Modified polynucleotide aptamers and methods of use

Abstract

Provided herein are modified polynucleotide aptamer switches, methods of tuning interactions between modified polynucleotide aptamer switches aptamer and target analytes, methods of using and manufacturing modified polynucleotide aptamer switches, and methods of detecting target analytes with modified polynucleotide aptamer switches.

Description

MODIFIED POLYNUCLEOTIDE APTAMERS AND METHODS OF USECROSS REFERENCE[1] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 584,778 filed September 22, 2023, U.S. Provisional Patent Application No. 63 / 681,033 filed August 8, 2024, U.S. Provisional Patent Application No. 63 / 696,667 filed September 19, 2024, each of which is hereby incorporated by reference in their entirety.BACKGROUND[2] Oligonucleotide probes are widely used in various molecular biology applications for detecting and quantifying specific target analytes. However, controlling the properties of affinity-based probes can be challenging.SUMMARY[3] Oligonucleotide-based molecular switches have been proven to be powerful reagents for a wide range of applications in biotechnology and medicine. However, it is important that the properties of the molecular switch (e.g., kinetics, thermodynamics, signal gain) are appropriately matched to the needs for a given application Unfortunately, there are limited methods available to tune the properties of oligonucleotide-based molecular switches and efforts to tune molecular switches are often time-consuming and rely on trial-and-error. Therefore, there is a need for improved methods and techniques for tuning and / or modulating the properties of oligonucleotide-based molecular switches.[4] Provided herein are methods of detecting a target analyte with a polynucleotide aptamer switch comprising: providing a polynucleotide aptamer switch configured to undergo a conformation change upon binding to the target analyte, the polynucleotide aptamer switch comprises: one or more labels which are configured to produce a change in a signal when the polynucleotide aptamer switch is bound to the target analyte; and one or more modifications which alter a binding affinity between the polynucleotide aptamer switch and the target analyte, the modification comprising at least one of: incorporation or substitution of one or more modified nucleobase(s) in an aptamer domain, a switching domain, a linker region, or a stem region of the polynucleotide aptamer switch; incorporation or substitution of one or more sugar modifications in the polynucleotide aptamer switch; incorporation or substitution of one or more modified internucleoside linkage(s) in the polynucleotide aptamer switch; incorporation or substitution of one or more non-natural nucleobase, a non-natural sugar, anda non-natural internucleoside linkage in a displacement region of the polynucleotide aptamer switch; incorporation or substitution of one or more sugar moiety modification(s) in the polynucleotide aptamer switch, or any combination thereof; and contacting the polynucleotide aptamer switch with the target analyte, inducing a conformational change in the polynucleotide aptamer switch and producing the change in signal, thereby detecting the target analyte. A method of tuning an interaction between a target analyte and a polynucleotide aptamer switch comprising: modifying the polynucleotide aptamer switch to comprise one or more modifications which alter a binding affinity between the polynucleotide aptamer switch and the target analyte, the one or more modifications comprise at least one of: incorporation or substitution of one or more modified nucleobase(s) in an aptamer domain, a switching domain, a linker region, or a stem region of the polynucleotide aptamer switch; incorporation or substitution of one or more sugar modifications in the polynucleotide aptamer switch; incorporation or substitution of one or more modified internucleoside linkage(s) in the polynucleotide aptamer switch; incorporation or substitution of one or more non-natural nucleobase, a non-natural sugar, and a non-natural internucleoside linkage in a displacement region of the polynucleotide aptamer switch; incorporation or substitution of one or more sugar moiety modification(s) in the polynucleotide aptamer switch; or any combination thereof. In some embodiments, the one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in a region of the polynucleotide aptamer switch where it hybridizes to itself, and comprise shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch. In some embodiments, one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in an aptamer domain of the polynucleotide aptamer switch, thereby shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch. In some embodiments, the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising a linker domain, the one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in the linker domain, thereby shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch. In some embodiments, the polynucleotide aptamer switch comprises an aptamer domain which binds to the target analyte, the one or more modifications are in the aptamer domain andincrease the binding affinity between the aptamer domain and the target analyte relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more modifications increase a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more modifications increase the binding affinity between the aptamer domain and the target analyte by increasing a number of hydrogen bonds between the target analyte and the aptamer domain, increasing a number of salt bridges between the target analyte and the aptamer domain, increasing a number of hydrophobic interaction sites between the target analyte and the aptamer domain, increasing a number of electrostatic interaction sites between the target analyte and the aptamer domain, or combinations thereof, relative to an unmodified polynucleotide aptamer switch. In some embodiments, the polynucleotide aptamer switch comprises an aptamer domain which binds to the target analyte, the one or more modifications are in the aptamer domain and decrease the binding affinity between the aptamer domain and the target analyte relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more modifications decrease a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more modifications decrease the binding affinity between the aptamer domain and the target analyte by decreasing a number of hydrogen bonds between the target analyte and the aptamer domain, decreasing a number of salt bridges between the target analyte and the aptamer domain, decreasing a number of hydrophobic interaction sites between the target analyte and the aptamer domain, decreasing a number of electrostatic interaction sites between the target analyte and the aptamer domain, or combinations thereof, relative to an unmodified polynucleotide aptamer switch. In some embodiments, the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising an aptamer domain covalently attached by a linker region to a switching region, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising an aptamer domain covalently attached by a linker region to a switching region. In some embodiments, the one or more modifications are in the switchingregion, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications are in the linker region, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications are in the linker region, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications are in the linker region, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications are in the aptamer domain, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications are in the aptamer domain, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. The method of claims 1-5, the polynucleotide aptamer switch is a beacon switch comprising an aptamer domain and a stem region, optionally, the aptamer domain is a loop region. In some embodiments, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte byincreasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, a bound state of the beacon switch to the target analyte occurs upon the hybridization or pairing of the stem region to itself, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, an bound state of the beacon switch to the target analyte occurs upon the hybridization or pairing of the stem region to itself, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, a bound state of the beacon switch to the target analyte occurs upon disruption of the hybridization or pairing of the stem region to itself, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, a bound state of the beacon switch to the target analyte occurs upon disruption of the hybridization or pairing of the stem region to itself, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications comprising incorporation or substitution of one or more modified nucleobase(s) in the polynucleotide aptamer switch comprise incorporation or substitution of one or more nucleobase(s) which differs from adenine, cytosine, guanine, thymine, or uracil. In some embodiments, the one or more modified nucleobase(s) comprise one or more of: Iso-dG-iso deoxyguanosine, 2-aminopyrimidine, 2-MethoxyEthoxy A, 2-MethoxyEthoxy MeC, 2- MethoxyEthoxy G, 2-MethoxyEthoxy T, 2'-O-Methyl RNA Bases (G,C,U,A), Fluoro C, Fluoro U, Fluoro A, Fluoro G, 2-Aminopurine, 5-Bromo dU, deoxyUridine, 2,6- Diaminopurine (2-Amino-dA), Dideoxy -C, deoxyinosine, Hydroxymethyl dC, Inverted dT, Iso-dGm, Iso-dC, Inverted Dideoxy-T, 5-Methyl dC, 5-Nitroindole , 5-hydroxybutynl-2’- deoxyuridine, 8-aza-7-deazaguanosine, deoxyinosine, or combinations thereof. In some embodiments, the polynucleotide aptamer switch is an aptamer beacon switch and the incorporation or substitution of one or more one or more modified nucleobase(s) are in a stemregion of the aptamer beacon switch, or the polynucleotide aptamer switch is intramolecular strand displacement switch and the incorporation or substitution of one or more one or more modified nucleobase(s) are in a switching domain or an aptamer domain of the polynucleotide aptamer switch. In some embodiments, the one or more modifications further comprise increasing or decreasing a number of nucleobases in a region of the polynucleotide aptamer switch where it hybridizes to itself. In some embodiments, the one or more modifications comprising incorporation or substitution of one or more sugar modifications in the polynucleotide aptamer switch comprise incorporation or substitution of one or more different furanose(s) into the polynucleotide aptamer switch. In some embodiments, the different furanose(s) comprises one or more of a: LNA-locked nucleic acid, TNA-threose nucleic acid, CeNA-cyclohexane nucleic acid, FANA-2’-fluoro-aribinonucleic acid, HNA- hexitol nucleic acid, GNA-glycol nucleic acid, L-DNA, or combinations thereof. In some embodiments, the polynucleotide aptamer switch is an aptamer beacon switch and the incorporation or substitution of one or more LNA-locked nucleic acid(s) are in a stem region of the aptamer beacon switch, In some embodiments, the polynucleotide aptamer switch is intramolecular strand displacement switch and the incorporation or substitution of one or more LNA-locked nucleic acid(s) are in a switching domain or an aptamer domain of the polynucleotide aptamer switch. In some embodiments, the one or more modifications comprising incorporation or substitution of one or more modified intemucleoside linkage(s) in the polynucleotide aptamer switch comprise incorporation or substitution of one or more internucleoside linkage(s) different from a phosphate linkage. In some embodiments, the internucleoside linkage different from a phosphate linkage comprises: phosphorothioate (PS), morpholino, PNA-peptide nucleic acids, glycol linkers, PEG (polyethylene glycol)-linkers, and hexa-ethyleneglycol linkers, or combinations thereof. In some embodiments, the one or more modifications comprising incorporation or substitution of one or more modifications are in a displacement region of the polynucleotide aptamer switch and comprise incorporation or substitution of one or more of: a non-natural nucleobase, a non-natural sugar, a non-natural internucleoside linkage, or combinations thereof. In some embodiments, the one or more modifications in the displacement region of the polynucleotide aptamer switch comprise: Hoogsteen and Wobble base-pairing, non-Watson and Crick base pairing, Z / P base pairing, incorporation of 2-aminopurine, substitution of adenine with 2-aminopurine, a duplex stabilizing modification, a duplex de-stabilizing modification, incorporation or substitution of PNA, incorporation or substitution of 5-hydroxybutynl-2’ -deoxyuridine, or combinations thereof. In some embodiments, the one or more modifications comprising incorporation orsubstitution of one or more sugar moiety modification(s) in the polynucleotide aptamer switch comprise: incorporation or substitution of a non-naturally occurring sugar or non- naturally occurring polymer into the polynucleotide aptamer switch, incorporation or substitution of four-carbon threose sugars, incorporation or substitution of peptide nucleic acids (PNAs), or combinations thereof. In some embodiments, the polynucleotide aptamer switch is an aptamer beacon switch, or the polynucleotide aptamer switch is intramolecular strand displacement switch, or the polynucleotide aptamer switch is an anchored displacement strand switch. In some embodiments, the one or more labels comprise a fluorophore and quencher, a redox reporter, or an electrochemical label. In some embodiments, the fluorophore and quencher comprises an acceptor fluorophore and donor fluorophore, contacting the polynucleotide aptamer switch with the target analyte comprises inducing a ratiometric FRET readout. In some embodiments, the polynucleotide aptamer switch is comprised in a device comprising one or more members which are configured to penetrate the skin of a subject, contacting the polynucleotide aptamer switch with the target analyte at least partially occurs by contacting an interstitial fluid of the subject.