Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/682—Signal amplification

Definitions

• This application generally relates to systems and methods to detect nucleic acid and/or small molecule targets and/or to provide a readily understandable readout indicating the presence or absence of the target(s).
• DESCRIPTION OF RELATED ART There is a need for simple and readily understandable, low-cost, non-invasive, semi- continuous use, accumulated and/or real-time multi-sensing of markers from a broad set of sources including human bodies, animals, object surfaces, environments without requiring the use of additional electronic devices, and with accuracy.
• a person is exposed to a range of environments on a daily basis.
• the conditions of these environments and the length of exposure to these conditions may impact a person’s mental and/or physical state. Several of these conditions may go undetected. Further, their impact on a person exposed to these conditions are not immediately apparent. For example, the microbiome present on a person’s skin may be indicative of the individual’s health and is not immediately apparent.
• Human bodies are continuously exposed to viruses and/or microbial cells and their byproducts which can include toxic metabolites. Circulation of toxic metabolites may contribute to the onset of cancer. In addition, microbes may migrate throughout the human body and become associated with tumor development.
• microflora in a microbiome has been found to be associated with various health conditions including cancer, chronic inflammation, hydration levels, skin hydration levels, immune system disfunction, atopic dermatitis, psoriasis, acne vulgaris, skin ulcers, and conditions associated with aging. These microbiomes include those from a subject’s gut, skin, and other topical areas of the body. [0007] Additionally, a person commonly comes in contact with a myriad of infectious agents including microbial cells such as bacterial cells and virions. An immediately pressing example of an environmental condition that is not immediately apparent is the presence of viral components, such as those of the novel corona virus (e.g., SARS-CoV-2).
• SARS-CoV-2 novel corona virus
• sequences e.g., specific single nucleotide polymorphisms (SNPs)
• genetic mutations e.g., BRCA1/2 mutations
• SNPs single nucleotide polymorphisms
• BRCA1/2 mutations genetic mutations
• specific cell free circulating mircroRNA signatures are associated with the onset and severity of Alzheimer’s Disease, for example. Detection of these sequences originating from a subject would provide diagnostic and prognostic benefits.
• diagnostic devices used for, e.g., nucleic acid detection are bulky, battery-powered, expensive, and/or difficult to learn to use.
• NAT nucleic acid testing
• a number of fluorescence-based assays for detecting viral nucleic acids use CRISPR technology. At least one case (Fozouni P, Son S, Diaz de Leon Derby M, Knott GJ, Gray CN, et al. 2020. Cell) specifically uses the Cas13 protein without a preamplification step. Cas13 is a protein that targets single-stranded (ss) RNA substrates.
• Cas13 is complexed with a so-called CRISPR RNA (crRNA) containing a programmable spacer sequence to form a nuclease- inactive ribonucleoprotein complex (RNP).
• crRNA CRISPR RNA
• RNP nuclease- inactive ribonucleoprotein complex
• HEPN higher eukaryotes and prokaryotes nucleotide-binding domain
• Target RNA binding and subsequent Cas13 cleavage activity can be detected with a fluorophore-quencher pair linked by an ssRNA, which fluoresces after cleavage by active Cas13.
• the reporter ssRNA was 5’-FAM-rUrUrUrUrU-IowaBlack FQ-3’, where FAM is a fluorophore and IowaBlack FQ is a quencher.
• Other assays use the Cas12 protein, which cleaves single- or double-stranded DNA upon complexation of a target nucleic acid, and which can be employed analogously to the Cas13.
• the Cas13 assay is reasonably sensitive, its limit of detection was shown to be about 100 copies of the target RNA per ⁇ l. The Cas13 assay thus appears attractive for detecting pathogen nucleic acids in human body fluids, or on surfaces etc.
• nucleic acid peroxidases a type of peroxidase-mimic composed in part of nucleic acids
• the function of split G quadruplex nucleic acid peroxidase probes is strongly dependent on the immediate sequence context (see, e.g., Connelly RP, Verduzco C, Farnell S, Yishay T, Gerasimova YV. 2019. ACS Chem Biol 14:2701-12).
• the present system or method disclosed herein may be directed towards a system and methods for detecting the presence of a target analyte such as a specific nucleic acid or a small molecule.
• the system or method further may include a method of signal amplification and/or a readily understandable readout.
• the invention is a system comprising a translation module, an amplification module, and a detection module.
• the translation module comprises a first nucleic acid comprising a first polynucleotide, and a second nucleic acid comprising a second polynucleotide.
• the second polynucleotide is configured to reversibly hybridize the first polynucleotide.
• the translation module is configured to accept one or more input signals, the one or more input signals comprise one or more target nucleic acids, and the one or more target nucleic acids comprise a target polynucleotide.
• the first polynucleotide is configured to hybridize the target polynucleotide
• the second polynucleotide is configured to dissociate from the first polynucleotide in the presence of the target polynucleotide to provide a translator output.
• the amplification module comprises one or more sensing modalities. In some embodiments, the amplification module is configured to accept the translator output. In some embodiments, the one or more sensing modalities are configured to detect the translator output. In some embodiments, the one or more sensing modalities are configured to provide an amplifier output upon detecting the translator output. In some embodiments, the detection module is configured to accept the amplifier output.
• the detection module comprises one or more substrates and one or more activatable nucleic acid peroxidases.
• the one or more activatable nucleic acid peroxidases are configured to be converted into one or more active nucleic acid peroxidases in the presence of the amplifier output.
• the one or more active nucleic acid peroxidases are configured to convert the one or more substrates into one or more products.
• the one or more sensing modalities comprises one or more of: one or more isothermal chemical ligation-hybridization and chemical cross replication modules configured to replicate the translator output as the amplifier output, one or more hairpin-chain reaction modules configured to replicate the translator output as the amplifier output, one or more duplicator gate cascade modules configured to replicate the translator output as the amplifier output, one or more CRISPR-Cas13 modules configured to provide an active Cas13 complex as the amplifier output, one or more CRISPR-Cas12 modules configured to provide an active a Cas12 complex as the amplifier output, or one or more nucleated polymerization module configured to provide a polymer as the amplifier output.
• the one or more activatable nucleic acid peroxidases are one or more of: one or more caged nucleic acid peroxidases comprising a digestible region, one or more nucleic acid peroxidases in tension, and/or one or more first peroxidase polynucleotides, and one or more second peroxidase polynucleotides.
• the amplifier output is configured to digest the digestible region of the one or more caged nucleic acid peroxidases, and the one or more caged nucleic acid peroxidases are configured to be converted to the one or more active nucleic acid peroxidases upon digestion of the digestible region.
• the amplifier output is configured to relax the one or more nucleic acid peroxidases in tension, and the one or more nucleic acid peroxidases are configured to be converted to the one or more active nucleic acid peroxidases upon relaxation.
• the one or more first peroxidase polynucleotide and the one or more second peroxidase polynucleotide are configured to form the one or more active nucleic acid peroxidases when the one or more first peroxidase polynucleotide are in proximity to the one or more second peroxidase polynucleotide.
• the one or more activatable nucleic acid peroxidases are configured to bring the one or more first peroxidase polynucleotides and the one or more second peroxidase nucleotides into proximity when the amplifier output is present.
• the one or more sensing modalities comprises one or more of one or more CRISPR-Cas13 modules configured to provide an active Cas13 complex as the amplifier output or one or more CRISPR-Cas12 modules configured to provide an active Cas12 complex as the amplifier output;
• the one or more activatable nucleic acid peroxidases are one or more first peroxidase polynucleotides, and one or more second peroxidase polynucleotides.
• the one or more first peroxidase polynucleotide and the one or more second peroxidase polynucleotide are configured to form the one or more active nucleic acid peroxidases when the one or more first peroxidase polynucleotide are in proximity to the one or more second peroxidase polynucleotide.
• the detection module is configured to bring the one or more first peroxidase polynucleotides and the one or more second peroxidase nucleotides into proximity when the amplifier output is present. [0021] In some embodiments of any of the systems, the system further comprising one or more reporter nucleic acids.
• the one or more reporter nucleic acids are configured to hold the one or more first peroxidase polynucleotides separate from the one or more second peroxidase polynucleotides when the one or more reporter nucleic acid is not cleaved.
• the one or more activatable nucleic acid peroxidases are configured to bring the one or more first peroxidase polynucleotides into proximity with the one or more second peroxidase polynucleotides when the one or more reporter nucleic acids is cleaved.
• the amplifier output is configured to cleave the one or more reporter nucleic acids.
• the system comprises a plurality of activatable nucleic acid peroxidases.
• each activatable nucleic acid peroxidase comprises one of the one or more first peroxidase polynucleotide and one of the one or more second peroxidase polynucleotide.
• the detection module comprises: a first portion comprising a first surface, the one or more first peroxidase polynucleotides attached to the first surface, a second portion comprising a second surface, the one or more second peroxidase polynucleotides attached to the second surface, and a hinge connecting the first portion and the second portion.
• the hinge is configured to hold the detection module in an open form when the one or more reporter nucleic acids is uncleaved or the one or more reporter nucleic acids is single stranded, and the hinge is configured to hold the detection module in a closed form when the one or more reporter nucleic acids is cleaved or the one or more reporter nucleic acids is double stranded.
• the one or more first surface and the second surface are configured to separate the one or more first peroxidase polynucleotides and the one or more second peroxidase polynucleotides when the detection module is in the open form, and the first surface and the second surface are configured to bring the one or more first peroxidase polynucleotides into proximity with the one or more second peroxidase polynucleotides when the detection module is in the closed form.
• the one or more sensing modalities comprises one or more of one or more CRISPR-Cas13 modules configured to provide an active Cas13 complex as the amplifier output or one or more CRISPR-Cas12 modules configured to provide an active a Cas12 complex as the amplifier output; and the one or more activatable nucleic acid peroxidases are one or more nucleic acid peroxidases in tension.
• the amplifier output is configured to relax the one or more nucleic acid peroxidases in tension, and the one or more nucleic acid peroxidases are configured to be converted to the one or more active nucleic acid peroxidases upon relaxation.
• the system further comprises one or more reporter nucleic acids.
• the one or more reporter nucleic acids are configured to hold the one or more nucleic acid peroxidases in tension when the one or more reporter nucleic acid is not cleaved, and the amplifier output is configured to cleave the one or more reporter nucleic acids.
• the system comprises a plurality of activatable nucleic acid peroxidases
• the detection module comprises a first portion comprising a first surface, the one or more nucleic acid peroxidases are attached to the first surface; a second portion comprising a second surface, the the one or more nucleic acid peroxidases are attached to the second surface; and a hinge connecting the first portion and the second portion, the hinge is configured to hold the detection module in an open form when the one or more reporter nucleic acids is uncleaved, and the hinge is configured to hold the detection module in a closed form when the one or more reporter nucleic acids is cleaved.
