected as soon as possible. The ATP level of bacterial cells was regulated by incubating with 50 pM ATP or 10 p.M Venturicidin A in the culture media. DIR was added to the culture media at a concentration of 0.5 pM 20 min before detection.

[0166] Flow cytometry assay of bacterial cells'. The fluorescence intensities of the cells were recorded with a DB LSRFortessa Flow Cytometer, where either the APC or the PE-Cy5 channel was employed for analysis. When processing the data from the flow cytometer, bacterial cells were gated by size based on side scattering (SSC) and forward scattering (FSC) readings. As a result, only singlets were isolated from the bacterial cell population. Following this, cells exhibiting fluorescence intensity above 103were classified as fluorescent cells, while those below 103were categorized as non- fluorescent cells.

[0167] Express DASA sensors in mammalian cells: pBK-CMV plasmid was used as the backbone to construct the mammalian cell expression system. The ssDNA expression systems that contain sequence coding P protein, sequences of the dDASA sensor, and reverse transcriptase sequence were obtained by doubly digesting the plasmids using Xbal and Avril, and by agarose gel purification to collect the correct DNA fragments. Next, pBK-CMV plasmid was linearized by PCR with a 30-bp homologous sequence added to each of the two ends. The primers used to linearize pBK-CMV plasmid were SEQ ID NO: 1 and SEQ ID NO: 2.

[0168] NEBuilder HiFi DNA Assembly Master Mix (NEB) was then used to assemble the linearized pBK-CMV back-bone with the DNA fragments that contain the ssDNA expression system. NEB 5-ahpla competent E. coli cells were used for transformation and plasmids werepurified using QIAprep Miniprep Kit (QIAGEN). 300 ng of the correctly constructed plasmids were transfected into HeLa cells with the same protocol described in the ATP level imaging in the mammalian cell sensing section. After 4 hrs of transfection, the medium was changed to DMEM, and the cells were cultured overnight. Right before the sensing experiment, the ATP level was up-regulated and down-regulated with 5 mM CaCh and 10 pM oligomycin in opti- MEM medium, respectively. 30 min after manipulating the ATP level, the cells were incubated in 1 pM DIR and 1 : 1000 Hoechst 33342 for 30 min and washed with PBS twice before imaging with a Nikon spin disk confocal microscope at 37°C in the opti-MEM medium. Cell images were acquired in three channels, where the 640 nm channel is for imaging the DIRFA -based sensors in the cytoplasm, the 488 nm channel is for imaging eGFP in the cytoplasm, and the 408 nm channel is for imaging the Hoechst dyes in genomic DNA.

[0169] For tracking the real-time ATP levels, the cells were cultured in regular DMEM overnight after 4 hrs of transfection. Right before the sensing experiment, the cells were incubated in 1 pM DIR for 1 hr, and then ATP level was regulated with 5 mM CaCh or 10 pM oligomycin in opti-MEM medium, or 25 mM glucose in DMEM and glucose-free DMEM. The same cell populations were recorded with ZEISS microscopy during the treatments.

[0170] Statistical analysis'. The LOD values were calculated using the formula LOD = 3.3 x (Sy / S), where Sy is the standard deviation of the fluorescence signal response and S is the slope of the calibration curve

[0123] . All experiments were repeated at least three times independently. The standard error of the mean was used as the error bars. GraphPad was used to analyze and present data. Fluorescence intensity in the cellular images was quantified using Fiji (ImageJ

[0124] ) by measuring the mean intensity of each cell. For each treatment group, five cells per image were analyzed across five images. Cells exhibiting red fluorescence signals above background levels, indicating successful sensor transfection, were randomly selected from the four corners and center of each image. Two-tailed distribution and two-sample equal variance t-tests were used for analyzing significance, used to quantify if the differences between two different groups were significant or not.Results and Discussion

[0171] Development and Characterization of DIRFA-Based Split Aptamer (DASA) Sensors: To develop a Anorogenic DNA sensor for intracellular sensing and imaging, the study started with the Lettuce aptamer

[0064] and the dimethylindole red (DIR) specific Fluorogenic DNA Aptamer DIRFA

[0071] . The Lettuce aptamer exhibited a 5-fold and 7-fold increase in Auorescence at 505 nm when bound to DFHBI in HEPES buffer (40 mM HEPES, pH 7.5, adjusted with NaOH; 100 mM KC1; and 1 mM MgCh) and Tris buffer (100 mM NaCl; 20 mMTris-HCl pH 7.4; 2 mM MgCh; 5 mM KC1; 1 mM CaCh), respectively, compared to the mutated control in which two conserved bases were altered to prevent target binding.64 However, in PBS buffer, which displays pH and salinity closer to intracellular conditions, the fluorescence enhancement was minimal (FIGS. 1A-1B). In contrast, DIRFA showed a 7- to 11 -fold increase in fluorescence when compared to its mutated control in PBS buffer (FIG. IB). These findings signify the potential of the DIRFA / DIR complex for cellular sensor applications.

[0172] To compare the performance and stability of the DIRFA with well-established RNA aptamers, the study included the broccoli fluorogenic RNA as a benchmark

[0066] . The study tested the fluorescence intensity of DIRFA and broccoli in 50% cell lysates in PBS buffer. A decrease in fluorescence intensity was observed for the broccoli aptamer starting within 40 min, while the fluorescence intensity of the fluorogenic aptamer DIRFA remained stable over a 4 h tracking period (FIG. 21 A). The linear regression of the fluorescence signal revealed a significant decrease in slope for the RNA Broccoli aptamer compared to the DNA DIRFA aptamer, suggesting higher stability for the DIRFA aptamer under the tested conditions (FIG. 21B). Consequently, the DIRFA was selected as the signal output throughout this article.