[5] Aspects disclosed herein provide polynucleotide aptamer switches configured to undergo a conformation change upon binding to the target analyte, the polynucleotide aptamer switch comprises: one or more labels which are configured to produce a change in a signal when the polynucleotide aptamer switch is bound to the target analyte; and one or more modifications which alter a binding affinity between the polynucleotide aptamer switch and the target analyte, the modification comprising at least one of: incorporation or substitution of one or more modified nucleobase(s) in an aptamer domain, a switching domain, a linker region, or a stem region of the polynucleotide aptamer switch; incorporation or substitution of one or more sugar modifications in the polynucleotide aptamer switch; incorporation or substitution of one or more modified internucleoside linkage(s) in the polynucleotide aptamer switch; incorporation or substitution of one or more non-natural nucleobase, a non-natural sugar, and a non-natural intemucleoside linkage in a displacement region of the polynucleotide aptamer switch; incorporation or substitution of one or more sugar moiety modification(s) in the polynucleotide aptamer switch; or any combination thereof. In some embodiments, the one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in a region of the polynucleotide aptamer switch where it hybridizes to itself, thereby shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch. In someembodiments, one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in an aptamer domain of the polynucleotide aptamer switch, thereby shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch. In some embodiments, the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising a linker domain, the one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in the linker domain, thereby shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch. In some embodiments, the polynucleotide aptamer switch comprises an aptamer domain which binds to the target analyte, the one or more modifications are in the aptamer domain and increase the binding affinity between the aptamer domain and the target analyte relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more modifications increase a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte relative to an unmodified polynucleotide aptamer switch. The polynucleotide of claims 46-47, the one or more modifications increase the binding affinity between the aptamer domain and the target analyte by increasing a number of hydrogen bonds between the target analyte and the aptamer domain, increasing a number of salt bridges between the target analyte and the aptamer domain, increasing a number of hydrophobic interaction sites between the target analyte and the aptamer domain, increasing a number of electrostatic interaction sites between the target analyte and the aptamer domain, or combinations thereof, relative to an unmodified polynucleotide aptamer switch. In some embodiments, the polynucleotide aptamer switch comprises an aptamer domain which binds to the target analyte, the one or more modifications are in the aptamer domain and decrease the binding affinity between the aptamer domain and the target analyte relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more modifications decrease a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more modifications decrease the binding affinity between the aptamer domain and the target analyte by decreasing a number of hydrogen bonds between the target analyte and the aptamer domain, decreasing a number of salt bridges between the target analyte and the aptamer domain, decreasing a number of hydrophobic interaction sites between the targetanalyte and the aptamer domain, decreasing a number of electrostatic interaction sites between the target analyte and the aptamer domain, or combinations thereof, relative to an unmodified polynucleotide aptamer switch. In some embodiments, the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising an aptamer domain covalently attached by a linker region to a switching region, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising an aptamer domain covalently attached by a linker region to a switching region. In some embodiments, the one or more modifications are in the switching region, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications are in the linker region, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications are in the linker region, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte In some embodiments, the one or more modifications are in the linker region, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications are in the aptamer domain, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and thetarget analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications are in the aptamer domain, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the polynucleotide aptamer switch is a beacon switch comprising an aptamer domain covalently attached to a stem region, optionally, the aptamer domain is a loop region. In some embodiments, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, a bound state of the beacon switch to the target analyte occurs upon the hybridization or pairing of the stem region to itself, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, a bound state of the beacon switch to the target analyte occurs upon the hybridization or pairing of the stem region to itself, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, a bound state of the beacon switch to the target analyte occurs upon disruption of the hybridization or pairing of the stem region to itself, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In some embodiments, the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, a bound state of the beacon switch to the target analyte occurs upon disruption of the hybridization or pairing of the stem region to itself, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte. In someembodiments, the one or more modifications comprising incorporation or substitution of one or more modified nucleobase(s) in the polynucleotide aptamer switch comprise incorporation or substitution of one or more nucleobase(s) which differs from adenine, cytosine, guanine, thymine, or uracil. In some embodiments, the one or more modified nucleobase(s) comprise one or more of: Iso-dG-iso deoxyguanosine, 2-aminopyrimidine, 2-MethoxyEthoxy A, 2- MethoxyEthoxy MeC, 2-MethoxyEthoxy G, 2-MethoxyEthoxy T, 2'-O-Methyl RNA Bases (G,C,U,A), Fluoro C, Fluoro U, Fluoro A, Fluoro G, 2-Aminopurine, 5-Bromo dU, deoxyUridine, 2, 6-Di aminopurine (2-Amino-dA), Dideoxy-C, deoxyinosine, Hydroxymethyl dC, Inverted dT, Iso-dGm, Iso-dC, Inverted Dideoxy-T, 5-Methyl dC, 5-Nitroindole , 5- hydroxybutynl-2’ -deoxyuridine, 8-aza-7-deazaguanosine, deoxyinosine, or combinations thereof. In some embodiments, the polynucleotide aptamer switch is an aptamer beacon switch and the incorporation or substitution of one or more one or more modified nucleobase(s) are in a stem region of the aptamer beacon switch, or the polynucleotide aptamer switch is intramolecular strand displacement switch and the incorporation or substitution of one or more one or more modified nucleobase(s) are in a switching domain or an aptamer domain of the polynucleotide aptamer switch. In some embodiments, the one or more modifications further comprise increasing or decreasing a number of nucleobases in a region of the polynucleotide aptamer switch where it hybridizes to itself. In some embodiments, the one or more modifications comprising incorporation or substitution of one or more sugar modifications in the polynucleotide aptamer switch comprise incorporation or substitution of one or more different furanose(s) into the polynucleotide aptamer switch. In some embodiments, the different furanose(s) comprises one or more of a: LNA-locked nucleic acid, TNA-threose nucleic acid, CeNA-cyclohexane nucleic acid, F ANA-2’ -fluoro- aribinonucleic acid, HNA-hexitol nucleic acid, GNA-glycol nucleic acid, L-DNA, or combinations thereof. In some embodiments, the polynucleotide aptamer switch is an aptamer beacon switch and the incorporation or substitution of one or more LNA-locked nucleic acid(s) are in a stem region of the aptamer beacon switch, In some embodiments, the polynucleotide aptamer switch is intramolecular strand displacement switch and the incorporation or substitution of one or more LNA-locked nucleic acid(s) are in a switching domain or an aptamer domain of the polynucleotide aptamer switch. In some embodiments, the one or more modifications comprising incorporation or substitution of one or more modified intemucleoside linkage(s) in the polynucleotide aptamer switch comprise incorporation or substitution of one or more internucleoside linkage(s) different from a phosphate linkage. In some embodiments, the intemucleoside linkage different from aphosphate linkage comprises: phosphorothioate, morpholino, PNA-peptide nucleic acids, glycol linkers, PEG (polyethylene glycol)-linkers, and hexa-ethyleneglycol linkers, or combinations thereof. In some embodiments, the one or more modifications comprising incorporation or substitution of one or more modifications are in a displacement region of the polynucleotide aptamer switch and comprise incorporation or substitution of one or more of: a non-natural nucleobase, a non-natural sugar, a non-natural internucleoside linkage, or combinations thereof. In some embodiments, the one or more modifications in the displacement region of the polynucleotide aptamer switch comprise: Hoogsteen and Wobble base-pairing, non-Watson and Crick base pairing, Z / P base pairing, incorporation of 2- aminopurine, substitution of adenine with 2-aminopurine, a duplex stabilizing modifications, a duplex de-stabilizing modification, incorporation or substitution of PNA, incorporation or substitution of 5-hydroxybutynl-2’-deoxyuridine, or combinations thereof. In some embodiments, the one or more modifications comprising incorporation or substitution of one or more sugar moiety modification(s) in the polynucleotide aptamer switch comprise: incorporation or substitution of a non-naturally occurring sugar or non- naturally occurring polymer into the polynucleotide aptamer switch, incorporation or substitution of four-carbon threose sugars, incorporation or substitution of peptide nucleic acids (PNAs), or combinations thereof. In some embodiments, the polynucleotide aptamer switch is an aptamer beacon switch, or the polynucleotide aptamer switch is intramolecular strand displacement, or the polynucleotide aptamer switch is an anchored displacement strand switch. In some embodiments, the one or more labels comprise a fluorophore and quencher, a redox reporter, or an electrochemical label. In some embodiments, the fluorophore and quencher comprises an acceptor fluorophore and donor fluorophore, contacting the polynucleotide aptamer switch with the target analyte comprises inducing a ratiometric FRET readout. In some embodiments, the polynucleotide aptamer switch is comprised in a device comprising one or more members which are configured to penetrate the skin of a subject, contacting the polynucleotide aptamer switch with the target analyte at least partially occurs by contacting an interstitial fluid of the subject.BRIEF DESCRIPTION OF DRAWINGS[6] Figure 1 : Examples of oligonucleotide modifications: The figure provides examples of chemical modifications that can be made to nucleic acids. These range from simple site-specific atomic substitutions to more exotic molecular replacements.Modifications can be made to the nucleobases (green shaded areas), sugar ring component(red shaded areas), and other backbone modifications such as the phosphate group and phosphodiester linkage (examples shaded in blue).[7] Figure 2: Examples of polynucleotide aptamer switches: This figure demonstrates two different types of aptamer switch polynucleotides. These aptamer switch polynucleotides can be configured as optically responsive switches by attaching a fluorophore and quencher, or as electrochemically active switches by attaching redox reporter-labeled aptamer switches on an electrode surface. FIG. 2 at a) shows the intramolecular strand displacement (ISD) switch having an aptamer region which is attached covalently attached to a switching domain (also known as a ‘displacement strand’) through a linker region. When unbound to the target molecule, the switching domain is hybridized to the aptamer region. When bound to the target the aptamer switch undergoes a conformational change in structure resulting in the switching domain is displaced from the aptamer region. The target molecule is denoted by the red circle. FIG. 2 at b) shows the aptamer beacon switch having a stem region and a binding region (also known as loop region, or an aptamer domain). In this example, when unbound to the target the aptamer beacon adopts a structure where the stem is stabilized and both ends of the aptamer switch (i.e., 3-prime and 5-prime ends) are brought into close proximity. When bound to the target, the stem loop region is destabilized and the stem region is not bound, which results in the two ends of the aptamer increasing in distance. The target molecule is denoted by the red circle. Although in this example the aptamer beacon forms the stem region in the unbound state, aptamer beacons can also be configured to form the stem region in the bound state. FIG 2 at c) shows an anchored displacement strand (ADS) aptamer switch, having an aptamer region which is attached to a switching domain (also termed ‘displacement strand’) via hybridization between an anchor region appended to the aptamer region and an anchor complement region contained within a second switching strand that contains the switching domain and a linker. When unbound to the target molecule, the switching domain is hybridized to the aptamer region. When bound to the target the aptamer switch undergoes a conformational change in structure resulting in the switching domain being displaced from the aptamer region. The target molecule is denoted by the red circle.[8] Figure 3: Decreasing the apparent dissociation constant of an cortisol -binding anchored displacement strand (ADS) aptamer switch with Phosphorothioate (PS) modifications: This figure demonstrates tuning the thermodynamics of a cortisol-binding anchored displacement strand aptamer switch by introducing intemucleoside phosphorothioate modifications into the portion of the switching strand that competes with aptamer-target binding. Panel (a) illustrates different ADS aptamer switch designs that employ an existingcortisol -binding aptamer paired with switching strands that contain various 15nt partially complementary switching domains. The switching domains maintain an identical nucleotide sequence with varied numbers of PS intemucleoside linkages (0, 4, 8, and 15 with the remainder being natural phosphodiester linkages), such that increased numbers of PS linkages provide greater destabilization and larger decreases in dissociation constant. Panel (b) shows the fluorescent cortisol binding responses of these varying aptamer switch designs, illustrating the decreases in dissociation constant imparted by the addition of PS linkages. As the number of PS bases in the switching strand increases, hybridization of the switching domain is destabilized and the apparent dissociation constant of the aptamer switch decreases from KD > 300 pM for the natural DNA strand to 153 pM, 51 pM, and 4.6 pM for the strands containing 4, 8, and 15 PS modifications respectively. Data shown represents the mean and standard deviation of n=4 independent replicate measurements.[9] Figure 4: Decreasing and Increasing the background signal and binding behavior of a glucose-binding ISD aptamer switch with Phosphorothioate (PS) Modifications: This figure demonstrates tuning the thermodynamics of a glucose-binding ISD aptamer switch by introducing internucleoside phosphorothioate modifications into the DS or a flanking region of the aptamer sequence in order to stabilize or destabilize DS-aptamer hybridization and thus decrease or increase target-free background signal and aptamer binding affinity. Panel (a) illustrates different ISD aptamer switch designs that employ an existing glucose-binding aptamer covalently linked to 9nt DS domains through a 20nt poly-T linker domain. The DS domains contain identical nucleobase sequences, but are augmented to contain either the natural sequence, 3 flanking PS-modified dT bases on the 5’ and 3’ end of the stem formed by aptamer-DS hybridization, or the DS sequence containing 6 PS modified linkages. Panel (b) shows the normalized fluorescent glucose binding responses of these varying aptamer switch designs, illustrating the modulation of background and concentration sensitivity range by the addition of PS modifications. As flanking PS modified dT bases are added, DS hybridization is stabilized by base stacking leading to lower background and shifted switch sensitivity to higher target concentrations. As PS modifications are added directly within the DS, DS hybridization is destabilized, leading to higher background and shifted switch sensitivity to lower target concentrations. Data shown represents the mean and standard deviation of n=4 independent replicate measurements.