• the one or more first surface and the second surface are configured to hold the one or more nucleic acid peroxidases in tension when the detection module is in the open form, and the first surface and the second surface are configured to hold the one or more nucleic acid peroxidases in tension when the detection module is in the closed form.
• the amplification module further comprises one or more activatable elements.
• the amplifier output is configured to activate the one or more activatable elements to provide one or more activated elements.
• the one or more activated elements are configured to detect one or more nucleic acids, and the one or more sensing modalities are configured to provide a second amplifier output upon detecting the one or more nucleic acids; and the detection module is further configured to accept the second amplifier output, and the one or more activatable nucleic acid peroxidases are configured to be converted into the one or more active nucleic acid peroxidases in the presence of the second amplifier output.
• the one or more activatable elements is a masked nucleic acid, and the one or more activated elements is not masked.
• the second amplifier output and the amplifier output are the same.
• the one or more activated elements and the translator output are the same.
• the one or more input signals is provided by dissociating the one or more target nucleic acids of the one or more input signals from another polynucleotide.
• the invention is a method for detecting one or more target nucleic acids, the one or more target nucleic acids comprises a target polynucleotide.
• method comprises: providing a sample to a translation module, the translation module comprising: a first nucleic acid comprising a first polynucleotide, and a second nucleic acid comprising a second polynucleotide, the second polynucleotide is configured to reversibly hybridize the first polynucleotide; hybridizing the first polynucleotide to the target polynucleotide; dissociating the second polynucleotide from the first polynucleotide to provide a translator output; providing the translator output to an amplification module, the amplification module comprises one or more sensing modalities; detecting the translator output by the one or more sensing modalities; providing an amplifier output from the one or more sensing modalities; providing the amplifier output to a detection module, the detection module comprises one or more substrates and one or more activatable nucleic acid peroxidases; converting the one or more activatable nucleic acid peroxidases into one
• the step of providing the amplifier output from the one or more sensing modalities comprises one or more of: replicating the translator output by isothermal chemical ligation-hybridization and chemical cross replication, replicating the translator output by hairpin-chain reaction, replicating the translator output with duplicator gate cascades, providing an active Cas13 complex, providing an active a Cas12 complex, or providing a polymer by nucleated polymerization.
• the step of converting the one or more activatable nucleic acid peroxidases into one or more active nucleic acid peroxidases comprises one or more of: digesting a digestible region of one or more caged nucleic acid peroxidases, relaxing one or more nucleic acid peroxidases in tension, or bringing one or more first peroxidase polynucleotides and one or more second peroxidase polynucleotides into proximity.
• the step of providing the amplifier output from the one or more sensing modalities comprises providing one or more of an active Cas13 complex and an active a Cas12 complex; and the step of converting the one or more activatable nucleic acid peroxidases into one or more active nucleic acid peroxidases comprises one or more of one or more first peroxidase polynucleotides and one or more second peroxidase polynucleotides into proximity.
• the step of converting the one or more activatable nucleic acid peroxidases into one or more active nucleic acid peroxidases comprises cleaving one or more reporter nucleic acids with the active Cas13 complex or active Cas12 complex, and the one or more reporter nucleic acids are configured to hold the one or more first peroxidase polynucleotides separate from the one or more second peroxidase polynucleotides when the one or more reporter nucleic acid is not cleaved.
• the step of providing the amplifier output from the one or more sensing modalities comprises providing one or more of an active Cas13 complex and an active a Cas12 complex; and the step of converting the one or more activatable nucleic acid peroxidases into one or more active nucleic acid peroxidases comprises relaxing one or more nucleic acid peroxidases in tension.
• the step of converting the one or more activatable nucleic acid peroxidases into one or more active nucleic acid peroxidases comprises cleaving one or more reporter nucleic acids with the active Cas13 complex or active Cas12 complex, and the one or more reporter nucleic acids are configured to hold the one or more activatable nucleic acid peroxidases in tension when the one or more reporter nucleic acid is not cleaved.
• the amplification module further comprises one or more activatable elements
• the method further comprises: activating the one or more activatable elements with the amplifier output, providing one or more activated elements, detecting one or more nucleic acids with the one or more activated elements, providing a second amplifier output, and providing the second amplifier output to the detection module.
• the one or more activatable elements is a masked nucleic acid, and the one or more activated elements is not masked.
• the second amplifier output and the amplifier output are the same.
• the one or more activated elements and the translator output are the same.
• the method further comprises dissociating the one or more target nucleic acids of the one or more input signals from another polynucleotide.
• the system is auto-catalytic.
• the system comprises an activated Cas complex, wherein the activated Cas complex is configured to activate one or more nucleic acid peroxidases, and wherein the activated Cas complex is configured to activate one or more activatable Cas complexes.
• the activated Cas complex is configured to cleave a hairpin loop of one or more cryptic nucleic acids, and a cryptic nucleic acid comprises a complement strand and a trigger strand.
• the system further comprises a primed Cas complex, wherein the primed Cas complex comprises a crRNA and a partial complement nucleic acid, and wherein the partial complement nucleic acid is capable of hybridizing to a first portion of the crRNA.
• the trigger strand is capable of hybridizing to a second portion of the crRNA.
• the primed Cas complex is activated when the partial complement nucleic acid is hybridized with the first portion of the crRNA and the trigger strand hybridizes with the second portion of the crRNA.
• the cryptic nucleic acid is a cryptic RNA.
• the cryptic RNA is a modified cryptic RNA.
• the modified cryptic RNA comprises one or more 2’- fluorination, one or more phosphorothioation, and/or one or more 2’-O-Methyl group.
• the modified cryptic RNA is flanked on one or both sides with DNA.
• an activatable Cas complex comprises a crRNA and a cryptic nucleic acid, wherein the crRNA comprises a first crRNA complementary sequence and a second crRNA complementary sequence, wherein the cryptic nucleic acid comprises a first complementary sequence, a second complementary sequence, a hairpin loop, and a cryptic sequence, wherein the first complementary sequence is capable of hybridizing to the first crRNA complementary sequence, wherein the second complementary sequence is capable of hybridizing to the second crRNA complementary sequence, wherein the second complementary sequence is capable of hybridizing to the cryptic nucleic acid, wherein the second complementary sequence and the cryptic nucleic acid are joined by the hairpin loop, wherein the activated Cas complex is configured to cleave the hairpin loop, and wherein the activatable Cas complex is activated when the first complementary sequence hybridizes to the first crRNA complementary sequence and the second complementary sequence hybridizes to the second crRNA complementary sequence.
• the cryptic nucleic acid dissociates from the second crRNA complementary sequence after the hairpin loop is cleaved, and the second complementary sequence hybridizes to the second crRNA complementary sequence after the cryptic nucleic acid dissociates from the second crRNA complementary sequence.
• the cryptic nucleic acid is a cryptic RNA.
• the cryptic RNA is a modified cryptic RNA.
• the modified cryptic RNA comprises one or more 2’-fluorination, one or more phosphorothioation, and/or one or more 2’-O-Methyl group.
• the modified cryptic RNA is flanked on one or both sides with DNA.
• Figure 1 depicts an exemplary embodiment of a hinged system sensitive to nucleic acid strandedness.
• Figure 2 depicts an exemplary embodiment of a hinged system sensitive to nucleic acid strandedness.
• Figure 3 depicts an exemplary embodiment of a switchable hinged-beam nucleic acid nanodevice with a stretched (left) and relaxed (right) peroxidase.
• Figure 4 depicts an exemplary embodiment of a switchable hinged-beam nucleic acid nanodevice with a split peroxidase separated (left) and in proximity to one another (right).
• Figure 5 depicts an exemplary embodiment of a switchable hinged-beam nanodevice where the inactive state (left) possesses an uncleaved double-stranded nucleic acid and the active state (right) possesses a cleaved double-stranded nucleic acid.
• Figure 6 depicts a molecular rendering of a DNA holiday junction.
• Figures 7A-7B depict exemplary embodiments of a switchable hinged system possessing multiple peroxidases.
• Figure 7A depicts the exemplary embodiment with an uncleaved reporter nucleic acid and stretched peroxidases.
• Figure 7B depicts the exemplary embodiment a cleaved reporter nucleic acid and relaxed peroxidases.
• Figures 8A-8E depict an exemplary embodiment comprising a single nucleic acid strand including a peroxidase sequence.
• the peroxidase may be inhibited by complementary regions and/or the formation ( Figure 8A).
• a hairpin loop region may be cleaved by a nuclease ( Figure 8B) to produce an active peroxidase unit (Figure 8C).
• Figure 8D depicts stability of an exemplary hairpin sequence.
• Figure 8E depicts an exemplary embodiment activated by the introduction of an invader strand.
• Figure 9 depicts an exemplary embodiment of a G-plex (peroxidase) stretcher.
• Figures 10A-10B depict exemplary embodiments of a circularized G-plex (peroxidase) stretcher.
• Figures 11A-11E depict an exemplary embodiment of constrained G-plex (peroxidase) variants.
• Figures 12A-12B depicts an exemplary embodiment of a G-plex (peroxidase) inhibited by stretching.
• Figure 13 depicts an exemplary embodiment of a G-plex (peroxidase) inhibited by stretching.
• Figures 14A-14C depict exemplary embodiments of G-plexes (peroxidases) inhibited by stretching.
• Figure 15 depicts an exemplary embodiment of a card system for detecting an analyte.
• Figure 16 depicts an exemplary embodiment of a card system with a QR code for detecting an analyte.
• Figure 17 depicts an exemplary embodiment of a system for detecting an analyte present on one or more people in a public area.
• Figure 18 depicts an exemplary architecture overview of an exemplary embodiment of a card system for detecting an analyte.
• Figures 19A-19L depict performance modeling of an exemplary double-catalytic system comprising activated Cas13. Solid lines denote a positive output, and dashed lines denote a negative control reaction run in parallel.
• Figure 20 depicts exemplary data obtained with closed hairpin G4 quadruplex variants (dashed lines) and fully active variants (solid lines).
• Figures 21A-21B depict an exemplary architecture overview of an exemplary embodiment of a card system for detecting an analyte.
• Figure 22 depicts exemplary data obtained with closed hairpin G4 quadruplex variants (solid lines) and fully activated variants (dashed lines).
• Figure 23 depicts exemplary data obtained with caged G4 quadruplex variants (solid lines) and fully active variants (dashed lines).
• Figure 24 depicts exemplary data obtained with stretched G4 quadruplex variants (solid lines) and fully active variants (dashed lines).
• Figures 25A-25H depict G plex peroxidase duplexes.
• Figure 26 depicts an exemplary embodiment of an in-solution system.
• Figure 27 depicts an exemplary embodiment including a feedback loop using an auto- catalytic cascade.
• Figure 28 depicts an exemplary self-catalytic amplification scheme in general terms.
• Figures 29A-29B depict an exemplary system using split trigger fragments and primed crRNA.