[0173] To understand the function of stem DI, a truncational analysis was carried out by removing one base pair at a time (FIG. 2A). Notably, removing more than 3 base pairs led to an 80% reduced fluorescence intensity at 650 nm when compared to the canonical DIRFA (FIGS. 2B-2C). Based on this observation, it was hypothesized that the stem DI is critical for the light-up effect and could function as a transducer region when linked with a target recognition aptamer to develop a small-molecule sensor.

[0174] To test the above hypothesis, the study used the ATP aptamer as a proof of concept since the ATP aptamer has been extensively researched and applied in sensing fields [73-77]. The stem DI was replaced with a split ATP aptamer [78,79] (FIG. 2D). In the absence of ATP, the structure of stem D 1 was disrupted since the two strands of the split ATP aptamer were not perfectly base paired with each other. In contrast, the binding of ATP stabilized this region, and hence, restored the fluorogenic property of the DIRFA. This design was termed as DIRFA- based Split Aptamer (DASA) sensor and showed a 244% fluorescence increase at 650 nm upon the addition of 5 mM ATP when excited at 618 nm and tested with a fluorometer (FIGS. 2E- 2F, using the same parameters hereafter unless specified otherwise). Introducing point mutations in either the split ATP aptamer, DIRFA, or both led to reduced fluorescence signal changes (FIGS. 2E-2F). These results demonstrated that the DASA sensor is capable of sensing ATP in solution.

[0175] To optimize the performance of the DASA sensors for cellular applications, the study systematically varied the length of the stem DI region from 7 base pairs (bp) to 5, 3, and 2 bp, named DASA-1, DASA-2, DASA-3, and DASA-4, respectively (FIGS. 3A-3D) and compared the increase of fluorescence signals upon adding 5 mM ATP in PBS buffer (FIGS. 3E-3H). When the length was reduced from 7 bp to 5 bp and then to 3 bp, the fluorescence upon ATP binding increased from 25% to 168% and 256%, respectively. Further shortening the stem from 3 bases to 2 bases reduced the fluorescence enhancement to 194%. Notably, as the stem DI region is shortened, the fluorescence intensity of the sensor decreases, reaching its lowest point at 3 base pairs. Surprisingly, the fluorescence partially recovers when the stem is further reduced to only 2 base pairs (FIGS. 3A-3H). The study used UNAfold to evaluate the potential conformations of the sensor under the conditions of 137 mM Na+and 0 mM Mg2+, matching the salt condition of the PBS buffer used in the experiments. The results indicate that as the stem shortens, the AG of the DIRFA structure increases, and the melting temperature decreases (TABLE 2), which suggests that the formation of the DIRFA structure is more difficult with shorter stems, consistent with the observed decrease in fluorescence and sensor response. However, the 3-base pair variant (DASA-3) is an exception, as it did not favor the DIRFA structure. The DIRFA structure was absent from the top 13 predicted conformations returned by the software, which may explain its lower fluorescence intensity compared to the 2-base pair design. Given the software’s limitations, particularly its inability to account for ATP binding and concentration, further investigation is needed to fully understand this behavior.TABLE 2. Summary of predicted structural stability and melting temperatures for DASA variants.

[0176] The linear ranges of fluorescence intensity showed a small difference between DASA-2 (0.05-5 mM ATP), DASA-3 (0.05-25 mM ATP), and DASA-4 (0.05-2.5 mM ATP)(FIGS. 4A-4C). The LOD values for DASA-2, DASA-3, and DASA-4 were 0.7 mM, 1.0 mM, and 0.6 mM, respectively.

[0177] To evaluate sensor specificity for ATP over other nucleotides, the DASA sensors were incubated with UTP, CTP, and GTP. Minimal fluorescence was observed for these nucleotides, while ATP incubation produced a 9-fold increase in fluorescence intensity at 650 nm, demonstrating strong selectivity for ATP over other nucleotide triphosphates (FIGS. 4D- 4F). This selectivity aligns with previous findings for ATP-specific aptamers

[0075] . However, it is important to note that the sensor may also interact with other adenosine-containing molecules, as the ATP aptamer primarily recognizes the adenosine moiety of ATP

[0072] . Given that this ATP aptamer is well-characterized, the study used it as a proof of concept to demonstrate the potential of the sensing platform.

[0178] The study further assessed the performance of DIRFA and DASA sensors at 37°C for potential cellular applications. The DIRFA showed a 23 -fold fluorescence increase when compared with the mutated DRIFA at 37°C (FIGS. 5A-5J). This increase is higher than the 8- fold increase at room temperature, indicating a better signal-to-noise ratio at 37°C. While the fluorescence intensity of the D1RFA / D1R complex at 650 nm is comparable between both temperatures, most of the DASA sensors exhibited a 3-8-fold decrease in fluorescence intensity at 37°C than at room temperature. Moreover, the fluorescence enhancement of DASA-2, DASA-3, and DASA-4 with ATP incubation was 20%-90% lower at 37°C relative to room temperature. This reduction may be attributed to the compromised stability of the ATP aptamer structure at a higher temperature due to thermostability

[0080] .

[0179] Dimerization of the DASA Sensors'. To enhance the stability, binding affinity, and brightness of DASA sensors for cellular applications, the study explored the potential improvements achieved by dimerizing the sensors, linking two monomer units together [81-85] (FIG. 6A). As a proof of concept, the study dimerized DIRFA (FIG. 22A) to evaluate the performance of the dimeric DIRFA (dDIRFA) relative to its monomeric form. In PBS buffer, dDIRFA demonstrated a 215% increase in fluorescence intensity at 650 nm compared to the mutated DIRFA. This increase mirrors the fluorescence difference observed between the monomeric DIRFA and its mutated form, suggesting that dimerization did not significantly affect fluorescence generation (FIG. 22B).