[0010] Figure 5: Increasing the apparent dissociation constant of a glucose-binding ISD aptamer switch with Locked Nucleic Acids (LNA): This figure demonstrates tuning the thermodynamics of a glucose-binding ISD aptamer switch by LNA bases into the DS theaptamer sequence in order to stabilize DS-aptamer hybridization and thus increase the dissociation constant of the ISD aptamer switch. This figure also demonstrates the ability to simultaneously introduce offsetting PS and LNA modifications to achieve more highly modified DS sequences with smaller impacts on the dissociation constant of the aptamer switch than would be observed if either modification was introduced alone. Panel (a) illustrates different ISD aptamer switch designs that employ an existing glucose-binding aptamer covalently linked to 9nt DS domains through a 20nt poly-T linker domain. The DS domains contain identical nucleobase sequences, but are augmented to contain either the natural sequence, 2 LNA bases, or 3 LNA bases and 6 PS intemucleoside linkages. Panel (b) shows the normalized fluorescent glucose binding responses of these varying aptamer switch designs, illustrating the modulation of the ISD dissociation constant by introducing LNA bases with and without PS modifications. As LNA bases are added, DS hybridization is stabilized due to stronger LNA base pairing, leading to a shift in dissociation constant from KD = 19 mM for the natural DNA sequence (AS 38) to KD = 760 mM for the LNA-modified DNA sequence (AS 6). LNA bases added alongside PS modifications introduce offsetting effects, such that adding 3 LNA bases and 6 PS modifications alters the dissociation by a smaller amount than 2 LNA bases alone (KD = 143 mM for AS 69). Data shown represents the mean and standard deviation of n=4 independent replicate measurements.