• Figure 30 depicts an exemplary embodiment of a cleavable loop wherein the cryptic trigger RNA sequence located with the double-stranded hairpin stem can is formed with chemically modified RNA bases. These chemical modifications may be any chemical modification, preferably wherein the chemically modified trigger strand element is capable of triggering collateral Cas13 activity when hybridizing with crRNA, and most preferably also not being a substrate for Cas13 cleavage.
• Figures 31A-31C depict an exemplary self-catalytic amplification scheme in general terms employing a previously cryptic trigger RNA sequence segment that becomes free to fully hybridize with a pre-complexed crRNA.
• Figures 32A-32C depict exemplary graded biosensors and genetic signal graded output designs.
• 32A depicts UV (left) and hydration (right) biosensors that display multiple sensor output levels.
• 32B depicts a genetic signal with graded readout exemplified for Covid-19.32C depicts graded test outputs. These may enable enhanced guidance for when users should self- isolate, and when they may safely return to the workplace or school.
• Exemplary biosensors are high sensitivity, low cost tests.
• the present system or method is directed towards systems and/or methods for detecting one or more analytes using molecular beacons to provide one or more detectable signals.
• the detectable signal is provided by use of a peroxidase that is configured to switch from an inactive state to an active state.
• a peroxidase that is configured to switch from an inactive state to an active state.
• each molecule of the analyte corresponds to multiple molecules contributing to the detectable signal, e.g., each molecule of the analyte may correspond to two, tens, hundreds, thousands, or tens of thousands of molecules contributing to the detectable signal.
• An analyte can be any detectable molecule of interest including but not limited to a nucleic acid and a small molecule.
• the analyte is a nucleic acid, for example a DNA or an RNA.
• An analyte may be obtained from any appropriate source including, but not limited to, saliva, exhalation, sweat, the skin microbiome, or an object’s surface.
• An analyte may be extracted by any suitable method known in the art. For example, extraction of a nucleic acid may be achieved by including a lysis buffer such as 10% protease K, 0.7 M NaCl, 0.1% Hexadecyl trimethyl ammonium Bromide (CTAB) and MES at pH 5.0. Other methods known in the art suitable for nucleic acid extraction are contemplated. [0070]
• the sample may be prepared in any suitable way.
• the methods or systems directly accept analytes (e.g., nucleic acids) from bodily fluids such as saliva.
• DNA and RNA nucleases present in a raw sample or other external sources may be deactivated.
• the sensor spot contains a sufficiently high concentration of broad-band nuclease inhibitors to efficiently deactivate nucleases.
• the sensor contains two initially physically separate sensor areas. The first area comprises a hydrogel matrix containing sufficiently high concentrations of proteinase K to deactivate nucleases.
• a sample e.g., saliva is applied directly to the hydrogel matrix of the first area.
• a sample e.g., saliva
• a sample is applied by first unpeeling a cover which will expose the application spot to air. Oxygen in the air will activate an exothermic chemical reaction in a heat pad layer positioned underneath the sample application spot.
• the heat pad is configured to locally heat the area underneath the sample application spot to an appropriate temperature. In some embodiments, the temperature is approximately 90°C.
• proteins present in a sample (e.g., saliva) applied to the application spot will be deactivated by proteinase K provided by the form. In some embodiments, the proteinase K will be denatured by the heating reaction.
• the processed sample present at the first area is provided to the second area.
• a suitable period of time is approximately 10 minutes.
• the second area comprises sensor components (e.g., modules or layers).
• the analyte may undergo a pre-detection amplification step such as whole genome amplification. This amplification increases the amount of a genome (e.g., a viral genome) available that may possess the analyte of interest. Such whole genome amplification may increase the likelihood that there is sufficient analyte available to generate detectable signal without the use of an additional instrument (e.g., by the naked eye).
• Exemplary whole genome amplification systems may be based on Phi29 or any known polymerase.
• the polymerase is isothermal and is enzymatically active at skin temperature (e.g., Phi29).
• the generated signal undergoes an exponential amplification for subsequent detection.
• no analyte amplification step (e.g., whole genome amplification) may be needed.
• an analyte amplification step may precede signal amplification.
• the present system or method comprises a translation module, an amplification module comprising one or more sensing modalities, and a detection module configured to accept the amplifier output.
• the system or method is capable of detecting trace amounts of nucleic acid molecules with high sensitivity and specificity in a device-free system with naked- eye readable output. In some embodiments, they system or method uses a nuclease that can be programmatically activated to produce indiscriminate nuclease activity upon the initial detection of a specific nucleic acid sequence. In some embodiments the system or method is auto-catalytic.
• the form factor is a modular system for naked-eye readable nucleic acid detection. In some embodiments, the system comprises three signal amplification layers.
• Figures 18 and 21A-21B depict an architecture overview of an exemplary sensor as exemplified in a card-like form factor.
• the translation module comprises a first nucleic acid comprising a first polynucleotide, and a second nucleic acid comprising a second polynucleotide, wherein the second polynucleotide is configured to reversibly hybridize the first polynucleotide.
• the translation module is configured to accept one or more input signals.
• the one or more input signals comprise one or more target nucleic acids, wherein the one or more target nucleic acids comprise a target polynucleotide.
• the first polynucleotide is configured to hybridize the target polynucleotide
• the second polynucleotide is configured to dissociate from the first polynucleotide in the presence of the target polynucleotide to provide a translator output (e.g., an activating or first activating sequence).
• the plurality of nucleic acid input signals includes at least 1 target, at least 2 targets, at least 5 targets, at least 10 targets, at least 25 targets, at least 50 targets, at least 75 targets, at least 100 targets, at least 250 targets, at least 500 targets, at least 750 targets, or at least 1,000 targets.
• the plurality of nucleic acid input signals includes one or more targets of a viral or bacterial nucleic acid.
• the plurality of nucleic acids of the system are derived from the genome of SARS-CoV-2.
• the translator module employs DNA strand displacement techniques.
• a translator gate binds to a unique genetic sequence (ssDNA or ssRNA).
• the translator gate is configured to output a ssRNA or ssDNA trigger strand with sequence X.
• the translator gate is configured to output a double-stranded (ds) DNA trigger or dsRNA trigger with sequence Y.
• the translator gate is a nucleic acid (e.g., a first nucleic acid) and the output trigger is a second nucleic acid.
• An array of unique translator gates can be formed, each targeting different regions along a genome of interest. Arrays may be configured to increase the production of trigger strands X or Y. Wang B, Thachuk C, Ellington AD, Winfree E, Soloveichik D.2018. Proc Natl Acad Sci U S A 115:E12182-E91.
• the system of method may be altered to detect different or new analytes only by altering the translation module
• the translation module is the first layer of a multilayer architecture.
• translator output is an RNAse Cas13 configured to cleave an RNA component of the activatable peroxidase thereby activating (e.g., ungating) the peroxidase in the detector layer.
• translator output is an DNAse Cas12 configured to cleave an DNA component of the activatable peroxidase thereby activating (e.g., ungating) the peroxidase in the detector layer.
• Amplification Module [0084] In some embodiments, the amplification module is configured to accept the translator output (e.g., the activating or first activating sequence).
• the one or more sensing modalities are configured to detect the translator output, and the one or more sensing modalities are configured to provide an amplifier output (e.g., an active or first active nuclease) upon detecting the translator output.
• the amplifier output e.g., an active or first active nuclease
• the amplifier output is an active Cas protein (e.g., an active Cas13 protein).
• an inhibited nuclease or first inhibited nuclease is activated to provide the amplifier output (e.g., an active or first active nuclease).
• the inhibited nuclease or first inhibited nuclease is an inactive Cas protein, inactive Cas13 protein, or inactive CRISPR-Cas13 complex and the amplifier output (e.g., an active or first active nuclease) is an active Cas protein, active Cas13 protein, or active CRISPR-Cas13 complex.
• the Cas protein in any embodiment may be any suitable wildtype or modified Cas protein including but not limited to Cas12, Cas13, Cas14, subtypes of any of these Cas proteins (e.g., Cas13a).
• a system may use more than one Cas protein (e.g., a mixture of Cas12 and Cas13 proteins).
• a suitable Cas protein may have transcleavage activity.
• the target of the active Cas protein may be a single stranded RNA (ssRNA), a single stranded DNA (ssDNA), a double stranded DNA (dsDNA), or any other appropriate nucleic acid.
• the amplification module further comprises one or more activatable elements.
• the one or more activatable elements are one or more masked activatable nucleic acid sequences.
• the one or more masked activatable nucleic acid sequences may possess one or more cryptic or hidden recognition sequences or strands.
• the one or more cryptic or hidden recognition (i.e., masked) strands or sequences may be masked such that a Cas protein (e.g., Cas13) is unable to recognize the strand or sequence, bind to the strand or sequence, and become active. If the sequence or strand is unmasked or released it is no longer cryptic or hidden. Such unmasked strands or sequences may then be recognized by a Cas protein (e.g., Cas13), bind to the protein, and the protein is able to become active.
• a Cas protein e.g., Cas13
• the amplifier output (e.g., an active or first active nuclease) is configured to activate the one or more activatable elements to provide one or more activated elements (e.g., an activating or second activating sequence).
• the one or more activated elements (e.g., an activating or second activating sequence) are configured to detect one or more nucleic acids.
• the one or more sensing modalities are configured to provide a second amplifier output (e.g., an active or second active nuclease) upon detecting the one or more nucleic acids.
• the second amplifier output e.g., an active or second active nuclease
• the amplifier output e.g., an active or first active nuclease
• the one or more activated elements e.g., an activating or second activating sequence
• the one or more input signals are the same.
• the one or more activated elements e.g., an activating or second activating sequence
• the translator output e.g., an activating or first activating sequence
• the amplifier output comprises a nuclease that is activated upon binding an ssDNA, an ssRNA, a dsDNA or a dsRNA translator output (e.g., from the translator layer).
• the amplifier output comprises one or more of: one or more isothermal chemical ligation-hybridization and chemical cross replication modules configured to replicate the translator output (see, e.g., Edeleva et al, Chem.
• one or more hairpin-chain reaction modules configured to replicate the translator output
• one or more duplicator gate cascade modules configured to replicate the translator output
• one or more CRISPR-Cas13 modules configured to provide an active Cas13 complex
• one or more CRISPR-Cas12 modules configured to provide an active a Cas12 complex
• the amplifier output comprises one or more duplicator gate cascade modules, wherein a set of DNA strands is configured such that a single signal (e.g., an input signal nucleic acid) can hybridized the duplicator gate.
• the hybridization of the signal to the duplicator gate promotes dissociation of two or more of the strands of the duplicator gate resulting in an amplified output signal.
• the output signal comprises different sequences.
• the output signal comprises the same sequence.
• the amplification module is the second layer of a multilayer architecture.
• the amplifier layer comprises a nuclease.
• the nuclease of the amplifier layer is and RNAse.
• the RNAse is RNAse A.
• the RNAse A is configured to be activated by the translator output.
• the RNAse A is coupled to a nucleic acid configure to allow its activation by the translator output.