[0180] To evaluate whether the dimerization has the potential to enhance the stability and performance of the Anorogenic aptamer under cellular environments, the study compared the dimeric DIRFA (dDIRFA) and monomeric DIRFA in a 20% cell lysate environment. A 40% improvement in the signal-to-noise ratio, with a 35% increase in Auorescence intensity, wasobserved for dDIRFA compared to DIRFA (FIGS. 22C-22D). To assess the stability of these sensors in cells, the study delivered them into HEK293 cells. The signal-to-noise ratio, which measures the fluorescence difference between cells transfected with aptamers and those transfected with empty vehicles was compared. A significantly higher signal-to-noise ratio was observed for the Anorogenic DNA aptamers than that of the RNA aptamer. Specifically, the signal-to-noise ratios were 1.25 for broccoli RNA aptamer and 7.62 for DIRFA (FIGS. 23A- 23F, 0 h). Similar to the results observed in cell lysis, the dimerization of DIRFA resulted in an improved signal-to-noise ratio (9.59, FIGS. 23A-23F, 0 h). To evaluate the stability of these Auorogenic aptamers in living cells, the study monitored Auorescence over time. Seven hours post-transfection, the broccoli RNA no longer produced a signal above the noise level, while DIRFA and dDIRFA maintained signal-to-noise ratios of 6.08 and 10.09, respectively (FIGS. 23A-23F, 7 h). At 21 h, DIRFA’s ratio had decreased to 3.35, but dDIRFA remained stable with a ratio of 10.03 (FIGS. 23A-23F, 21 h). These results demonstrate that the DNA-based aptamer (DIRFA) provides greater stability than the RNA-based broccoli aptamer, and that dimerization further enhances DIRFA’s stability. Given its superior performance, the study dimerized DASA sensors (dDASA) for intracellular applications.

[0181] Recognizing that Mg2+ion concentrations can influence the fluorescence signals of Auorogenic aptamers [86,87], the study evaluated the behavior of the dDASA sensor in the presence and absence of Mg2+. The in test tube characterizations, conducted in PBS without additional Mg2+, demonstrated that the sensing performance of dDASA is independent of Mg2+, unlike some Auorogenic RNA aptamers

[0088] . While increasing Mg2+concentrations at physiologically relevant levels caused minor fluctuations in overall Auorescence intensity (FIGS. 24A-24B), the ratio of the 5 mM ATP signal to the background (without ATP) remained relatively stable (FIGS. 24C-24D). These findings highlight the robustness of the dDASA sensor, making it well-suited for complex biological environments, such as cellular systems, where Mg2+levels may vary. However, given that Auorescence intensity can be inAuenced by Mg2+concentrations, it remains essential to include appropriate controls when analyzing small metabolite concentrations in such settings. These controls ensure accurate interpretation of results and enhance the sensor’s reliability in practical applications.

[0182] To optimize the design of dDASA, the study evaluated the dimers of DASA-1 to - 4 (called dDASA- 1 to -4 hereafter) by comparing their Auorescence spectra with and without ATP at either room temperature (22°C) or 37°C (FIGS. 7A-7H). When compared between FIGS. 5A-5J and FIGS. 7A-7H, the Auorescence intensity of the DASA sensors under both temperatures increased from 15% to a few folds after dimerization. In evaluating ATP sensingcapabilities, dDASA-1 showed limited discrimination between ATP presence and absence at both temperatures. In contrast, dDASA-2 effectively distinguished ATP presence across both temperatures. While dD ASA-3 produced a significant signal increase in the presence of ATP at room temperature, its response weakened at 37°C, showing reduced signal strength and differentiation. Additionally, although dDASA-4 demonstrated strong fluorescence and differentiation in its monomeric form, its sensing ability diminished post dimerization. Based on the sensing performance of DASA monomers and dimers across both temperatures, dDASA-2 was selected for further investigation.

[0183] To evaluate if the length of the linker between monomers will influence the sensor performance, the study tested 4-nt, 8-nt, 10-nt, and 13-nt linkers, and found that the 10-nt linker (dDASA 2-C) exhibited strong fluorescence and substantial fluorescence enhancement when detecting ATP in PBS buffer at both temperatures (FIGS. 6B-6C, FIGS. 8A-8H, FIGS. 25A- 25B). Therefore, the study used dDASA 2-C for further experiments.

[0184] Introducing point mutations into the ATP aptamer to disrupt its binding affinity resulted in approximately 2-fold lower fluorescence intensity in the mutated dDASA. The mutated aptamer exhibited a small but statistically insignificant fluorescence response upon ATP addition (FIGS. 6B-6C, FIGS. 25A-25B). This small response could be attributed to the single-point mutation affecting only one binding site

[0089] . To evaluate the performance of the dDASA 2-C under both temperature conditions, the study measured its detection range and observed nearly 2 orders of magnitude dynamic range (linear range between 0.05 and 2.5 mM of ATP at room temperature and 0.05-5 mM ATP at 37°C, respectively, FIGS. 6D-6E). Moreover, the dDASA 2-C sensor showed good selectivity over other rNTPs under both temperatures (FIGS. 6F-6G).

[0185] The study further evaluated the performance of both dimeric and monomeric forms of DASA sensors, specifically using the top-performing sequences dDASA 2-C and DASA-2 (hereafter referred to as dDASA and DASA, respectively), at room temperature through fluorescence microscopy

[0090] . The study tested the performance of the probes under an excitation wavelength of 635 nm and an emission range of 685 / 40 nm, as these conditions are optimal for near-infrared fluorescent probes used in cellular imaging. This wavelength setup was selected to reduce phototoxicity and minimize spectral crosstalk with probes excitable by visible light. Additionally, these parameters were chosen to decrease scattering and absorption within mammalian tissues, thereby improving imaging clarity and accuracy

[0091] . Under these conditions, dDASA demonstrated a 155% increase in fluorescence intensity when sensing 5mM ATP, compared to a 43% increase observed for DASA, further suggesting that dimerization enhanced the ATP sensing capability of the DASA sensor (FIGS. 26A-26B).