[0011] Figure 6: Modulating oligonucleotide switch affinity with non-natural linkers: This figure demonstrates utilizing a non-natural linker in order to alter the thermodynamics of an aptamer switch towards a complementary oligonucleotide target molecule. Panel (a) illustrates how switch constructs that respond with fluorescent signaling to a target short oligonucleotide are designed to containing linkers of various lengths of natural poly thymidine residues (T5, T10, T15, T20, and T25) and non-natural hexa-ethyleneglycol spacers (Spl8, 3xSpl8, 5xSpl8). Panel (b) shows the normalized fluorescence target-binding responses of these switch constructs obtained from fluorescent plate reader experiments for n=4 independent replicate measurements. Aptamer switches containing hexa-ethyleneglycol-based linkers exhibit stabilized DS hybridization and higher dissociation constants as compared to aptamers with DNA linkers of similar length. For example, the design with a single hexa-ethyleneglycol linker (Spl8, length ~30 angstroms) has a dissociation constant of KD = 647 nM, while the equivalent DNA linker (T10, length ~34 angstroms) has KD = 470 nM. The design with a three hexaethyleneglycol linker (3xSpl8, length >60 angstroms) has a dissociation constant of KD = 412 nM, while the equivalent DNA linker (T20, length ~68 angstroms) has KD = 226 nM. The design with a five hexa-ethyleneglycol linker (5xSpl8, length >90 angstroms) has adissociation constant of KD = 369 nM, while the closest equivalent DNA linker (T25, length ~85 angstroms) has KD = 138 nM. These results illustrate that substituting non-natural polymer linkers offer a method to modulate the dissociation constant of an aptamer switch (see Figure 6b and example sequences).DETAILED DESCRIPTION

[0012] Molecular switches that undergo conformational changes upon binding to a target analyte are useful for a wide range of applications. Such applications include (but are not limited to) point-of-care diagnostics, real-time molecular monitoring, drug delivery, forensics, medical imaging, and monitoring of chemical processes. In particular, aptamers have proven to be useful as molecular switches due to their ability to bind target analytes with great affinity and specificity, high thermal stability, facile chemical synthesis, and the ability to be readily modified (e g., with small molecule fluorophores, quenchers, conjugation handles, etc ). Aptamer switch polynucleotides can be used with a variety of detection schemes such as optical, electrochemical, colorimetric, and sequencing readouts. As used herein, the term “aptamer switch polynucleotide” refers to a polynucleotide that changes conformation upon binding to a target analyte (from a non-reporting state to a reporting state).

[0013] For aptamer switch polynucleotides to be optimized for a given application, it is important that the kinetics and thermodynamics of the molecular switch are appropriately tuned. For example, it is often desirable that the apparent dissociation constant of a molecular switch is matched to the expected concentration of the analyte within the sample that will be measured. If the apparent dissociation constant of a molecular switch is much lower or higher than the sample concentration, the molecular switch will not be sensitive to changes in analyte concentration. Unfortunately, there are limited methods available to tune the properties of oligonucleotide-based molecular switches and efforts to tune molecular switches are often time-consuming and rely on trial-and-error. Conventional means of tuning aptamer switches offer coarse-grain control and primarily rely on altering the sequence of the natural oligonucleotide structure and either stabilizing or destabilizing regions of the aptamer switch by altering the length of duplexed regions or introducing mismatches. Therefore, there is a need for improved methods and techniques for tuning and / or modulating the properties of oligonucleotide-based molecular switches.

[0014] Two key properties of aptamer switch polynucleotides are the binding kinetics (i.e., binding on-rates and binding off-rates) and apparent binding dissociation constants (i.e.,affi nities) that are observed when challenged against the analyte of interest. These properties can be controlled by tuning the energetic states and structures of the reporting state and / or the non-reporting state of the aptamer switch. As an example, we can consider an intramolecular strand displacement (ISD) aptamer switch which consists of an aptamer (i.e. the aptamer domain) attached to a displacement strand (also known as a switching domain (SD)) through an oligonucleotide linker. The displacement strand is at least partially complementary to a portion of the aptamer and binds to the aptamer in the non-reporting state. When the target analyte is added at sufficient concentrations, the aptamer binds to the analyte, and displaces the displacement strand, thereby achieving the reporting state.

[0015] By adding optical labels (such as small molecule fluorophores or quenchers) to either end of the ISD aptamer switch, a change in optical signal is obtained when the ISD aptamer switch binds to the target analyte molecule, since the binding induced conformational change of the aptamer switch changes the distance between the two optical labels. The change in optical signal can comprise an increase in fluorescence emission by changing the distance between the two optical labels, or a decrease in fluorescence emission by changing the distance between the two optical labels. There are several mechanisms by which the distance of the optical labels results in a change in fluorescence. For example, in Fluorescence Resonance Energy Transfer (FRET) the fluorescence signals of the donor and acceptor molecules both change as a function of the distance between the reporters. There are also several mechanisms by which the fluorescence of a reporter molecule can be quenched (i.e., reduced in intensity) such as by FRET, dexter electron transfer, static quenching, and collisional quenching. By changing the composition of the displacement strand (e.g., sequence) the strength of the interaction between the aptamer and displacement strand can be altered which can be used to tune both the kinetic and thermodynamic properties of the molecular switch. Additionally, the length of the oligonucleotide linker can also be used to control the effective concentration of the displacement strand which can also be used to control the kinetic and thermodynamic properties of the molecular switch.