• the RNAse of the amplifier layer is coupled to a surface and it is configured to be activated when released from the surface by nucleic acid strand displacement.
• the amplification layer of the system comprises a nucleic acid circuit.
• the nucleic acid circuit is configured to create an amplifier output upon addition of a translator output.
• the amplifier output is one or more ssRNA, ssDNA, or partially duplex species. Partially duplex species include dsDNA, dsRNA, or dsDNA/RNA hybrids.
• the nucleic acid circuit of the system creates an amplifier output that is at least 10 times the concentration of the translator output. In some embodiments, the nucleic acid circuit of the system creates an amplifier output that is at least 100 times the concentration of the translator output. In some embodiments, the nucleic acid circuit of the system creates an amplifier output that is at least 1000 times the concentration of the translator output. [0092] In some embodiments, the amplification module as the second layer responds to the output from the translator module as the first layer.
• the second layer is responsive to one or more of a ssDNA, a ssRNA, a dsDNA, a dsRNA, or a hybrid dsDNA/RNA molecule with specified sequence from the first layer (e.g., the translator output or trigger).
• the second layer comprises a plurality of sensing modalities. The sensing modalities may be distinct. At least one or more of the sensing modalities is responsive to the trigger. These modalities can be used individually, in parallel, or in sequence.
• the second layer uses a protein free nucleic-acid strand amplification cascade responsive to the trigger as a sensing modality.
• the amplification cascade is configured to output an amplified signal (amplified output).
• the amplified output is one or more of a ssDNA, a ssRNA, a dsDNA, a dsRNA or a hybrid dsDNA/RNA molecule or motifs.
• the amplified output may have the same sequence as a trigger or may have a sequence distinct from trigger.
• the amplification cascade yields N copies of output molecules or motifs per 1 trigger (input) signal provided to the amplifier module or layer.
• N may be at least 1, at least 10, at least 100, or at least 1000. The expected N depends on the details of the cascade and the time allotted for running the cascade.
• nucleic-acid strand amplification cascades examples include so-called hairpin-chain reaction cascades (Choi HM, Beck VA, Pierce NA. 2014. ACS Nano 8:4284-94) and isothermal chemical ligation-hybridization cycle (Edeleva E, Salditt A, Stamp J, Schwintek P, Boekhoven J, Braun D. 2019. Chem Sci 10:5807-14). These approaches can be adapted to amplify any output strand species in response to a trigger strand motif.
• the sensing modality utilizes a CRISPR-Cas based nucleic acid detection.
• the CRISPR-Cas complex is configured to detect a target nucleic acid molecule using a specifically designed crRNA.
• the Cas protein is a Cas13 protein.
• the Cas13 protein Upon complexing with the specifically designed crRNA, the Cas13 protein becomes activated and acts as a nuclease that indiscriminately digests single-stranded RNA. Fozouni P, Son S, Diaz de Leon Derby M, Knott GJ, Gray CN, et al. 2020. Cell.
• the Cas13/crRNA ribonucleoprotein (RNP) complex is designed to respond to trigger which is output from the first layer (e.g., the trigger or translator signal).
• each trigger molecule yields an activated Cas13 protein capable of degrading ssRNA molecules with the rate k cas .
• the Cas13 RNP is designed to respond to an amplified output produced by a nucleic-acid strand amplification cascade provided by the amplification module (e.g., the second layer).
• a Cas12a protein can be utilized. Cas12a indiscriminately digests single-stranded DNA molecules once activated.
• the output of a second layer may be one or more of: M activated Cas13 or Cas12 molecules per trigger strand produced from the first layer when only using Cas13 or Cas12; N amplified output molecules or motifs per trigger strand from the first layer when only using a nucleic acid amplification cascade; or N*M activated Cas13 or Cas12 when the Cas13 or Cas12 RNP are designed to respond to amplified output produced by a nucleic acid amplification cascade.
• Detection Module [0099] In some embodiments the detection module comprises one or more substrates and one or more activatable nucleic acid peroxidases.
• the one or more substrates and one or more activatable nucleic acid peroxidases may be an activatable colorimetric actuator.
• the one or more activatable nucleic acid peroxidases are configured to be converted into one or more active nucleic acid peroxidases in the presence of the amplifier output (e.g., an active or first active nuclease).
• the one or more substrates and one or more activated nucleic acid peroxidases may be an activated colorimetric actuator.
• the one or more active nucleic acid peroxidases are configured to convert the one or more substrates into one or more products.
• the detection module is further configured to accept the second amplifier output (e.g., an active or second active nuclease).
• the one or more activatable nucleic acid peroxidases are configured to be converted into the one or more active nucleic acid peroxidases in the presence of the second amplifier output (e.g., an active or second active nuclease).
• the systems or methods employ one or more switchable nanostructures.
• the switchable nanostructure can be constructed from nucleic acids (e.g., DNA and/or RNA).
• a DNA holiday junction ( Figure 6) is a simple hinged- beam nanodevice.
• the holiday junction comprises two near vertical DNA helices that are constrained by a separator helix at the helices’ lower portions.
• the constraint transduces strain on a single-stranded DNA motif at the top of the holiday junction.
• Split Probes [0102]
• the systems or methods employ enzymatic units split into two inactive subunits that are configured to become active when the two subunits come into proximity with one another.
• the split enzymatic units are nucleic acids.
• the Deoxyribozyme is a peroxidase-mimicking G-quadruplex Deoxyribozyme (DNAzyme) (PDz) that catalyzes generation of a colorimetric signal.
• PDz peroxidase-mimicking G-quadruplex Deoxyribozyme
• Any suitable Deoxyribozyme (DNAzyme) or Ribozyme (RNAzyme) that is capable of producing a detectable signal may be used.
• the system or method may include any cofactors (e.g., hemin).
• the two inactive subunits are portions of a G quadruplex probes that become an active G quadruplex when the subunits come into proximity with one another (e.g., the probes are in a closed state).
• the system may contain an integer multiple of a subunit (n) and an integer multiple of a second subunit (m).
• the first subunits and the second subunits may be brought into proximity with one another such that an integer multiple of active enzymatic units are formed (x).
• x equals n.
• x equals m.
• n, m, and x are all equal.
• the probes are active when a target nucleic acid stabilizes a closed state of the nanostructures. In a closed state, a pair of subunits will be in proximity to one another. Such a pair may recombine, thereby producing an active enzymatic unit.
• the pair may be in proximity to one another at any suitable distance that permits recombining of the two paired subunits.
• the two subunits are in proximity when they are about 0.1 nm to about 20 nm, about 0.5 nm to about 15 nm, about 1 nm to about 10 nm, about 2 nm to about 10 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm about 13 nm about 14 nm, or about 15 nm from one another.
• the immediate neighborhood of an active enzymatic unit when the nanostructure is in a closed state will be duplex DNA.
• the neighboring duplex DNA will assist in reducing interference with single-stranded elements.
• one or more subunit of a subunit pair may be a blunt-end subunit. Without being bound by theory, it is believed that switchable or dimerizable DNA nanostructures in which blunt-end DNA interfaces are being brought into proximity with one another assists in increasing positioning accuracy of the subunits. See Gerling T, Wagenbauer KF, Neuner AM, Dietz H. 2015. Science 347:1446-52.
• the systems or methods use signal amplification.
• the signal amplification is implemented by nucleated polymerization of nanostructures.
• a nucleus form upon addition of a target strand, templating the growth of a filamentous or dendritic structure where upon monomers may be integrated.
• Each monomer integrated into the filament is configured to generate one or more new active enzymatic unit (e.g., G-quadruplex peroxidase). For example, Minev D, Wintersinger, C.M., Ershova, A., Shih, W.M. 2019.
• bioRxiv demonstrate nucleated growth of ribbons including triggering the growth of ribbons upon detection of a 192nt long target nucleic acid motif.
• a pair of G-quadruplex subunits are configured as binding domains between the crisscross slat units of the ribbon.
• the system or method comprises two nucleic acid (e.g., DNA) based bricks attached to each other by their edges to form a clamshell structure (e.g., Figures 1, 2, and 7A-7B).
• the clamshell structure comprises a first portion comprising a first surface with one or more first peroxidase polynucleotides attached, a second portion comprising a second surface with one or more second peroxidase polynucleotides attached, and a hinge connecting the first portion and the second portion.
• the hinge is configured to hold the clamshell (i.e., the detection module) in an open (i.e., inactive) form when one or more reporter nucleic acids is uncleaved, and the hinge is configured to hold the clamshell (i.e., the detection module) in a closed (i.e., active) form when the one or more reporter nucleic acids is cleaved.
• the two bricks may be part of a single DNA structure, or covalently attached through the hinge, or fused in any other ways.
• the bricks are constructed using the methods of DNA origami, by other DNA nanotech approaches (e.g., tile assembly), or any other appropriate manner.
• each brick is decorated on its interior face (e.g., surface) by multiple segments of a split enzymatic unit (e.g., a G-quadruplex peroxidase).
• a split enzymatic unit e.g., a G-quadruplex peroxidase
• A denotes a 3’ segment of a split G-quadruplex peroxidase
• B denotes the 5’ segment of the same split G-quadruplex peroxidase
• the first and second surfaces i.e., the two opposing brick surfaces
• the closed state of the brick switch e.g., the clamshell
• the closed state of the brick switch may constitute a global free energy minimum.
• the clamshell possesses a high energy barrier in its open, inactive state to limit conversion into a closed, active state in the absence of a trigger, kinetically trapping the brick in an open conformation.
• the open conformation of the clamshell is limited from converting into a closed, active state in the absence of a trigger using steric occlusion.
• electrostatic repulsion between the like-charged polyanionic surfaces of the brick switch may constitute a force that counteracts closure in the absence of a trigger.
• a molecular, spring-loaded hinge mechanism may counteract closure in the absence of a trigger. Funke JJ, Ketterer P, Lieleg C, Schunter S, Korber P, Dietz H. 2016. Sci Adv 2:e160097.
• single or multiple single-stranded DNA springs may connect the bricks exerting similar hinge edges, thereby maintaining the clamshell in the open form in the absence of a trigger.
• the spring single-stranded DNA may further comprise secondary structure elements such as a hairpin motif.
• the clamshell’s one or more springs provides a force to pulling each brick surface away from each other in its inactive state. Accordingly, the clamshell is prevented from closing and activating the split enzymatic units decorating the interior surfaces of the bricks.
• a target nucleic acid interacts directly with the spring single- stranded DNA strands by, for example, forming a straight duplex DNA ( Figures 1 and 2).
• the formation of a duplex DNA resolves a hairpin motif of the single- stranded spring DNA.
• the duplex DNA allows for or promotes the bricks of the clamshell to form a closed clamshell configuration.
• the closed configuration the paired enzymatic subunits (e.g., G-quadruplex peroxidase segments) separated on the two interior faces of the clamshell are brought into proximity to one another.