[0186] Temporal Control of ATP Sensing in Living Cells: After optimizing the dDASA sensor in test tubes, the study transfected it into HeLa cells using the TurboFect transfection reagent and assessed its performance. The cells were then treated either with 5 mM CaCh to increase intracellular ATP levels [75,92] or 10 pM oligomycin, an ATP synthase inhibitor [93,94], to decrease ATP levels. Cells exposed to CaCh exhibited higher fluorescence intensity, while those treated with oligomycin showed reduced intensity (FIGS. 9A-9B). Introducing point mutations to disrupt the ATP binding pocket in dDASA (Mutated dDASA) resulted in displaying lower fluorescence intensity compared to the canonical dDASA (FIG. 9A, FIG. 9C). More importantly, Mutated dDASA displayed minimal changes with CaCh or oligomycin (FIG. 9D), indicating that its fluorescence intensity variations were associated with ATP binding activity. The study further confirmed the modulation of cellular ATP levels with these treatments using a Sigma adenosine 5 ’-triphosphate bioluminescent somatic cell assay kit (FIG. 27). Hence, it was concluded that dDASA could effectively sense ATP levels in living cells.

[0187] To evaluate the possibility of temporally controlled sensing, the study evaluated how fast the sensor can respond to ATP or DIR by recording the fluorescence change with a fluorometer before and immediately after adding ATP or DIR. The fluorescence signal reached a plateau in <7s (FIGS. 10A-10D), the minimal handling and instrument response time that could be achieved with the fluorometer. This fast response suggests that the design has a potential for temporally controlled sensing. To evaluate if DIR can permeate cell membranes at a desired time point to initiate ATP sensing, the study used DIRFA to assess how fast the aptamer displays observable fluorescent signals after incubating the DIR with cells. The study delivered the DIRFA into HeLa cells and recorded the fluorescence signal of the cells with a microscope (FIGS. 11A-11C). The cell showed a 52% increase in fluorescence intensity as fast as being incubated with 1 pM DIR for 1 min. The fluorescence intensity increased over time and plateaued at 20 min, with no significant statistical differences observed beyond this point (FIG. 11A, FIG. 11C).

[0188] For comparison, the study cotransfected the same amount of DIRFA and DIR as used in the post-transfection incubation study. This cotransfection resulted in a fluorescence intensity comparable to that observed 4 min after the addition of DIR post-transfection (p - 0.1136, FIGS. 11B-11C). However, this intensity was significantly lower than the plateau level observed after 30 min post-transfection incubation period (p = 0.0028, comparing the 30 minpost- transfection incubation with the cotransfection of DIR). These findings suggest that DIR efficiently penetrates cells and enables rapid sensing within as little as 1 min, while a stabilized signal is achieved after 20-30 min of incubation.

[0189] Real-Time ATP Tracking in Living Cells: To assess whether the dDASA sensor can monitor ATP levels in real-time in living cells, the study modulated intracellular ATP levels by alternating glucose concentrations in the cell culture media, given that glucose is a key energy source for ATP production

[0095] . After delivering the dDASA sensor into HeLa cells, the study changed the concentration of glucose alternatively. A higher fluo-rescence signal was observed in cells cultured in high-glucose media (25 mM added glucose) and a reduced signal in cells exposed to glucose-free media (FIG. 12A, FIG. 12C), indicating elevated ATP levels in high-glucose conditions and lower ATP levels in glucose-deprived conditions. These findings are consistent with previous studies

[0096] and corroborate results obtained from the ATP bioluminescent assay kit (FIG. 27). To further validate these observations, the study used dDASA to monitor ATP levels by alternately treating cells with 10 pM oligomycin and 5 mM CaCh. A decrease in fluorescence was observed with oligomycin treatment, indicating reduced ATP levels, while an increase in fluorescence followed CaCh treatment, reflecting elevated ATP levels (FIG. 12B, FIG. 12D). These results confirm that dDASA can reversibly track ATP levels in real-time. This also reinforces the conclusion that the fluorescence increase of the dDASA sensor upon ATP binding is reversible and can effectively reflect ATP dynamics in living cells.

[0190] Genetically Encoded Fluorogenic DNA Aptamer (GEFDA) Sensors for ATPDetection in Bacterial Cells: To expand the application of the DASA sensor, the study transformed dDASA and its variant into DH10B bacterial cells and incubated them with DIR and 1 mM ATP. A marginal fluorescence increase was noticed in comparison to both ATP- absent condition (3.9%) and bacterial cells transformed with a mutated ATP aptamer (4.8%) (FIG. 13A), presumably due to the fast division of bacterial cells and subsequent dilution of the aptamer sensors. This observation is also consistent with previous reports that multiple copies of the RNA aptamers are necessary for imaging target molecules in E. coli cells

[0097] .

[0191] It was hypothesized that consistently generating small-molecule sensors in bacterial cells would address the above issue. To synthesize dDASA in DH10B cells, the study used reverse transcriptase-based systems because, unlike the retron system [98-100], it produces only the desired DNA without generating additional byproducts. The study tested both a three- plasmid system

[0101] and a modified single-plasmid system adapted from Chen et al. [102-106]. As a proof of concept, the study tested the system with a 72-nt single-strandedDNA (ssDNA) but initially observed no DNA product (FIGS. 28A-28B). It was hypothesized that the ssDNA might be rapidly degraded, as ssDNA binding proteins (SBPs) are critical for stabilizing single-stranded regions during replication or transcription [107,108]. To address this, the study introduced the |3 protein to enhance ssDNA stability [109-112]. With the addition of protein, distinct ssDNA bands became visible (FIGS. 28B-28C). To further enhance DNA yield, the study constructed a high-copy plasmid, which led to a substantial increase in ssDNA production (FIGS. 28D-28E).