[0016] Although in the case of an ISD switch the switching domain is covalently attached through an oligonucleotide linker to the aptamer domain, the aptamer domain and switching domain can also be brought into proximity through a number of alternative means. These switches can broadly be considered strand displacement polynucleotide switches. For example, in an anchored displacement strand (ADS), a duplex is formed via Watson-Crick base pairing between two complementary oligonucleotide anchor regions which results in the aptamer domain (which is conjugated to one anchor strand) being attached to the linker andswitching domains (which are conjugated to the other complementary anchor strand) through the stable oligonucleotide duplexed ‘anchor’ region. The aptamer domain and switching domains can also be brought into proximity by being conjugated nearby to each other on a surface, or through other macromolecules, such as peptides or proteins.

[0017] Like ISD aptamer switches, the kinetic and thermodynamic properties of aptamer beacon switches can also be controlled by altering the length and / or composition of aptamer beacon hairpin. In aptamer beacon switches there exists a hairpin structure which is either formed or disrupted when the aptamer region binds to the target molecule of interest. The formation of disruption of the hairpin can be leveraged for either optical readouts (such as fluorescence intensity with a fluorophore and quencher), or electrochemical readouts by labeling the aptamer beacon switch with a redox reporter (e.g., methylene blue). By changing the length of the hairpin to increase or decrease the duplex stability, the sensitivity and timeresolution of the aptamer beacon can be altered.

[0018] A number of chemical modifications to oligonucleotides can also be used to alter the kinetic and thermodynamic properties of aptamer switches. In some cases, the chemical modifications are simply site-specific atomic substitutions, while in other cases more exotic molecular replacements are used which bear little resemblance to the natural structures. It is well known that such modifications can impact the local conformations and chemical reactivity of the oligonucleotides, thus altering the structures and dynamics of the nucleic acids. Therefore, the strength (i.e., Gibbs free energies) and kinetics (e.g., on-rates and off- rates) of the base-pairing interactions of the chemical modifications is altered relative to natural DNA or RNA oligonucleotides. These chemical modifications can cause an increase or decrease DNA and / or RNA duplex stability when substituted for natural bases. These changes in duplex stability are often measure as changes in melting temperatures (i.e., Tm), which is the temperature at which 50% of the oligonucleotide is duplexed with its complement and 50% is free in solution. As the stability of a duplexed oligonucleotide increases, so does its melting temperature.

[0019] This disclosure describes utilizing non-natural materials (e.g. non-natural bases, non-natural sugar backbones, non-natural linkers) to control or alter the kinetics, thermodynamics, structure, and / or conformational states of oligonucleotide-based aptamer switch polynucleotide. The use of non-natural materials, rather than conventional nucleotide bases, allows greater control and precision over the kinetics and thermodynamics (i.e. effective binding affinity) between the aptamer switch’s reporting and non-reporting states. This fine-tuned control enables the creation of highly optimized aptamer switches for a variety of applications.

[0020] Tuning molecular switches via non-natural modifications

[0021] As described above, there is a substantial need for improved methods for tuning and / or modulating the properties of oligonucleotide-based molecular switches, in terms of binding affinity and binding kinetics. There is a substantial need for methods to achieve more fine-grained control, and coarse grained control over a broader range than can be achieved through conventional means of tuning switches by adding or removing additional natural DNA bases or introducing mismatches. Further, these conventional methods have a large degree of constraints on how tuning can be achieved which may lead to limitations, for example to decrease switch affinity there is a need to strengthen switching domain hybridization and thus increase the length of the switching domain which changes the underlying length of the DNA construct which may cause issues where a specific length is required for an application. This may be an important factor in applications where the aptamer binding pocket is small, and thus the region of bases that afford effective competition between target and switching domain binding is limited so sequences cannot be extended arbitrarily. Incorporation of mismatches also introduces risks of increased off-target hybridization of the switching domain to other regions within the aptamer switch as the complementarity within the desired displacement region is reduced. With regard to these limitations, the ability to modulate aptamer switch binding properties via the introduction of non-natural modifications enables the tuning of properties such as affinity without the need to modify the sequence identity of the switching domain. Non-natural modifications can instead be used to weaken or strengthen the interactions between the switching domain and aptamer based on small or large changes in hybridization strength upon introduction of the modification. This allows finer-grained variation of switch affinities than the introduction of mismatches or removal of bases, as base-pairing can be maintained but with a slightly reduced free-energy from a modified base. This also allows tuning over a much broader range without the need to make substantial changes to switching domain lengths as high degrees of modification can impart large shifts in free energy within the same length of hybridization region. Finally, the use of non-natural modifications fortuning aptamer switches may also confer advantages for in vivo use or application in complex biofluids where nuclease degradation of natural DNA may occur. By introducing non-natural modifications the resulting aptamer switches may exhibit substantially reduced nuclease susceptibility, thus enabling measurements to be made with longer operational lifetimes orlonger duration of stability as less nuclease degradation will occur for a highly-modified oligonucleotide.

[0022] Aptamer switch polynucleotides undergo a conformational change in state between a binding-competent or “bound state” and an “unbound state”. The thermodynamic properties (e.g., apparent dissociation constant) and kinetic properties of the aptamer switch are dependent on the free energies and activation energies of the various bound, unbound, and transition states of the aptamer switch. By introducing chemical modifications that alter the energetics of one or more states of the aptamer switch (e.g., stabilizing either the bound or unbound aptamer switch state), the thermodynamic and / or kinetic properties of the aptamer switch can be altered relative to an aptamer switch containing only natural nucleotides. In some cases, this can be done by introducing chemical modifications that stabilize or destabilize oligonucleotide duplex structures.

[0023] For example, in the case of aptamer beacons where a hairpin region is formed upon analyte binding, the addition of modifications that stabilize (i.e., increase the melting temperature) of the hairpin region will promote the bound state of the aptamer switch and decrease the apparent dissociation constant of the aptamer switch (i.e., increase affinity). Instead, if the modifications are introduced to the hairpin region that destabilize (i.e., decrease the melting temperature) of the hairpin region will promote the unbound state of the aptamer switch and increase the apparent dissociation constant of the aptamer switch (i.e., decrease affinity).

[0024] In the case of an aptamer beacon switch where the hairpin is disrupted upon binding, the addition of modifications that stabilize (i.e., increase the melting temperature) of the hairpin region will promote the unbound state of the aptamer switch and increase the apparent dissociation constant of the aptamer switch (i.e., decrease affinity). Instead, if the modifications are introduced to the hairpin region that destabilize (i.e., decrease the melting temperature) of the hairpin region will promote the bound state of the aptamer switch and decrease the apparent dissociation constant of the aptamer switch (i.e., increase affinity).

[0025] In the case of aptamer switch beacons, modifications can also be incorporated into other regions of the aptamer that either stabilize or destabilize the formation of the aptamer beacon hairpin structure.

[0026] In the case of aptamer switches that rely on competitive binding of a “switching domain” to a region of the aptamer domain (e.g., ISDs, anchored displacement strands, bubble switches), the modifications can be incorporated into the switching domain, aptamer domain, or linker regions of the aptamer switch. A modification in the switching domain that stabilizesthe interaction between the switching domain with a region of the aptamer domain promotes the unbound state (where the switching domain is bound to the aptamer region), and results in an increased dissociation constant of the switch (i.e., decreased affinity). Likewise, a modification in the switching domain that destabilizes the interaction between the switching domain with a region of the aptamer domain promotes the bound state (where the switching domain is not bound to the aptamer region), and results in a decreased dissociation constant of the switch (i.e., increased affinity). Modifications can also be made within the linker region that stabilize or destabilize the interaction between the switching domain and the aptamer regions. When such a modification stabilizes the binding of the switching domain to the aptamer region, the unbound state is promoted which results in an increased apparent dissociation constant and a decreased affinity. When such a modification destabilizes the binding of the switching domain to the aptamer region, the bound state is promoted which results in a decreased apparent dissociation constant and an increased affinity. Finally, modifications can also be incorporated directly in the aptamer region of the switch. As in the other cases, when a modification stabilizes the state where the switching domain is bound to the aptamer region, the unbound state is stabilized, and the dissociation constant is increased which results in a decrease in apparent affinity of the switch. When a modification in included in the aptamer region that destabilizes the state where the switching domain is bound to the aptamer region, the bound state is promoted, and the dissociation constant is decreased which results in an increase in the apparent affinity of the switch.