• the bricks are designed to be permeable to cofactors, substrates, and/or other molecules. Such cofactors, substrates, and/or other molecules may promote or be necessary for the active enzymatic unit (e.g., G-quadruplex peroxidase) to perform its enzymatic function (e.g., its peroxidase function).
• a closed clamshell with active enzymatic units is configured as a seed nucleus.
• the active form of the clamshell templates the formation of filaments configured to recombine integer multiples of enzymatic units (e.g., G-quadruplex peroxidases) to form per monomer integration into the filament.
• multiple clamshells may be configured to cause amplification of the signal from a trigger strand through a cascade. For example, in some embodiments, closure of a clamshell structure leads to the display of one or more DNA motifs.
• the deactivating DNA motif is formed upon clamshell closure through recombination of fully complementary DNA duplex, thereby releasing one or more DNA motifs holding the clamshell opened.
• These DNA motifs are configured to deactivate the spring system on a second clamshell brick in the open and inactive form. This deactivation removes of the kinetic barrier preventing closure of the second clamshell and permits it to convert into a closed and active form.
• the closed form of the second clamshell may further display one or more DNA motifs configured to deactivate the spring system of additional open and inactive clamshell.
• the systems and methods may use any number of clamshells in a signaling cascade.
• the number of active enzymatic units may be increased by many multiples in the presence of a single copy of the target strand.
• the spring mechanism of any of the clamshell configurations disclosed herein may be provided in a number of ways. Methods in addition to those mentioned above, may include using DNA toehold and strand displacement techniques. In such configurations, the initial inactive spring (S) is displaced on one of its ends by a target strand. The target sequence hybridizes with S to form a longer spring (S’) that is partially duplex.
• the single stranded gap is hybridized by a smaller complementary strand that is present in the system, forming a more stable and stronger duplex spring (S’’) and adding further stability to the closed clamshell state.
• the use of DNA toehold and strand displacement is combined with nucleus polymerization as described above.
• the nucleic acid peroxidases of the systems or methods is a DNAzyme called peroxidase-like deoxyribozymes (PDz).
• the PDz may be incorporated to provide an optical read out. See Connelly RP, Verduzco C, Farnell S, Yishay T, Gerasimova YV.2019. ACS Chem Biol 14:2701-12.
• the active PDz generates a color change due to its catalytic activity.
• the color change is from one color to another color (e.g., from green to red).
• the color change is from a substantially clear or colorless state to a substantially opaque or colored state (e.g., from clear or colorless to blue).
• the color change is from a substantially opaque or colored state to a substantially clear or colorless state (e.g., from blue to clear or colorless).
• the color change is a darkening of a color (e.g., from light yellow to dark yellow).
• the color change is a lightening of a color (e.g., from dark yellow to light yellow).
• the PDz activity is that of G-quadruplex (G4) DNA which uses hemin as a cofactor to catalyze peroxidation of colorless organic molecules to produce a colored oxidation product.
• G4 DNA which uses hemin as a cofactor to catalyze peroxidation of colorless organic molecules to produce a colored oxidation product.
• substrates include ABTS (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) DQG ⁇ 70% ⁇ ⁇ -Tetramethylbenzidine).
• the system or methods employ split PDz (sPDz) as split probes for colorimetric detection.
• two paired portions of a split PDz are brought into proximity of one another in the presence of an analyte to be detected (e.g., a target nucleic acid molecule).
• the paired portions are brought together by recruitment of the two portions by partial sequence complementarity with a target nucleic acid molecule.
• the paired portions are attached to sequences complementary to sequences of a target nucleic acid molecule, and the uncomplimentary sequences of the paired portions are configured to recombine to form an active PDz. In the absence of the target nucleic acid, the split PDz pair is not brought into proximity with one another and is inactive.
• systems or methods use a hinge-like molecular device that can be prepared in at least two structurally distinct configurations.
• one or more integrated PDzs are rendered inactive (e.g., by separation of a pair of split probes or by holding a PDz in tension).
• the one or more multiple integrated PDzs are activated (e.g., the pair of split probes are brought into proximity with one another or the PDz is relaxed) ( Figures. 3-5).
• the inactive PDz is under tension through physically stretching of the nucleic acid strands comprising the PDz sequences ( Figures 3 and 5).
• the inactive PDz is a pair of physically separated nucleic acids wherein each of the pair comprises one portion of a PDz (i.e., an sPDz or split probe) ( Figure 4).
• Figure 3 depicts an exemplary schematic illustration of a switchable hinged-beam DNA nanodevice. The depicted beams are be composed of one or more DNA double-helices connected by a pivot junction. Gerling T, Wagenbauer KF, Neuner AM, Dietz H.2015. Science 347:1446-52. In the inactive state (left), the nanodevice is constrained by a reporter single- stranded RNA (ssRNA).
• ssRNA reporter single- stranded RNA
• the reporter ssRNA is flanked by ssRNA segments (anchors) configured to base pair into specific sites on the hinged-beam device (e.g., regions of ssDNA or ssRNA of the beam).
• ssRNA segments anchors
• the reporter ssRNA may be cleaved by any suitable enzymatic unit, for example, an active Cas13 complexed with an appropriate crRNA. Upon cleavage of the reporter ssRNA, the tension exerted upon the PDz is released, and the PDz can relax.
• FIG. 5 depicts an exemplary schematic illustration of a switchable hinged-beam nanodevice similar to that depicted in Figure 3.
• the inactive state is constrained by a reporter double-strand DNA (dsDNA).
• the reporter dsDNA is flanked by nucleic acid segments (anchors) (e.g., ssDNA or ssRNA) configured to base pair into specific sites on the hinged-beam device (e.g., regions of ssDNA or ssRNA of the beam).
• the reporter dsDNA may be cleaved by any suitable enzymatic unit, for example, an active Cas12 complexed with an appropriate crRNA.
• the PDz is inactive under tension (left), and upon cleavage of the reporter dsDNA, the tension exerted upon the PDz is released, and the PDz can relax. The relaxed PDz to refold into the active PDz state (right).
• a ssRNA or dsDNA reporter anchored to the nanodevice may also be used to separate two paired portions of an sPDz ( Figure 4). Upon cleavage of the reporter, the two paired portions of the sPDz can come into proximity with one another and complex to form an active PDz (right).
• the active state may be reached by a conformational change that leads to relaxation of the device into another conformation that allows refolding of the active PDz ( Figures 3 and 5) or recombination of a split PDz ( Figure 4). These refolded or recombined PDzs may then become catalytically active.
• the state changes can be triggered by cleavage of a reporter ssRNA or a reporter ssDNA or dsDNA.
• the reporter ssRNA can be firmly anchored in the device by using sufficiently long overhangs.
• the system is extended to substantially simultaneous activation of multiple PDzs per cleavage event, by using larger switchable DNA origami bricks.
• the switchable hinged beam device in which the beams of the device are comprised of multiple DNA helices.
• the nanodevice has room for positioning multiple copies of PDz which are each inactive or active according to the conformation of the object.
• the nanodevice is constrained in a specific conformational state by a single nucleic acid molecule.
• the nanodevice is a static closed variant stabilized by single DNA oligonucleotides.
• the nanodevice is a static open variant stabilized by single DNA oligonucleotides.
• the nanodevice is configured to enhance the sensitivity of an associated enzymatic unit (e.g., a Cas13 protein).
• the nanodevice is configured such that the limit of detecting target nucleic acids using a PDz remains comparable or even exceeds (e.g., the capacity of detecting even fewer copies of target nucleic acids) that of the conventional, fluorometric assay.
• N 10 activated PDz per cleaved hinge device may be used, such that C READ will be around 40 minutes.
• the substrate ssRNA is in large excess over Cas13 and is cleaved by Cas13.
• Any suitable concentrations of cleaving protein e.g., Cas13
• Suitable concentrations may be in the range of 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, or 1mM.
• Cas13 is present in the range of about 400nM.
• the concentration of a PDz substrate is present in a concentration similar to that of the catalytic converter device.
• the substrate is present at about 100nM, 250nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1mM, 1.25mM, 1.5mM, 2mM, 2.5mM, 3mM, 4mM, 5mM, 6mM, 7mM, 7.5mM, 8mM, 9mM, 10mM, 20mM, 25mM, 30mM, 40mM, 50mM, 60mM, 70mM, 75mM, 80mM, 90mM, 100mM, 200mM, 250mM, 300mM, 400mM, 500mM, 600mM, 700mM, 750mM, 800mM, 900mM, or 1000mM.
• the substrate is present in the mM range. In preferred embodiments the substrate is present at about 1mM-10mM.
• the PDz motifs may be held in present in stretched or split states as part of a clamshell device ( Figures 7A-7B). In some embodiments, the inactive state of the clamshell can be converted into an active state by a single reporter ssRNA cleavage event.
• the systems or methods employ a single nucleic acid strand comprising a PDz sequence ( Figure 8A). In some embodiments, the PDz’s catalytic activity is inhibited by a partially self-complementary region of the strand ( Figure 8A).
• the strand also includes a hairpin loop region that is composed of RNA or DNA nucleotide bases that may be targeted by an enzymatic cleaving unit (e.g., a CRISPR-Cas13 or -Cas12 system) ( Figure 8B).
• an enzymatic cleaving unit e.g., a CRISPR-Cas13 or -Cas12 system
• the cleavage of the hairpin loop permits is triggered by activation of the enzymatic unit (e.g., an amplifier output).
• the cleavage permits dissociation of the PDz sequence from the complementary sequence, allowing the PDz to refold into a catalytically active state (Figures 8C-8D).
• dissociation is encouraged by the self-complementary region not being fully self-complementary.
• the self-complementary region may possess one or more base pair mismatches or abasic sites ( Figures 8A-8C).
• the active PDz is configured to produce a colorimetric output visible with the unaided eye ( Figure 8C).
• the amplifier output may be in the form of a nucleic acid that is complementary or semi-complementary to a portion of the hairpin loop (e.g., an invader strand).
• the system or method does not require an enzymatic unit (e.g., an active CRISPR-Cas complex).
• the invader strand is a single stranded nucleic acid that binds to the hairpin loop promoting dissociation of the PDz sequence and the self-complementary sequence ( Figure 8E).
• the dissociation is similar to hybridization chain reaction systems.
• the detection of different analytes provides the same detectable signal (e.g., the same colorimetric signal).
• a positive detection of the molecule is determined by all sensors (e.g., reservoirs) providing a positive signal.
• the detection of analytes provides a different detectable signal (e.g., detection of a first analyte provides a first colorimetric signal and detection of a second analyte provides a second colorimetric signal).
• the different detectable signals contrast with one another.
• the detection using different detectable signals is provided for in the same reservoir.
• detection of the target molecule is determined by a composite signal.
• the detection module is the third layer of a multilayer architecture.
• the detector layer comprises an activatable (e.g., gated) peroxidase.
• the peroxidase is gated and is configured to become an active peroxidase upon cleavage of the gating mechanism.
• an active peroxidase reacts enzymatically with one or more substrates to provide one or more products.