[0192] To assess the impact of SBP on the formation of the DfRFA / DTR complex, the study tested two commercially available SBPs, ET SSB and Rec A. In the presence of SBP, the DIRFA / DIR complex exhibited a slight increase in fluorescence intensity compared to conditions lacking SBPs (FIG. 28F). This observation aligns with previous findings indicating that SBP can enhance aptamer functionality [113-115]. To evaluate if the plasmid can express functional GEFDA in bacterial cells, the study replaced the 72-nt sequence with DIRFA or dDIRFA (GEFDA in the scheme in FIG. 14A). The plasmid contains the following elements: 1) A rrnB T1 terminator for termination of transcription; 2) An IPTG inducible reverse transcriptase from Moloney mouse leukemia virus for reverse-transcription; 3) A val-tRNA binding sequence for initiation of reverse transcription (val-tRNA primer) in E. colv, 4) A hairpin structure for termination of reverse transcription; and 5) an IPTG inducible p. The study transformed the plasmids into DH10B competent cells to express DIRFA or dDIRFA. The plasmids were transformed into DH10B cells and induced by IPTG. In DH10B cells transformed with the dDIRFA plasmid, over 40% displayed fluorescence levels above 103, while fluorescence at this level was nearly absent in the nontransformed control group (FIGS. 14B-14C). This result confirms both the successful production and functional aptamer activity of dDIRFA in bacterial cells. When comparing dDIRFA with DIRFA, cells expressing the monomeric DIRFA showed only about 20% fluorescence-positive cells, suggesting that the dimeric form is likely brighter or more stable than the monomer in this cellular context (FIGS.29A-29C).

[0193] To evaluate the sensing ability of the dDASA sensor as a GEFDA sensor, the study replaced the DIRFA sequence in the plasmid with the dDASA sensor. After transformation, the bacterial cells were divided into three groups and incubated with either cell culture media alone, media supplemented with 50 pM ATP, or media containing 10 pM Venturicidin A (VentA), an ATP-synthase complex inhibitor that depletes intracellular ATP levels in bacterial cells

[0116] . Following treatment, the cells were incubated with DIR, and their fluorescence intensities were measured by flow cytometry. The results showed that DIR incubation producedonly a minor increase in background fluorescence (FIG. 14D, No DIR vs No IPTG), while IPTG induction significantly raised the fluorescence signal (FIG. 14D, No IPTG vs Medium), indicating successful detection of intrinsic ATP levels in bacteria expressing the dDASA plasmid. Fluorescence intensity further increased in the bacteria incubated with 50 pM ATP (FIG. 14D, Medium vs 50 pM ATP) and decreased in those treated with VentA (FIG. 14D, Medium vs 10 pM VentA). These findings confirm that the dDASA plasmid can effectively detect intracellular ATP levels (FIG. 14D, FIG. 14F).

[0194] Interestingly, two distinct bacterial subpopulations appeared under the “medium” condition (FIG. 14D), suggesting that the sensor could differentiate subpopulations at singlecell resolution, offering insights into cellular responses to treatments. One subpopulation showed fluorescence similar to ATP-treated cells, while the other resembled VentA-treated cells, implying potential variability in ATP levels or sensor activity across the population. Although factors like transformation efficiency or expression variability may contribute to this distribution, the increase in fluorescence within the lower-intensity subpopulation upon ATP treatment suggests that the sensor was successfully delivered and synthesized in both subpopulations. This unique capability highlights the sensor’s potential for resolving and monitoring ATP variability at single-cell resolution, offering valuable insights into cellular heterogeneity and response dynamics that warrant further investigation.

[0195] To confirm that the observed signal changes were specifically due to dDASA expression in response to intracellular ATP levels, the study introduced point mutations into the ATP-binding site of dDASA and expressed the mutated version with the same system. Although adding DIR increased the baseline fluorescence of the mutated dDASA plasmid (FIG. 14E, No DIR vs No IPTG), neither IPTG induction nor ATP level changes produced any additional fluorescence changes (FIGS. 14E-14F). This suggests that the fluorescence intensity variations in the unmutated sensor were indeed due to ATP recognition and binding. Collectively, these results demonstrate the successful development of GEFDA sensors capable of detecting ATP levels in bacterial cells.

[0196] GEFDA Sensors for Monitoring ATP in Mammalian Cells: To adapt the GEFDA sensor for ATP detection in mammalian cells, the study cloned all essential components - inducible reverse transcriptase, T1 terminator, hairpin structure, val-tRNA binding sequence, and the protein - into the pBK-CMV plasmid. This plasmid utilizes the CMV promoter to drive protein expression in mammalian cells, replacing the original plasmid backbone, which was driven by an E. coli lac promoter specific to prokaryotes.

[0197] To validate this system, the study used dDIRFA to compare plasmid performance with and without the p protein. Following transfection with the plasmid expressing dDIRFA, cells showed increased fluorescence upon DIR incubation, whereas DIR incubation alone, without plasmid transfection, did not result in any fluorescence increase (FIGS. 30A-30B). Notably, the addition of protein significantly enhanced fluorescence intensity (FIGS. 31A- 31B) These experiments confirm the successful expression and functionality of dDIRFA in mammalian cells.

[0198] To assess the performance of the GEFDA sensor, the study replaced dDIRFA with dDASA in the plasmid containing the P protein. After transfecting cells with this plasmid, the study modulated cellular ATP levels using 10 pM oligomycin or 5 mM CaCh. Fluorescence intensity increased in cells treated with CaCh and decreased in those treated with oligomycin. When the ATP-binding site was mutated, fluorescence intensity was no longer affected by these treatments (FIGS. 32A-32C). These experiments confirmed the successful ATP sensing capability of the GEFDA-dDASA sensor.