[0027] Tuning with non-natural modifications can be applied to develop aptamer switches from parent aptamers with a broad range of properties including affinities. Tuning can be applied adapt parent aptamers with affinities ranging from 1 pM to 100 mM into aptamer switches. These switches can be tuned using non-natural modifications to achieve dissociation constants that vary between equal to the dissociation constant of the parent aptamer up to 10,000-fold higher than the dissociation constant of the parent aptamer. In some embodiments, the dissociation constant of the complex increases by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1,000%, at least about 5,000%, at least about 10,000%, at least about 50,000%, at least about 100,000%, or at least about 500,000% compared to a complex comprising an aptamer switch that does notcomprise the one or more non-naturally occurring modifications to the displacement region. In some embodiments, the dissociation constant of the complex increases by from about 5% to about 10%, from about 5% to about 15%, from about 5% to about 20%, from about 5% to about 25%, from about 5% to about 30%, from about 5% to about 35%, from about 5% to about 40%, from about 5% to about 45%, from about 5% to about 50%, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 25%, from about 10% to about 30%, from about 10% to about 35%, from about 10% to about 40%, from about 10% to about 45%, from about 10% to about 50%, from about 15% to about 20%, from about 15% to about 25%, from about 15% to about 30%, from about 15% to about 35%, from about 15% to about 40%, from about 15% to about 45%, from about 15% to about 50%, from about 20% to about 25%, from about 20% to about 30%, from about 20% to about 35%, from about 20% to about 40%, from about 20% to about 45%, from about 20% to about 50%, from about 30% to about 40%, from about 30% to about 50%, or from about 40% to about 50% compared to a complex comprising an aptamer switch that does not comprise the one or more non-naturally occurring modifications to the displacement region. In some embodiments, the dissociation constant of the complex decreases by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 100%, at least about 1,000%, at least about 5,000%, at least about 10,000%, at least about 50,000%, at least about 100,000%, or at least about 500,000% compared to a complex comprising an aptamer switch that does not comprise the one or more non-naturally occurring modifications to the displacement region. In some embodiments, the dissociation constant of the complex decreases by from about 5% to about 10%, from about 5% to about 15%, from about 5% to about 20%, from about 5% to about 25%, from about 5% to about 30%, from about 5% to about 35%, from about 5% to about 40%, from about 5% to about 45%, from about 5% to about 50%, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 25%, from about 10% to about 30%, from about 10% to about 35%, from about 10% to about 40%, from about 10% to about 45%, from about 10% to about 50%, from about 15% to about 20%, from about 15% to about 25%, from about 15% to about 30%, from about 15% to about 35%, from about 15% to about 40%, from about 15% to about 45%, from about 15% to about 50%, from about 20% to about 25%, from about 20% to about 30%, from about 20% to about 35%, from about 20% to about 40%, from about 20% toabout 45%, from about 20% to about 50%, from about 30% to about 40%, from about 30% to about 50%, or from about 40% to about 50% compared to a complex comprising an aptamer switch that does not comprise the one or more non-naturally occurring modifications to the displacement region.

[0028] In some embodiments, the dissociation constant of the complex increases by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% compared to a complex comprising an aptamer switch that does not comprise the one or more non-naturally occurring modifications to the sequence that is at least partially complementary to the analyte . In some embodiments, the dissociation constant of the complex increases by from about 5% to about 10%, from about 5% to about 15%, from about 5% to about 20%, from about 5% to about 25%, from about 5% to about 30%, from about 5% to about 35%, from about 5% to about 40%, from about 5% to about 45%, from about 5% to about 50%, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 25%, from about 10% to about 30%, from about 10% to about 35%, from about 10% to about 40%, from about 10% to about 45%, from about 10% to about 50%, from about 15% to about 20%, from about 15% to about 25%, from about 15% to about 30%, from about 15% to about 35%, from about 15% to about 40%, from about 15% to about 45%, from about 15% to about 50%, from about 20% to about 25%, from about 20% to about 30%, from about 20% to about 35%, from about 20% to about 40%, from about 20% to about 45%, from about 20% to about 50%, from about 30% to about 40%, from about 30% to about 50%, or from about 40% to about 50% compared to a complex comprising an aptamer switch that does not comprise the one or more non-naturally occurring modifications to the sequence that is at least partially complementary to the analyte . In some embodiments, the dissociation constant of the complex decreases by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% compared to a complex comprising an aptamer switch that does not comprise the one or more non-naturally occurring modifications to the sequence that is at least partially complementary to the analyte . In some embodiments, the dissociation constant of the complex decreases byfrom about 5% to about 10%, from about 5% to about 15%, from about 5% to about 20%, from about 5% to about 25%, from about 5% to about 30%, from about 5% to about 35%, from about 5% to about 40%, from about 5% to about 45%, from about 5% to about 50%, from about 10% to about 15%, from about 10% to about 20%, from about 10% to about 25%, from about 10% to about 30%, from about 10% to about 35%, from about 10% to about 40%, from about 10% to about 45%, from about 10% to about 50%, from about 15% to about 20%, from about 15% to about 25%, from about 15% to about 30%, from about 15% to about 35%, from about 15% to about 40%, from about 15% to about 45%, from about 15% to about 50%, from about 20% to about 25%, from about 20% to about 30%, from about 20% to about 35%, from about 20% to about 40%, from about 20% to about 45%, from about 20% to about 50%, from about 30% to about 40%, from about 30% to about 50%, or from about 40% to about 50% compared to a complex comprising an aptamer switch that does not comprise the one or more non-naturally occurring modifications to the sequence that is at least partially complementary to the analyte .

[0029] Types of non-naturally occurring modifications

[0030] The displacement region can comprise one or more non-naturally occurring modifications. In some embodiments, the one or more non-naturally occurring modifications occur on at least 1 nucleotide base, at least 2 nucleotide bases, at least 3 nucleotide bases, at least 4 nucleotide bases, at least 5 nucleotide bases, at least 6 nucleotide bases, at least 7 nucleotide bases, at least 8 nucleotide bases, at least 9 nucleotide bases, at least 10 nucleotide bases, at least 11 nucleotide bases, at least 12 nucleotide bases, at least 13 nucleotide bases, at least 14 nucleotide bases, or at least 15 nucleotide bases. In some embodiments, the one or more non-naturally occurring modifications decrease at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 hydrogen bonds between the target analyte and the aptamer domain relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more non-naturally occurring modifications decrease at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 salt bridges between the target analyte and the aptamer domain relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more non-naturally occurring modifications decrease at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 hydrophobic interaction sites between the target analyte and the aptamer domain relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more non-naturally occurring modifications increase at least 1, at least2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 electrostatic interaction sites between the target analyte and the aptamer domain relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more non-naturally occurring modifications increase at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 hydrogen bonds between the target analyte and the aptamer domain relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more non-naturally occurring modifications increase at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 salt bridges between the target analyte and the aptamer domain relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more non-naturally occurring modifications increase at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 hydrophobic interaction sites between the target analyte and the aptamer domain relative to an unmodified polynucleotide aptamer switch. In some embodiments, the one or more non-naturally occurring modifications increase at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 electrostatic interaction sites between the target analyte and the aptamer domain relative to an unmodified polynucleotide aptamer switch.

[0031] Sugar Modification

[0032] A sugar modified nucleotide is structurally different from the canonical furanose structure of DNA and RNA.

[0033] Examples of sugar modifications, but are not limited to: LNA-locked nucleic acid, TNA-threose nucleic acid, CeNA-cyclohexane nucleic acid, FANA-2’-fluoro-aribinonucleic acid, HNA-hexitol nucleic acid, GNA-glycol nucleic acid, L-DNA. LNA, FANA, and HNA all increase DNA and RNA duplex stability. CeNA and GNA both stabilize RNA duplexes, but de stabilize DNA duplexes. TNA can both stabilize or destabilize DNA duplexes depending on the purine content (low TNA purine content is less stable). L-DNA cannot bind to R-DNA or R-RNA, but can bind to PNA.