• the peroxidase is a G-quadraplex peroxidase (G4).
• the peroxidase of the system reacts with ABTS or TMB to create a color change visible to the naked eye.
• the third layer is the detection module comprising one or more caged nucleic acid peroxidase enzyme.
• the one or more caged nucleic acid peroxidase enzyme is capable of catalyzing the conversion of an initially colorless compound into a colored compound to produce a naked-eye readable output signal once activated.
• the caged enzyme peroxidase can be activated only through amplified output strands generated in the second layer. In this case, the N output strands from the second layer generated per trigger molecule generate N active enzyme peroxidases.
• the number of colored compound molecules produced in the third layer grows linearly in time with N*k cat *t, where k cat is the catalysis rate of the active peroxidase.
• a split G-quadruplex DNAzyme probe is used.
• a nucleic acid motif recruits the two split pieces of the probe, thereby recombining the active G quadruplex.
• the caged enzyme peroxidase can be uncaged by the activity of Cas proteins activated in the second layer. Over time, Cas proteins will activate more and more peroxidases. If M active Cas molecules have been produced in the second layer, the third layer is capable of generating M*k cat *k cas *t 2 colored molecules per trigger molecule, provided that sufficient reactants (e.g., substrate and reaction co-factors) are present.
• the second layer uses a nucleic-acid cascade and Cas proteins in series. N*M active Cas molecules per trigger molecule will be generated.
• the third layer is capable to then generate N*M*k cat *k cas *t 2 colored molecules per trigger molecule, provided that sufficient reactants (e.g., substrate and reaction co-factors) are present.
• a G-quadruplex sequence motif sequestered in a DNA hairpin stem is used. By hiding the functional G- quadruplex sequence in the hairpin it cannot function as a peroxidase. The G-quadruplex enzyme motif is therefore inhibited as long as it stays buried in the hairpin loop.
• the loop in the hairpin can be RNA or DNA.
• trigger-strand activated Cas13 or Cas12a proteins from the second layer can digest the loop.
• the hairpin stem strands will dissociate, and the G-quadruplex sequence will be able to fold into the active G-quadruplex structure.
• hybridization of DNA strand motifs created by protein-free paths in the second layer invade the hairpin loop or regions of the hairpin stem such that the G quadruplex motif can refold and become active.
• the caged enzyme peroxidase can be activated either through amplified output strands from the second layer via strand-displacement reactions or by ssRNA or ssDNA degrading activity from activated Cas proteins produced in the second layer.
• the third layer will be capable of generating N*k cat *t + M*k cat *k cas *t 2 colored molecules per trigger molecule.
• a G-quadruplex sequence motif is stretched out on a DNA duplex rack, which inhibits formation of the active G-quadruplex structure. For example, see design v1.3.2 at Example 3 ( Figures 14A-14C). The peroxidase is inhibited as long as it stays stretched on the DNA duplex rack.
• the stretched-out G-plex can be flanked with a single-stranded (e.g., UUUUU) region.
• the trigger-strand activated Cas13 or Cas12a proteins from the second layer can cut the single-strand (e.g., UUUUU).
• the G-plex when not under tension, is able to relax and fold into the active G- quadruplex structure.
• hybridization of DNA strand motifs created by protein-free paths in the second layer strand-displace the stretched-out G4 from the DNA rack such that the G quadruplex motif is able to fold and become active.
• the modules are configured to provide an auto-catalytic cascade to amplifying the signal from a target.
• the system or method employs one or more of the following steps: introduction of a first inhibited nuclease (e.g., an activatable Cas protein (e.g., Cas12 or Cas13) or an activatable Cas protein complexed with CRISPR) and introduction of a first activating sequence (e.g., a polynucleotide released as a translator output) to yield a first active nuclease (e.g., an active Cas proteins (e.g., Cas12 or Cas13) or an active Cas protein complexed with CRISPR); introduction of a first active nuclease (e.g., an active Cas protein (e.g., Cas12 or Cas13) or an active Cas protein complexed with CRISPR) and introduction of an inhibited colorimetric actuator (e.g., an activatable nucleic acid peroxidase, substrates, and cofactors) to yield an active colorimetric actuator (e
• the Cas protein is a Cas12 protein, Cas13 protein, subvariant protein thereof. In some preferred embodiments, the Cas protein is a Cas12a protein. In some embodiments, the system or method provides programmatic activation of one or more RNAse proteins in the presence of one or more activating sequences. [0140] In some embodiments, amplifying hairpin probes (Figures 27D and 27H) may be provided in high concentrations. In some embodiments, amplifying hairpin probes are provided in the hundreds of nanomolar range.
• the amplifying hairpin probes are provided at about 100nM or greater, about 200nM or greater, about 300nM or greater, about 400nM or greater, about 500nM or greater, about 600nM or greater, about 700nM or greater, about 800nM or greater, about 900nM or greater, about 1,000nM or greater.
• the architecture is a layered (tiered) system for detecting nucleic acids comprising a translation layer, an amplification layer, and a detection layer ( Figure 18).
• the translation layer receives a plurality of nucleic acid input signals and converts them into a single signal (e.g., the translator output).
• the nucleic acid input signals may include ssRNA or ssDNA.
• the translator output may include ssDNA, ssRNA, dsDNA, dsRNA, and/or a partially duplexed dsDNA, dsRNA, or RNA/DNA hybrid.
• the amplification layer amplifies the translator output and creates a second signal (e.g., the amplifier output).
• the detection layer converts the amplifier output into an optical output (e.g., a colorimetric change).
• the three layers of the detection system are incorporated into a lateral flow assay. In some embodiments, the three layers of the detection system are incorporated into a paper strip assay.
• the detection system is incorporated by means of gel, polymer, desiccation, lyophilization or some combination thereof the lateral flow assay. In some embodiments, the incorporation is achieved by means of soaking, impression printing, inkjet printing, or some combination thereof.
• the substrate of incorporation is a polymer film, nonwoven, paper, or fiber glass. In some embodiments, the incorporation results in immobilization of the detector layer at a specified immobilization site. In some embodiments, the immobilization of the detector layer is reversible upon release by the amplifier output and is carried away through diffusion or capillary action into a runoff site.
• the location of the optical output designates the presence of the measured nucleic acid wherein a negative result is designated by an optical output at the original immobilization site and a positive result is designated by an optical output in the runoff site.
• the immobilization of the detector layer is reversible upon release by diffusion or capillary action.
• the amplifier output destroys the detector layer wherein a negative result is designated by an optical output and a positive result is designated by the absence of an optical output.
• the immobilization of the detector layer is irreversible.
• a negative result is designated by the absence of an optical output and a positive result is designated by an optical output.
• the system or method employ a solid state system.
• the system or methods may incorporate agarose gels, polyacrylamide gels, and other similar materials. These gels may be cured into a well, extruded into film, soaked into absorbent material (e.g., nonwoven, cloth, fibers), or prepared in another appropriate manner.
• the system or method may incorporate surface immobilization, including but not limited to, nanoparticles and acrylamide copolymerization.
• the system or method may incorporate techniques, including but not limited to, physical adsorption, covalent attachment, bioaffinity immobilization of some nucleic acids, LFA, and surface modified chips.
• RNA molecules tethered to a G4 on one end and the surface on the other may permit Cas to cut the tether as the sample flows.
• the G4s will be carried to where the hemin, ABTS, peroxide is and react. This can result in a localized color change at the end of the strip if positive. If diffusion is an issue, a color change may still occur in the middle which could signify a negative result [0145] In some embodiments, the readout results from where the color change occurs.
• a positive result may be indicated by a color change near the end of the form and a negative result may be indicated by a color change near the middle of the form.
• an RNA based G4 may be tethered in near the middle of the form, and the ABTS, Hemin, and peroxide is provided the end of the form.
• an activated Cas protein digests the G4, preventing a color.
• an inactive Cas protein does not digest the G4, and a color occurs near the middle of the strip.
• the system may incorporate powder, for example, lyophilized components. These may be used for surface immobilization.
• the powder may be added to an adhesive attached to some absorbent material or pad which carries the sample past the powder. If all systems can be rehydrated into an active state, the sample is capable of activating the powdered components. In some embodiments, a coating powder may be incorporated onto nonwoven forms.
• the system or method employ a non-solid state system, including, for example, in-solution system ( Figure 26).
• the system or method provides for detection of multiple analytes. In some embodiments, the detection of multiple analytes is provided separately in a single form-factor (e.g., in separate reservoirs) ( Figures 15-18).
• the detection of multiple analytes is provided in together in a single form-factor (e.g., in a single reservoir).
• the second analyte is of a the same or a similar molecular type as the first analyte.
• the first analyte is a nucleic acid (e.g., a DNA or an RNA)
• the second analyte may also be a nucleic acid (e.g., a second DNA or RNA).
• the first analyte may be a virus protein and the second analyte may be antibodies associated with infection by the virus.
• the first and second analyte may be the same analyte (e.g., the same DNA consisting of the same DNA sequence).
• the system or method may provide for redundant detection of the same analyte by separate sensors. It will be readily understood that such redundant, independent detection of the same analyte provides an increased confidence in detections of the analyte.
• the second analyte is of a different molecular type as the first analyte.
• the second analyte may be a protein, polypeptide, or an oligonucleotide.
• the system or method detects an analyte associated with an infectious agent (e.g., a virus) and an analyte associated with an immune response to the infectious agent (e.g., an antibody).
• an infectious agent e.g., a virus
• an analyte associated with an immune response to the infectious agent e.g., an antibody
• the detecting the combination of an infectious agent analyte and an immune response analyte may be of critical importance in monitoring how quickly and where a vector-borne disease is spreading and correlating such information with the rate at which a population is able to become immune against it.
• any of the designs or methods disclosed herein may be in any appropriate form-factor or use-case including, but not limited to, a skin wearable, a sticker on an object’s surface, a two-dimensional applique, or a substrate (e.g., a polymeric film substrate).
• the form-factor or use-case may be a wearable temporary tattoo that displays exposure information.
• the exposure information may include, but is not limited to, cumulative UV exposure, real-time UV exposure, or a combination thereof.
• the form-factor or use-case may include one or more hydration sensors that detect electrolytes in sweat.
• the form-factor or use-case display visual readouts with graded output levels. Figure 32.
• the designs or methods disclosed herein allow for routine testing. In some embodiments, the designs or methods disclosed herein enabling semi-quantitative assessment(s) of various conditions or states.
• the designs or methods may provide assessments of viral loads to determine an infectious period, the degree of up/down regulation of an microRNA biomarker associated with one or more health condition of interest (e.g., Alzheimer’s Disease, Mild Traumatic Brain Injury, a type of cancer, or a comination thereof), or any combination thereof.
• the form-factor or use-case may further define one or more reservoir protected by a porous membrane.
• a reservoir may contain a biosensor system focused on a one or more specific analytes, for example a specific RNA or DNA sequence being targeted.