[0199] To evaluate whether the GEFDA-dDASA sensor can track real-time ATP dynamics in live cells, the study monitored fluorescence changes in the same cell population while modulating intracellular ATP levels using 25 mM glucose, 10 pM oligomycin, or 5 mM CaCh- A gradual increase in fluorescence was observed during glucose incubation, confirming that the GEFDA sensor effectively tracks ATP levels in individual cells (FIG. 33A, FIG. 33C). Interestingly, after the addition of 10 pM oligomycin, fluorescence initially increased before undergoing a marked decline (FIG. 33B, FIG. 33D), aligning with previously reported compensatory glycolysis immediately following oligomycin treatment

[0117] . After replacing oligomycin with CaCh, the fluorescence intensity in the same cell population gradually recovered, suggesting that ATPase inhibition by oligomycin may be reversible (FIG. 33B, FIG. 33D). These results confirm the successful detection and monitoring of intracellular ATP dynamics using the dDASA plasmid, highlighting its potential for future studies of cellular behavior through real-time event monitoring. To this end, this study has demonstrated the potential of using GEFDA sensors to trace ATP levels in both bacterial and mammalian cells.Conclusion

[0200] Although genetically encoded sensors based on fluorescent proteins and RNA aptamers have been widely used for visualizing cellular events, there is always a need for improved sensors to monitor metabolite concentrations in live cells. Taking advantage of the DNA aptamers which are more stable than RNA and can be easily reprogrammed, this study developed a new genetically encoded Anorogenic DNA aptamer (GEFDA) sensor termedDASA for real-time detection of ATP in E. coli and HeLa cells. By overcoming the issues of all the prelabeled DNA aptamer sensors such as sensor dilution, degradation, and low S / B ratio in live-cell detection, the GEFDA-dDASA sensor expresses the dimeric sensor which contains dual chromophore binding sites and dual-target recognition sites with a plasmid that contains a tRNA binding sequence, terminators, and sequences that encode IPTG inducible reverse transcriptase and single-stranded DNA binding protein. With the GEFDA sensors, the study monitored the ATP levels in both bacterial and mammalian cells. Utilizing the DNA aptamers allowed the expansion of the aptamer library from existing DNA aptamers and obtaining aptamers for new targets with SELEX. Therefore, this method signifies a major advancement in genetically encoded sensors by adding GEFDA as a new member, along with the fluorescent proteins and fluorogenic RNAs to detect small-molecule metabolites as valuable tools for monitoring the real-time dynamics of metabolites and other small molecules in biological systems.EXAMPLE ASPECTS

[0201] Example 1: A dual aptamer system for detection of a target analyte, the system comprising: a) a signal input aptamer, wherein said signal input aptamer can change a physical feature upon interaction with a target analyte; and b) a signal output aptamer, wherein said signal output aptamer changes a physical feature upon binding of a target analyte to the signal input aptamer, wherein said change of physical feature of the signal output aptamer allows for output of a detectable signal; wherein both the signal input aptamer and the signal output aptamer comprise nucleic acid, and further wherein both the signal input aptamer and the signal output aptamer are linked to each other.

[0202] Example 2: The dual aptamer system of any examples herein, particularly Example1, wherein the target analyte comprises a small molecule, nucleic acid, virus, protein, or peptide.

[0203] Example 3: The dual aptamer system of any examples herein, particularly Example2, wherein the small molecule comprises a metal ion, drug, or environmental pollution

[0204] Example 4: The dual aptamer system of any examples herein, particularly Example 2, wherein the small molecule comprises a metabolite.

[0205] Example 5 : The dual aptamer system of any examples herein, particularly Examples 1-3, wherein the signal input aptamer is an ATP aptamer and further wherein the target analyte comprises ATP.

[0206] Example 6: The dual aptamer system of any examples herein, particularly Examples 1-5, wherein the change of physical feature of the signal input aptamer comprises adifference in melting temperature, variation in sensitivity to pH or another environmental condition change, or change in conformation of the signal input aptamer such as a change in secondary or tertiary structure of the nucleic acid.

[0207] Example 7 : The dual aptamer system of any examples herein, particularly Examples 1-5, wherein the change of physical feature of the signal output aptamer comprises a difference in melting temperature, variation in sensitivity to pH or another environmental condition change, or change in conformation of the signal output aptamer such as a change in secondary or tertiary structure of the nucleic acid.

[0208] Example 8 : The dual aptamer system of any examples herein, particularly Examples 6 or 7, wherein the change of conformation comprises a change in a stem-loop structure.

[0209] Example 9: The dual aptamer system of any examples herein, particularly Example 8, wherein the change is in the stem structure.

[0210] Example 10: The dual aptamer system of any examples herein, particularly Examples 1-9, wherein the target analyte binds the signal input aptamer.

[0211] Example 11: The dual aptamer system of any examples herein, particularly Example 10, wherein binding of the target analyte increases level of detectable signal by at least 50%.

[0212] Example 12: The dual aptamer system of any examples herein, particularly Examples 1-11, wherein the signal output aptamer changes conformation in response to a change in conformation of the signal input aptamer.

[0213] Example 13: The dual aptamer system of any examples herein, particularly Example 12, wherein, upon a change of conformation of the signal output aptamer, the signal output aptamer is capable of interacting with a detection agent at a higher rate than before the change in conformation of the signal output aptamer.

[0214] Example 14: The dual aptamer system of any examples herein, particularly Example 13, wherein the detection agent is external to the system.

[0215] Example 15: The dual aptamer system of any examples herein, particularly Example 14, wherein the detection agent fluoresces, or fluoresces at a higher rate, upon interaction with the signal output aptamer.

[0216] Example 16: The dual aptamer system of any examples herein, particularly Examples 13-15, wherein the detection agent comprises dimethylindole red (DIR), magnesium green (MG), or 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI).

[0217] Example 17: The dual aptamer system of any examples herein, particularly Examples 1-16, wherein the signal input aptamer and the signal output aptamer do not naturally occur together.

[0218] Example 18: The dual aptamer system of any examples herein, particularly Example 17, wherein the signal input aptamer is truncated or otherwise engineered for detection optimization.