[0034] Nucleobase Modifications

[0035] Nucleobase modifications differs from the canonical pyrimidine and purine bases (adenine, cytosine, guanine, thymine, uracil).

[0036] Examples of nucleobase modifications include, but are not limited to: Iso-dG-iso deoxy guanosine, 2-aminopyrimidine, 2-MethoxyEthoxy A, 2 -Methoxy Ethoxy MeC, 2- MethoxyEthoxy G, 2-MethoxyEthoxy T, 2'-O~Methyl RNA Bases (G,C,U,A), Fluoro C, Fluoro U, Fluoro A, Fluoro G, 2-Aminopurine, deoxyUridine, 2,6-Diaminopurine (2-Amino- dA), Dideoxy-C, deoxy Inosine, Hydroxymethyl dC, Inverted dT, Iso-dGm, Iso-dC, Inverted Dideoxy -T, 5-Methyl dC, 5-Nitroindole, 5-hydroxybutynl -2’ -deoxyuridine, carboxy -modified nucleotides, and 8-aza-7-deazaguanosine. Modifications such as 2-aminopurines, 2- aminopyrimidines, 2-Methyoxy A, 2-MethoxyEthoxy MeC, 2-MethoxyEthoxy G, 2- MethoxyEthoxy T, 2 -O-Methyl RNA Bases (G,C,U,A), Fluoro C, Fluoro IJ, Fluoro A, Fluoro G, 2,6-Diaminopurine (2-Amino-dA), 5-Methyl dC, 5-carboyx C, and 5-hydroxybutynl-2’- deoxyuridine are known to stabilize oligonucleotide duplexes relative to their natural counterpart. Modifications such as 5-nitroindole, deoxyinosine, and other universal or sterically hindered modifications are known to destabalize oligonucleotide duplexes relative to a typical Watson-Crick base-pair interaction with a natural oligonucleotide.

[0037] In some embodiments, nucleobase modifications may include C8-alkyne-uracil, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifiuoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- deazaadenine and 3 -deazaguanine and 3-deazaadenine. Examples of non-natural nucleotides include, but are not limited to, 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, 0-6 substituted purines, 2-aminopropyladenine, 5- propynyluracil, 5-propynylcytosine, 5-methylcytosine, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5- propynyl cytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5 -trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyl adenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7- deazaadenine, 3 -deazaguanine, 3 -deazaadenine, tricyclic pyrimidines, phenoxazine cytidine([5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4- b][l,4]benzothiazin-2(3H)-one), phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H- pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2- one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5 -bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5- fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5 -fluorouracil, 5- iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl- uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5- hydroxycytosine, 2'-deoxyuridine, and 2-amino-2'-deoxyadenosine.

[0038] Internucleoside linkages modifications

[0039] A non-natural internucleoside linkages differs structurally from the canonical phosphate linkage in oligonucleotides.

[0040] Examples of nucleobase modifications include, but are not limited to: phosphorothioate, morpholino, PNA-peptide nucleic acids, glycol linkers, PEG (polyethylene glycol)-linkers, and hexa-ethyleneglycol linkers. PNA is known to increase the strength of duplexed DNA and RNA, while phosphorothioate is known to destabilize DNA duplexes.

[0041] Summary of modification effects on duplex stability

[0042] Summary of Effect of Modification Location on kd

[0043] Controlling kinetics and thermodynamics of aptamer switches with nonnatural nucleotides in the displacement region. One aspect of this disclosure is the use ofnon-natural nucleotides (which can include one or more of a non-natural nucleobase, a nonnatural sugar, and a non-natural internucleoside linkage) within the competitor region to alter the kinetics and thermodynamics of an aptamer switch polynucleotide. The thermodynamics of DNA hybridization can be modulated by replacing canonical Watson-Crick base pairing with alternative binding modes. Naturally occurring non-Watson Crick base pairing modes include Hoogsteen and Wobble base-pairing which can affect the strength of DNA duplexes by altering native hydrogen bonding patterns, and have influenced the development of artificially expanded genetic information systems (AGEIS). This expanded genetic alphabet introduced novel modified nucleobases, such as Z:P, which alter the hydrogen bonding patterns compared to standard pyrimidine (Py) and purine (Pu) bases. The Z base provides a donor, donor, acceptor pattern (PyDDA) with P (PuAAD), whereas naturally occurring C is characterized as Py DAA with G (PuADD), resulting in a base pair that is more stable than a normal G:C pair. Additionally, mismatches between Z / P and canonical bases are more stable than normal mismatched bases, establishing new modes of interaction and thus, changing the free energies of oligonucleotide sequences that incorporate them. These concepts can be applied to control the thermodynamics and kinetics of aptamer switches, where the number and identity of mismatches results in a modulation of the apparent affinity of the aptamer switch. The use of non-natural nucleotides provides a greater number of potential interactions between interacting domains within the aptamer switch polynucleotide, enabling more fine tune control of the free energies and thus the stability and kinetics of polynucleotide duplexes.

[0044] In some embodiments, the binding affinity of the aptamer switch can be increased or decreased by the addition of one or more non-natural nucleotides within the switching domain. In some embodiments, the affinity of the aptamer switch polynucleotide can be increased by replacing a natural nucleobase with a modified nucleobase to increase the affinity of the aptamer switch polynucleotide. For example, 2-aminopurine can substitute adenine base within the displacement strand of an intramolecular strand displacement aptamer to destabilize the duplex and slightly lower the melting temperature, resulting in weaker hybridization and shifting the equilibrium to the binding-competent state. Alternatively, duplex-stabilizing modifications like PNA could be used within the displacement strand in order decrease the affinity of the aptamer polynucleotide switch by increasing the stability of the displacement strand to a complementary region within the aptamer sequence, which will shift the equilibrium to the unbound state.

[0045] In some embodiments, the kinetics of the aptamer switch can be increased or decreased by the addition of one or more non-natural nucleotides within the switching domain. For example, 5-hydroxybutynl-2’-deoxyuridine is a modified base that increases the melting temperature of oligonucleotides and stabilizes oligonucleotide duplexes. If one or more modifications are added in the switching domain or the stem domain, this will shift the equilibrium to the unbound state of the aptamer switch and decrease the temporal resolution in adopting the binding competent state. Other modification either shifting the equilibrium to the unbound or bound state of the aptamer switch and decreasing or increasing the temporal resolution in adopting the binding competent state are possible, and are further summarized in Table 1 below.

[0046] Controlling kinetics and thermodynamics of aptamer switches with nonnatural linkers. Some common sugar moiety modifications include altering the 2’ position with fluoro (-F), amino (-NH2), azido (-N3), or methoxy / OMe (-0CH3) groups. It is known that these types of modifications can affect the hydrogen bonding and resulting sugar pucker conformations of polynucleotides composed of these modifications. The catalog of sugar modifications has expanded greatly in recent years with the field of xeno nucleic acids or XNA. Homo- and heteroduplexes comprised of XNA result in diverse structures and helical geometries which results in altered plasticity of the resulting polynucleotides. This variance in polynucleotide rigidity can be employed to alter the linker region of the aptamer switch to modulate affinity temporal resolution of the binding event. The incorporation of varying numbers of modified nucleotides can result in novel geometries that can provide more granular changes to the effective local concentration of the displacement strand, and thus, the thermodynamic and kinetics of the aptamer switch polynucleotide.

[0047] In some embodiments, the thermodynamics of the aptamer switch can be modulated by replacing one or more of the nucleotides in the linker with a non-natural sugar modification or polymer. For example, the linker region may be substituted with an analogous sequence of unnatural four-carbon threose sugars. The absence of one carbon per nucleotide linkage in a TNA strand results in increased rigidity, resulting in shorter lengths of these non-natural polynucleotides compared to their natural counterparts. The decreased length of the modified linker region will result in an increase in the effective concentration of the displacement strand and shift the equilibrium to the unfolded state of the aptamer, which in turn will increase in the apparent KD of the switch.