• Figure 17 depicts an exemplary form-factor comprising four separate reservoirs.
• the four reservoirs correspond to a biosensor system for detecting an analyte associated with SARS-CoV-2 (i.e., SARS-CoV-2), an analyte associated with viruses from the CoV family (pancoronavirus), an additional analyte target (RNAseP), and a control target.
• SARS-CoV-2 i.e., SARS-CoV-2
• an analyte associated with viruses from the CoV family pancoronavirus
• RNAseP additional analyte target
• the reservoirs act as homogeneous assays where all biosensor reactions occur concurrently.
• the reservoirs provide separation among the biosensor systems to avoid cross-reaction effects.
• the reservoir may be protected with a lid or seal that can be peeled or partially peeled back. In some embodiments, the lid or seal is replaced after a source (e.g., saliva deposited by a lick) is provided.
• the form-factor may comprise a control on the borders of one or more of the reservoirs that provides an indication upon the application of a source.
• the border may change color (e.g., from clear to blue) when presented with a detectable characteristic of the source (e.g., a pH associated with a source such as saliva).
• a detectable characteristic of the source e.g., a pH associated with a source such as saliva.
• the indication is instantaneous or near-instantaneous.
• the indication corresponds to a sufficient amount of source provided to the associated reservoir.
• the performance of the system or method may be modified by any suitable means.
• such modifications include concentrating a sample (e.g., saliva) in a solid state collector by spatially constraining it to some active area, activating multiple peroxidases (e.g., G4) per amplifier output (e.g., active Cas proteins such as Cas 12 and/or Cas13) cutting event, increasing the rate of substrate to product turn over (e.g., peroxidation) by using more substrate (e.g., ABTS and H 2 O 2 ), and reducing residual background activity of non-switchable (e.g., consistently active) peroxidases by, for example, purification.
• the chemistry of the sensor can be calibrated to be less or more sensitive to different concentrations of the target analyte.
• the system includes one, two, or more levels of quantification (e.g., energy bars). These levels of quantification may be used to finetune certain variables (e.g., the number of PDzs).
• Auto-Catalytic Embodiments and Split Trigger Fragments Any of the embodiments disclosed herein may include an auto-catalytic component or components. Some embodiments may use a DNAzyme called G4 peroxidase-like deoxyribozymes (PDz). In some of these embodiments, optical sensors based on G4 peroxidase have been widely explored for the detection of various analytes including nucleic acids (R. P. Connelly, C. Verduzco, S. Farnell, T. Yishay, Y.
• G-quadruplex (G4) DNA which uses hemin as a cofactor to catalyze peroxidation of colorless organic molecules to produce a colored oxidation product.
• G-quadruplex (G4) DNA which uses hemin as a cofactor to catalyze peroxidation of colorless organic molecules to produce a colored oxidation product.
• substrates include ABTS (2,’'-azino-bis(3- ethylbenzothiazoline-6-VXOIRQLF ⁇ DFLG ⁇ DQG ⁇ 70% ⁇ ⁇ -Tetramethylbenzidine).
• a target nucleic acid molecule may recruit the two parts of a split G4 peroxidase by partial sequence complementarity, where overhangs then recombine to form an active G4 peroxidase.
• the split PDz in preferred embodiments, in the absence of the target nucleic acid, the split PDz remains ideally inactive.
• a cryptic G4 peroxidase which is buried in the double-helical stem of a hairpin, may be used in such solutions.
• the hairpin loop e.g. when containing UUU, may be cut by the collateral activity of an active Cas protein. This hairpin loop cleavage may lead to strand dissociation and G4 peroxidase activity.
• the system can be successfully employed to report on and quantitatively measure Cas collateral activity as triggered by a specific RNA strand (or by an arbitrary target DNA or RNA strand via a translation module while keeping the trigger RNA strand the same).
• the Cas protein in an auto-catalytic embodiment may be any suitable wildtype or modified Cas protein including but not limited to Cas12, Cas13, Cas14, subtypes of any of these Cas proteins (e.g., Cas13a).
• a system may use more than one Cas protein (e.g., a mixture of Cas12 and Cas13 proteins).
• a suitable Cas protein may have transcleavage activity.
• the target of the active Cas protein may be a single stranded RNA (ssRNA), a single stranded DNA (ssDNA), a double stranded DNA (dsDNA), or any other appropriate nucleic acid.
• the Cas protein is a Cas13 protein.
• the Cas protein is a Cas13a protein.
• the system involves the collateral activity of a target-RNA activated Cas protein when directed not only onto a hairpin with G4 peroxidase sequence, but also onto another hairpin with a cryptic trigger sequence, which is not the target sequence.
• the unmasking of the cryptic trigger by hairpin-loop digestion frees the trigger molecule, which in turn may hybridize with suitable crRNA molecules to activate collateral activity of additional Cas enzymes. In some embodiments, this leads to feedback and allows for exponential amplification of the signal.
• Figure. 28 displays an example of self- catalytic amplification in general terms.
• the Cas protein targets an RNA. [0160] When burying complete trigger RNA sequences in hairpin structures, analogous to the cryptic G4 design, some hairpin stems may be too stable to dissociate after cleavage. In such situations, cryptic trigger RNA sequences remain cryptic, and limited or no self-catalysis may be achieved.
• split trigger fragments and primed crRNA may be used in any of the embodiments disclosed herein.
• Figures 29A-29B [0161]
• a trigger sequence is split into two sequence segments, e.g. in the middle, or as one-third and two-thirds, or as one-quarter and three-quarters or any other appropriate fragment. Fragmentation in degrees greater than two (three or more fragments make up a full trigger sequence e.g. as one-third each, or any appropriate length) may also be used.
• one or more of these segments is pre-mixed together with crRNA and Cas protein to form a complex (a pre-complexed fragment).
• the addition of the one or more missing, uncomplexed fragments of trigger RNA may result in activation of the collateral nuclease activity of Cas protein.
• the missing pieces of trigger RNA may be buried in the stems of hairpins with cleavable loops. Since these pieces are much shorter, the stability of the hairpin stems is also much reduced, meaning that the strand dissociation after cleavage actually occurs on practically relevant timescales.
• the collateral activity of already active Cas enzymes may lead to degradation of the newly generated trigger molecules before they are sequestered by crRNA, thus the feedback loop and exponential amplification may be attenuated limiting its practical benefit.
• the collateral activity of the already active Cas enzymes do not lead to degradation of the newly generated trigger molecules before they are sequestered by crRNA.
• Potential cannibalistic behavior (the tendency of the system to destroy components that are necessary for its desired function) is circumvented in preferred embodiments.
• the cleavable loop may contain canonical RNA bases that are easily and rapidly cleaved by Cas enzymes, e.g. UUUU.
• the cryptic trigger RNA sequence located with the double-stranded hairpin stem are formed from chemically modified RNA bases.
• RNA trigger is flanked with DNA to reduce likelihood of digestion of the RNA by already activated Cas protein.
• flanking RNA with DNA can be combined with incorporating modified RNA bases into the RNA trigger.
• the DNA flanking may be used in combination with any of the other modifications described herein. This setup enables systems wherein once cryptic trigger RNA molecules are rendered non-cryptic as a consequence of hairpin loop cleavage and strand dissociation, their degradation by already activated Cas enzymes is limited or eliminated, and can hybridize with crRNA (primed or not depending on the system) to activate additional Cas enzymes, thus continuing the desired feedback loop.
• the full trigger RNA sequence is partially masked within the double-stranded hairpin stem.
• the remaining, non- cryptic trigger sequence element may be pre-hybridized with crRNA ( Figures 29A-29B) to produce a primed crRNA in complex with a Cas protein which remains inactive in this configuration.
• the formerly cryptic trigger RNA sequence segment will be free to fully hybridize with the crRNA it is already in complex with. This will lead to Cas protein activation, thus continuing the desired feedback loop.
• Figures 31A-31C In some embodiments, there will be constant competition between hairpin masking of the cryptic trigger and the remainder of the free crRNA. In preferred embodiments, chemically modified RNA(s) are not employed.
• the RNA(s) may include any of the chemical modifications or combinations thereof disclosed herein.
• the tandem use may provide redundant and or enhanced protection.
• few or no single-strands are liberated into solution where they are subject to digestion by an active Cas protein.
• the freed trigger is adjacent to its desired destination. This may result in faster generation of newly activated Cas proteins.
• Equation 2 is rearranged to compute the time needed C READ for having reached a particular target concentration C READ of converted, and thus colored ABTS: [0173] Using the numbers for the rates as given above, 30 fmol/L for c target RNA (which corresponds to approximately 100 copies per microliter, and 0.5 mmol/l for C READ ,the following is arrive at: Example 2 System Performance [0174] Provided here is an illustration of the performance of an exemplary embodiment of the system or method comprising three layers.
• FIGs 19A-19L illustrate the performance of the combined action of the second layer (e.g., amplification module) and the third layer (e.g., detection module), as calculated with a simple numerical model.
• the model computes the concentration of catalytically active G quadruplex peroxidases (C G4ON ), the concentration of transparent molecules (C TRANS ) (e.g., a substrate) that are turned over by the peroxidase into “blue” molecules (C BLUE ) (e.g., a product).
• Active peroxidases are being generated by active Cas proteins (e.g., Cas13 or Cas12) with a rate that depends on the initial concentration of active Cas proteins and the catalysis rate k Cas .
• the initial concentration of active Cas proteins is assumed to be identical to the concentration of trigger strands (e.g., the translator output) produced by the first layer (e.g., translator module) or by an amplifying cascade provided by the second layer.
• the rate k G4 at which G quadruplex peroxidases turnover the substrate molecule into the product varies with time. k G4 depends on the current concentration of hydrogen peroxide and the concentration of available substrate molecules.
• Figures 19A-19L depict a simplified numerical model for computing the performance of the double-catalytic system consisting of activated Cas proteins (e.g., Cas12 or Cas13), which, in turn, activate accumulating G4 quadruplex peroxidases.
• k G4 is modeled at 10 per second ( Figures 19A-19D), 100 per second ( Figures 19E-19H), and 100 per second ( Figures 19I-19-L).
• k Cas is modeled at 600 per second ( Figures 19A-19H) and 1200 per second ( Figures 19I-19-L).
• Background active peroxidase (G4) is modeled at 5nM ( Figures 19A-19H) and 1nM ( Figures 19I-19-L).
• Trigger e.g., translator output concentration is modeled at 60 cps/ ⁇ l (0.1 fM) ( Figures 19A, 19E, and 19I), 600 cps/ ⁇ l (1 fM) ( Figures 19B, 19F, and 19J), 6,000 cps/ ⁇ l (10 fM) ( Figures 19C, 19G, and 19K), and 60,000 cps/ ⁇ l (100 fM) ( Figures 19D, 19H, and 19L).
• N amplification level
• the expected limit of detection corresponds to a target strand concentration of approximately 0.5 fmol/L (300 cps/ ⁇ l).