[0219] Example 19: The dual aptamer system of any examples herein, particularly Examples 16 or 17, wherein the signal output aptamer is truncated or otherwise engineered for detection optimization.

[0220] Example 20: The dual aptamer system of any examples herein, particularly Examples 1-19, wherein the system comprises at least one additional aptamer.

[0221] Example 21: The dual aptamer system of any examples herein, particularly Example 20, wherein the additional aptamer(s) is / are a signal input aptamer, a signal output aptamer, or both.

[0222] Example 22: The dual aptamer system of any examples herein, particularly Examples 1-21, wherein the system is reversible upon removal of the target analyte.

[0223] Example 23: The dual aptamer system of any examples herein, particularly Examples 1-22, wherein at least two different dual aptamer systems are linked together.

[0224] Example 24: The dual aptamer system of any examples herein, particularly Example 23, wherein the first dual aptamer system and the second dual aptamer system are joined via a linker.

[0225] Example 25: A vector encoding the dual aptamer system of any examples herein, particularly Examples 1-24.

[0226] Example 26: The vector of any examples herein, particularly Example 25, wherein the vector further comprises at least one additional component for expression of the dual aptamer system.

[0227] Example 27: The vector of any examples herein, particularly Example 26, wherein said at least one additional component comprises a reverse transcriptase, a terminator, a sequence that coded the dimer of the aptamers, a tRNA binding sequence, and / or a singlestranded DNA binding protein.

[0228] Example 28: A cell comprising the vector of any examples herein, particularly Examples 25-27.

[0229] Example 29: The cell of any examples herein, particularly Example 28, wherein the cell is a prokaryote or a eukaryote.

[0230] Example 30: A method of detecting a target analyte, the method comprising: providing the dual aptamer system of any examples herein, particularly Examples 1-24 in the presence of a target analyte, and detecting a signal via the signal output aptamer.

[0231] Example 31: The method of any examples herein, particularly Example 29, wherein said detection is reversible.

[0232] Example 32: The method of any examples herein, particularly Example 30, wherein said detection is carried out in real-time.

[0233] Example 33: The method of any examples herein, particularly Examples 29-31 , wherein said detection takes place in vivo, in vitro, or in cellulo.

[0234] Example 34: The method of any examples herein, particularly Examples 29-32, wherein the dual aptamer system is provided to the cell as nucleic acid.

[0235] Example 35: The method of any examples herein, particularly Example 34, wherein the nucleic acid is encoded in a vector.

[0236] Example 36: The method of any examples herein, particularly Example 34, wherein the cell is transformed with the nucleic acid.

[0237] Example 37: The method of any examples herein, particularly Examples 30-36, wherein the detection agent is provided to the system before, after, or during exposure to the target analyte.

[0238] Example 38: A method of screening to determine that a potential signal input aptamer can be used to detect a target analyte, the method comprising: a) providing a potential signal input aptamer, to determine if said signal input aptamer can change a physical feature upon interaction with a target analyte; b) providing a signal output aptamer, wherein said signal output aptamer changes a physical feature upon binding of a target analyte to the signal input aptamer, wherein said physical change of the signal output aptamer allows for output of a detectable signal; and c) determining whether the potential signal input aptamer can detect a target analyte by determining if a detectable signal is detected; wherein both the potential signal input aptamer and the signal output aptamer comprise nucleic acid, and further wherein both the potential signal input aptamer and the signal output aptamer are linked to each other.

[0239] Example 39: A method of screening to determine that a potential signal output aptamer can be used to detect a target analyte, the method comprising: a) providing a signal input aptamer, wherein said signal input aptamer can change a physical feature upon interaction with a target analyte; b) providing a potential signal output aptamer, and determining whether said signal output aptamer changes a physical feature upon binding of a target analyte to the signal input aptamer, wherein said physical change of the signal output aptamer allows foroutput of a detectable signal; and c) determining whether the potential signal output aptamer can detect a target analyte by determining if a detectable signal is detected; wherein both the potential signal input aptamer and the signal output aptamer comprise deoxyribonucleic acid (DNA), and further wherein both the potential signal input aptamer and the signal output aptamer are linked to each other.

[0240] Example 40: A dual aptamer system for detection of a target analyte, the system comprising: a) a signal input aptamer, wherein said signal input aptamer can change a physical feature upon interaction with a target analyte; b) a signal output aptamer, wherein said signal output aptamer changes a physical feature upon binding of a target analyte to the signal input aptamer, wherein said change of physical feature of the signal output aptamer allows for output of a detectable signal; and c) one or more additional components for expression selected from a reverse transcriptase, a sequence that codes a dimer form of one or both aptamers, an RNA polymerase terminator, a hairpin structure, a tRNA binding sequence, and a single- stranded DNA binding protein; wherein both the signal input aptamer and the signal output aptamer comprise nucleic acid, and further wherein both the signal input aptamer and the signal output aptamer are linked to each other.

[0241] Example 41: The dual aptamer system of any examples herein, particularly Example 40, wherein the signal input aptamer and / or the signal output aptamer comprise single stranded DNA.

[0242] Example 42: The dual aptamer system of any examples herein, particularly Example 40, wherein the signal input aptamer is an ATP aptamer, and wherein the target analyte comprises ATP.

[0243] Example 43: The dual aptamer system of any examples herein, particularly Example 40, wherein the change of physical feature of the signal input aptamer comprises a difference in melting temperature, variation in sensitivity to pH or another environmental condition change, or change in conformation of the signal input aptamer and / or the signal output aptamer such as a change in secondary or tertiary structure of the nucleic acid.

[0244] Example 44: The dual aptamer system of any examples herein, particularly Example 43, wherein the signal input aptamer and / or the signal output aptamer comprises a split structure having an unhybridized stem, and wherein the change of conformation causes the stem to hybridize.

[0245] Example 45: The dual aptamer system of any examples herein, particularly Example 40, wherein the target analyte binds the signal input aptamer.

[0246] Example 46: The dual aptamer system of any examples herein, particularly Example 45, wherein binding of the target analyte increases level of detectable signal by at least 50%.

[0247] Example 47: The dual aptamer system of any examples herein, particularly Example 40, wherein the signal output aptamer changes conformation in response to a change in conformation of the signal input aptamer.

[0248] Example 48: The dual aptamer system of any examples herein, particularly Example 47, wherein, upon a change of conformation of the signal output aptamer, the signal output aptamer is capable of interacting with a detection agent at a higher rate than before the change in conformation of the signal output aptamer.

[0249] Example 49: The dual aptamer system of any examples herein, particularly Example 48, wherein the detection agent fluoresces, or fluoresces at a higher rate, upon interaction with the signal output aptamer.

[0250] Example 50: The dual aptamer system of any examples herein, particularly Example 48, wherein the detection agent comprises dimethylindole red (DIR), magnesium green (MG), or 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHB1).

[0251] Example 51: The dual aptamer system of any examples herein, particularly Example 40, wherein the signal input aptamer and / or the signal output aptamer is truncated or otherwise engineered for detection optimization.

[0252] Example 52: The dual aptamer system of any examples herein, particularly Example 40, wherein the detectable signal is reversible upon removal of the target analyte.

[0253] Example 53: The dual aptamer system of any examples herein, particularly Example 40, wherein the first dual aptamer system and the second dual aptamer system are joined via a linker.

[0254] Example 54: The dual aptamer system of any examples herein, particularly Example 40, wherein the reverse transcriptase and the single-stranded DNA binding protein are IPTG-inducible.

[0255] Example 55: The dual aptamer system of any examples herein, particularly Example 40, wherein the RNA polymerase terminator is an rmB T 1 terminator.

[0256] Example 56: The dual aptamer system of any examples herein, particularly Example 40, further comprising the reverse transcriptase and the hairpin structure, and wherein the hairpin structure terminates reverse transcription of said reverse transcriptase.

[0257] Example 57 : The dual aptamer system of any examples herein, particularly Example 40, wherein the tRNA binding sequence is a val-tRNA binding sequence for initiation of reverse transcription.

[0258] Example 58: A vector encoding the dual aptamer system of any examples herein, particularly Example 40.

[0259] Example 59: A cell comprising the vector of any examples herein, particularly Example 58.

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Claims

CLAIMS1. A dual aptamer system for detection of a target analyte, the system comprising: a) a signal input aptamer, wherein said signal input aptamer can change a physical feature upon interaction with a target analyte; b) a signal output aptamer, wherein said signal output aptamer changes a physical feature upon binding of a target analyte to the signal input aptamer, wherein said change of physical feature of the signal output aptamer allows for output of a detectable signal; and c) one or more additional components for expression selected from a reverse transcriptase, a sequence that codes a dimer form of one or both aptamers, an RNA polymerase terminator, a hairpin structure, a tRNA binding sequence, and a single-stranded DNA binding protein; wherein both the signal input aptamer and the signal output aptamer comprise nucleic acid, and further wherein both the signal input aptamer and the signal output aptamer are linked to each other.

2. The dual aptamer system of claim 1, wherein the signal input aptamer and / or the signal output aptamer comprise single stranded DNA.

3. The dual aptamer system of claim 1, wherein the signal input aptamer is an ATP aptamer, and wherein the target analyte comprises ATP.

4. The dual aptamer system of claim 1, wherein the change of physical feature of the signal input aptamer comprises a difference in melting temperature, variation in sensitivity to pH or another environmental condition change, or change in conformation of the signal input aptamer and / or the signal output aptamer such as a change in secondary or tertiary structure of the nucleic acid.

5. The dual aptamer system of claim 4, wherein the signal input aptamer and / or the signal output aptamer comprises a split structure having an unhybridized stem, and wherein the change of conformation causes the stem to hybridize.

6. The dual aptamer system of claim 1 , wherein the target analyte binds the signal input aptamer.

7. The dual aptamer system of claim 6, wherein binding of the target analyte increases level of detectable signal by at least 50%.

8. The dual aptamer system of claim 1, wherein the signal output aptamer changes conformation in response to a change in conformation of the signal input aptamer.

9. The dual aptamer system of claim 8, wherein, upon a change of conformation of the signal output aptamer, the signal output aptamer is capable of interacting with a detection agent at a higher rate than before the change in conformation of the signal output aptamer.

10. The dual aptamer system of claim 9, wherein the detection agent fluoresces, or fluoresces at a higher rate, upon interaction with the signal output aptamer.

11. The dual aptamer system of claim 9, wherein the detection agent comprises dimethylindole red (DIR), magnesium green (MG), or 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI).

12. The dual aptamer system of claim 1, wherein the signal input aptamer and / or the signal output aptamer is truncated or otherwise engineered for detection optimization.

13. The dual aptamer system of claim 1, wherein the detectable signal is reversible upon removal of the target analyte.

14. The dual aptamer system of claim 1, wherein the first dual aptamer system and the second dual aptamer system are joined via a linker.

15. The dual aptamer system of claim 1, wherein the reverse transcriptase and the singlestranded DNA binding protein are IPTG-inducible.

16. The dual aptamer system of claim 1, wherein the RNA polymerase terminator is an rrnB T1 terminator.

17. The dual aptamer system of claim 1, further comprising the reverse transcriptase and the hairpin structure, and wherein the hairpin structure terminates reverse transcription of said reverse transcriptase.

18. The dual aptamer system of claim 1, wherein the tRNA binding sequence is a val- tRNA binding sequence for initiation of reverse transcription.

19. A vector encoding the dual aptamer system of claim 1 .

20. A cell comprising the vector of claim 19.

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