• Example 3 Activatable Peroxidase Designs
• Exemplary sequence designs include v1.2 G-plex (PDz) stretcher ( Figure 9).
• v1.2.6 and v1.2.7 are examples of two designs for deactivation and activation of circularized G-plex probes. Both are circularized G-plex (PDz) variants containing the G plex suppressor oligo ( Figures 10A-10B).
• v1.2.8 to v1.2.11 represent a total of 8 designs covering split and continuous G plex variants (in two sequence modifications). They present two slightly different ways of constraining the ends of the activatable G plex.
• Constrained G plex (PDz) variants are depicted in Figures.11A-11E and include v1.2.8- v1.2.11. Each of these variants include two subvariants.
• v1.2.8a is an example of a split G plex variant with its end backbone rotated proximally
• v1.2.8b is an example of a split G plex variant with its end backbone rotated distally ( Figure 11A).
• v1.2.9a is an example of a split G plex variant with its end backbone rotated distally
• v1.2.9b is an example of a split G plex variant with its end backbone rotated proximally
• Figure 11B v1.2.10a is an example of a continuous G plex variant with its end backbone rotated proximally
• v1.2.10b is an example of a continuous G plex variant with its end backbone rotated distally
• Figure 11C is an example of a continuous G plex variant with its end backbone rotated distally
• v1.2.11a is an example of a continuous G plex variant with its end backbone rotated distally
• v1.2.11b is an example of a continuous G plex variant with its end backbone rotated proximally ( Figure 11D).
• v1.2.12 G plex is an exemplary variant inhibited by stretching ( Figures 12A-12B).
• the test design creates a configuration with the termini of the G plex being held about 10 nm apart. Testing of the activity is performed using ordered strand combinations.
• the strand combinations may be strand 2; strand 2 then strand 3 ; strand 2, then strand 3, and then strand 1; strand 2, then strand 3, then strand 1, and then strand 3; or strand 2, then strand 3, and then 3 ( Figure 12B). These combinations are annealed together in order.
• Another annealing combination includes strand 1, strand 2, and then strand 3, and later annealed to strand 3 ( Figure 12B).
• v1.3.1 and v1.3.2 are exemplary simplified G plex (PDz) designs which are inhibited by stretching and include a poly-U site for Cas cutting ( Figures 13, 14A-14C).
• the test designs create a configuration with the termini of the G plex held about 13 nm apart.
• the nick site of v1.3.1 may allow for buckling, relieving mechanical stress, and may have a propensity to aggregate.
• v1.3.2 uses three strands to compensate for the potential nick-induced buckling of design v1.3.1. v1.3.2 may also form easily.
• Figure 24 depicts exemplary colorimetric data obtained stretched G4 quadruplex variants (v1.3.1 and v1.3.2).
• the dashed line represents fully active variants, and the solid line represents closed, inactive variants.
• the data provides absorbance measured by spectrophotometry.
• Closed variants were formed by heating non-G4 strands between 50° and 80°C for 5 to 20 minutes to anneal them together, cooling to room temperature with or without submerging in an ice bath for 10 to 90 minutes, adding the G4 strand before heating between 30° and 50°C for 5 to 30 minutes, and finally cooling to room temperature with or without submerging in an ice bath for 10 to 90 minutes.
• Open variants contain an unstretched system, indicative of the system after the stretched strand is released by Cas in the amplifier output.
• v8.1.0-v8.1.7 are examples of G plex caged duplexes ( Figures 25A-25H). These designs are similar to G plex caged hairpin designs ( Figures 8A-8E), however the system is composed of two strands.
• These variants may incorporate the reversible silencing of a G quadruplex peroxidase DNA molecule (G-plex strand) with a separate DNA molecule (silencing strand). To this end, the molecule containing the G quadruplex sequence motif is hybridized to a DNA oligonucleotide with complementary sequence.
• the silencing strand may contain several poly-U loops in the region of the duplex with the G plex sequence motif. These poly U loops can be digested by a Cas13 nuclease. Such produced strand fragments of the silencing strand are able to independently dissociate from the G plex strand, which in turn can then fold up into a functional G quadruplex structure that can act as a peroxidase.
• Figure 20 depicts exemplary colorimetric data obtained with G4 quadruplex variants (v8.1.0-v8.1.7).
• the dashed lines represent fully active variants, and the solid lines represent closed, inactive variants. Shading pair the “on” and “off” states of the same variant preparation. Different shades correspond to different design variants.
• the absorbance was measured by spectrophotometry, where an absorbance value of 4 (arbitrary units) is represented by a thin line (e.g., lines 2,14-1 and 2,14-1,2) and an absorbance value of 0.5 (arbitrary units) is represented by a thick line (e.g., lines 1,14-1 and 1,14-1,2).
• the residual activity of closed variants is attributed to ill-formed hairpins that can be removed by, for example, further purifying for refining production of strands.
• Open variants contain the G4 strand that will be displaced after the complementary strand is cleaved/destroyed by Cas protein in the amplifier output.
• Example 4 Exponential Increase of Indiscriminate Nuclease Activity
• a Cas13 protein is mixed with appropriately designed CRISPR RNA molecule (“crRNA”) to form a ribonucleoprotein complex.
• crRNA CRISPR RNA molecule
• a Cas13-cleavable caged G-quadruplex hairpin construct is provided in the system.
• the hairpin stem dissociates.
• One of the hairpin stem strands contains a G quadruplex sequence which can then fold into the G quadruplex and assumes peroxidase activity which can be used to generate a naked-eye readable colorimetric output.
• This is two-tiered enzyme cascade without feedback, in which target RNA activate Cas13 enzymes, which in turn catalyze the activation of peroxidases.
• the cascade system can be modified to include a feedback loop, which will generate exponential amplification.
• the exemplary system is autocatalytic.
• a crRNA-Cas13 complex is that responds to a target sequence (e.g. a SARS-Cov2 genomic RNA sequence motif) is used ( Figure 27A).
• a target sequence e.g. a SARS-Cov2 genomic RNA sequence motif
• Figure 27B A crRNA-Cas13 complex that utilizes a Cascade-crRNA featuring the RNA version of the G-quadruplex sequence motif is provided ( Figure 27B).
• a crRNA-Cas13 complex can be prepared that utilizes a cascade-crRNA that responds to a target RNA motif (e.g., another sequence) that can be, but does not need to be, fully independent of the viral target sequence (Figure 27C).
• a target RNA motif e.g., another sequence
• Figure 27H the system also provides another hairpin probe with a loop-stem structure as shown in Figure 27H.
• Digestion of this hairpin loop by collateral activity of Cas13 as seeded by a viral target RNA leads to strand dissociation where one of the resulting strands able to hybridize to the cascade-crRNA2-Cas13 complex in Figure 27C, which will lead to activation. Additional active Cas13 are thus generated that can cut both the amplifying hairpin probes (Figure 27H) and also the G quadruplex hairpin probes ( Figure 27D). Exponential self- amplification will strongly enhance the signal. This exemplary system will not present background peroxidase activity.
• the second target RNA motif e.g., another sequence
• Figure 27H may be the same sequence used to detect the initial SARS-Cov2 target in the Cas13 complex ( Figure 27A).
• an initial trigger RNA viral motif may have extremely low concentration (e.g., about 1 fmol/L or less)
• the amplifying hairpin probes (Figures 27D and 27H) may be provided in high concentrations (e.g., about 500 nmol/L). This means the system is provided with a large pool of secondary trigger molecules for Cas13 that will provide strong exponential amplification as soon as the cascade is triggered.
• Exemplary sequences for the cascade constructs include: [0201] These systems may be combined with one another to enhance sensitivity.
• Example 5 Use Cases [0202] Provided here is a non-exhaustive list of use cases and embodiments of the systems and methods.
• the user has a set of small thin cards at home.
• the user (1) takes out a card, (2) removes its cover, (3) applies saliva onto the microreservoirs on the card, (4) closes a lid covering the reservoirs, and (5) waits to learn if she’s positive or negative ( Figure 15).
• the microreservoirs may be empty before saliva is deposited on it or may comprise a gel-like material necessary for extraction, inactivation, or sensing.
• the sensors may be printed on the lid, protruding from its interior face. Thus, when the lid is closed, the sensors are in contact with the saliva, allowing the user to potentially lick the reservoirs knowing that there would be no contact with the sensor. A negative result will show the control sensor turn color only.
• a positive result will result in 2 or 3 or more sensors turning color.
• Self testing with voluntary mobile app reporting [0204] Similar to self-testing, but a computer vision mobile app scans the card and sends the results to a central location (e.g., for contact tracing).
• Self-testing with compulsory mobile app reporting [0205] Similar to self-testing with voluntary mobile app reporting. However, the meaning of each sensor is unknown to the user. Additional sensors and corresponding reservoirs may be added where the sensors may only need saliva to be activated (e.g., similar to an electrolyte sensor or a pH sensor). In some cases, there may be no sensor thus the material in contact with the saliva would never change color.
• the card may have a printed key using a barcode or QR code, or some other way that can be read by the mobile app (Figure 16).
• the test results and key are read by the mobile app and sent to a central location.
• the central location uses the key to determine the meaning of all the reservoirs and sends back the test results to the mobile app user. Even though the number of possible reservoir configurations is limited, the key can be unique to each card. Thus, knowledge of results of one card does not determine knowledge of the configuration of other cards.
• Supervised Test Similar to the above uses, but the test is done in the presence of someone approved to validate the test.
• a test may be done while being supervised by someone at the airline, school, stadium, etc. tasked to ensure that the test was correctly performed (Figure 17).
• This supervised test may be in addition to a self-test performed by the user earlier that day at home our just outside the venue being attended. Additionally, the job of the person tasked to ensure the test is correctly performed may include verifying that this person is who she says she is.
• the card may be placed on the user’s hand or some visible place and act as a health pass.
• Detection Targets Any of the embodiments described herein may be used to detect any suitable nucleic acid and/or small molecule targets.
• Suitable targets may originate from viruses (e.g., SARS- CoV-2), microbial cells (including those belonging to the microbiome of a subject), the subject’s cells, or may include any byproducts of these sources.
• Suitable targets may be associated with various health conditions including cancer (e.g., breast cancer), chronic inflammation, hydration levels, skin hydration levels, immune system disfunction, atopic dermatitis, psoriasis, acne vulgaris, skin ulcers, conditions associated with aging, and other medical conditions (e.g., Alzheimer’s Disease).
• the systems described herein identify and/or analyze the microbiome of a subject (or nucleic acids and/or small molecules thereof) and/or indicators of environmental exposures that may be deleterious (e.g., nucleic acids present in the SARS-CoV- 2 virion or produced by the human body as a consequence of exposure to SARS-CoV-2).
• the systems described herein detects nucleic acids derived from a subject’s own genome, for example, genomic fragment, microRNA, etc. from the subject.
• the systems detect and/or predict via correlation the occurrence of carcinogenic conditions, inflammation disorders, potential infections, and other health conditions.

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