[0048] In some embodiments, the kinetics of aptamer switch binding can be modulated by replacing a canonical base with a non-natural nucleotides within the linker region. In someembodiments, the temporal resolution may be increased by changing the polynucleotide monomers and linkage. For example, the composition of the linker can be replaced with an analogous sequence of PNA The repeating peptide units of PNA are known to adopt unique P-type helical geometries that are wound more loosely than canonical DNA duplexes. A linker composed of this unique geometry will increase the length and shift the equilibrium to the unfolded state, resulting in faster kinetics for the bound state in the presence of the target. Examples:

[0049] Example 1 : Decreasing the apparent dissociation constant of an cortisol-binding anchored displacement strand (ADS) aptamer switch with Phosphorothioate (PS) modifications.

[0050] An ADS aptamer switch that produces a change in fluorescent signal upon binding to cortisol is designed by hybridizing an aptamer sequence to a partially complementary switching strand through a conserved anchor sequence. A 15nt partially complementary switching domain within the switching strand hybridizes to the aptamer in the target-free state, and is displaced upon target binding, leading to fluorescent signaling of target binding. A switching domain is designed to contain only natural DNA bases and natural intemucleoside linkages. Further switching domains are designed to maintain an identical nucleotide sequence while introducing non-natural phosphorothioate (PS) intemucleoside linkages, which decrease the stability of switching domain hybridization to the aptamer, thus decreasing the dissociation constant of the aptamer switch (KD). Such switching domains are designed to incorporate varying numbers of PS linkages, including 4, 8, and 15, such that increased numbers of PS linkages provide greater destabilization and larger decreases in dissociation constant (see Figure 3a and example sequences).

[0051] The fluorescent corti sol -binding responses of aptamer switches employing these switching domains are experimentally characterized by: labeling the aptamer strand with an ATTO 643 fluorophore at its 5’ end and the various switching strands with Iowa Black FQ quenchers at their 3’ ends; hybridizing the aptamer and switching strands through temperature annealing at a concentration of 1 pM switching strand and 0.66 pM aptamer strand in binding buffer (20mM Tris-HCl, lOO mMNaCl, 5 mMKCl, 1 mMMgCh, ImM CaCl2, pH 7.5); adding various concentrations of cortisol (between -500 nM and 500 pM, as well as 0 M) at a concentration of 15 nM switching strand and 10 nM aptamer strand within a 50 uL volume in binding buffer in a black polystyrene 384-well plate in quadruplicate; incubating the sample for 30 minutes and reading the fluorescent signal of the samples with a fluorescence microplate reader; analyzing the dissociation constant of target-induced fluorescent signaling of theaptamer switch by fitting the resulting fluorescence values with a langmuir isotherm model (RFU = Amplitude*TargetConcentration / (KD+TargetConcentration) + background); and using the resulting KD from fitting to assess the impact of PS bond addition on aptamer switch function. The switching strand comprising all natural intemucleoside linkages (DS 33) exhibits relatively high dissociation constant of KD > 300 pM, while switching strands comprising increasing numbers of PS internucleoside linkages (DS 34, DS 35, and DS 36 containing 4, 8, and 15 PS intemucleoside linkages respectively) exhibit decreasing dissociation constants as the number of non-natural linkages is increased (153 pM, 51 pM, and 4.6 pM respectively), evidencing that non-natural internucleoside linkages can be used to predictably tune aptamer switch binding affinity (see Figure 3b and example sequences).

[0052] Example 2: Decreasing and Increasing the background signal and binding behavior of a glucose-binding ISD aptamer switch with Phosphorothioate (PS) Modifications

[0053] An ISD aptamer switch that produces a change in fluorescent signal upon binding to glucose is designed by covalently linking a glucose-binding aptamer sequence to a 9nt partially complementary displacement strand (DS) through a 20nt poly-T linker. The DS hybridizes to the aptamer in the target-free state, and is displaced upon target binding, leading to fluorescent signaling of target binding. An ISD switch (AS 38) is designed with a DS that contains only natural DNA bases and natural intemucleoside linkages. Another ISD switch (AS 62) is designed to augment the identical nucleotide sequence with 3 additional flanking dT bases linked by non-natural phosphorothioate (PS) intemucleoside linkages on both the 5’ and 3’ end of the ISD switch, which increase the stability of DS hybridization to the aptamer through additional base stacking, thus decreasing the background signal in the absence of target and shifting the binding curve of the aptamer switch to respond to higher target concentrations. Another ISD switch (AS 64) is designed to modify the identical nucleotide sequence of AS 38 to incorporate 6 non-natural PS intemucleoside linkages within the DS, which decrease the stability of DS hybridization to the aptamer, thus increasing the background signal in the absence of target and shifting the binding curve of the aptamer switch to respond to lower concentrations (see Figure 4a and example sequences).

[0054] The fluorescent glucose-binding responses of aptamer switches employing these switching domains are experimentally characterized by: labeling each ISD aptamer switch with an ATTO 643 fluorophore at its 5 ’ end and an Iowa Black FQ quencher at its 3 ’ end; annealing the aptamer switch at a concentration of 0.66 pM ISD aptamer strand in binding buffer (20mM Tris-HCl, 100 mM NaCl, 5 mM KC1, 1 mM MgCh, ImM CaCE, pH 7.5); adding various concentrations of glucose (between -500 pM and 500 mM, as well as 0 M) at a concentrationof 10 nM aptamer strand within a 50 uL volume in binding buffer in a black polystyrene 384- well plate in quadruplicate; incubating the sample for 30 minutes and reading the fluorescent signal of the samples with a fluorescence microplate reader; analyzing the background signal at 0 M glucose as well as the dissociation constant of target-induced fluorescent signaling of the aptamer switch by fitting the resulting fluorescence values with a langmuir isotherm model (RFU = Amplitude*TargetConcentration / (KD+TargetConcentration) + background); normalizing each binding curve by dividing by its amplitude; and using the resulting background and binding curve shape to assess the impact of PS bond addition on aptamer switch function. The aptamer switch comprising all natural internucleoside linkages (AS 38) exhibits an intermediate background signal, while the aptamer switch comprising flanking dT bases with PS linkages (AS 62) exhibits decreased target-free background signal and a right- shifted binding curve, indicating a higher dissociation constant and sensitivity shifted towards higher target concentrations. The aptamer switch comprising 6 PS linkages within the DS (AS 64) exhibits increased target-free background signal and a left-shifted binding curve, indicating a lower dissociation constant and sensitivity shifted towards lower target concentrations (see Figure 4b and example sequences).

[0055] Example 3: Increasing the apparent dissociation constant of a glucose-binding ISD aptamer switch with Locked Nucleic Acids (LNA)

[0056] An ISD aptamer switch that produces a change in fluorescent signal upon binding to glucose is designed by covalently linking a glucose-binding aptamer sequence to a 9nt partially complementary displacement strand (DS) through a 20nt poly-T linker. The DS hybridizes to the aptamer in the target-free state, and is displaced upon target binding, leading to fluorescent signaling of target binding. An ISD switch (AS 38) is designed with a DS that contains only natural DNA bases and natural intemucleoside linkages. Another ISD switch (AS 66) is designed to modify the identical nucleotide sequence of AS 38 to incorporate 2 nonnatural LNA bases within the DS, thus increasing the stability of DS hybridization due to the increased hybridization energy of LNA bases, leading to an increased dissociation constant of the ISD switch. Another ISD switch (AS 69) is designed to modify the identical nucleotide sequence of AS 38 to incorporate 6 non-natural PS internucleoside linkages and 3 non -natural LNA bases within the DS, with the increased hybridization stability from the LNA bases offsetting the decrease in hybridization stability imparted by the PS linkages, thus increasing the dissociation constant of the ISD switch, but with a smaller degree of change in dissociation constant than would be observed by adding either modification alone (see Figure 5a and example sequences).

[0057] The fluorescent glucose-binding responses of aptamer switches employing these switching domains are experimentally characterized by: labeling each ISD aptamer switch with an ATTO 643 fluorophore at its 5 ’ end and an Iowa Black FQ quencher at its 3 ’ end; annealing the aptamer switch at a concentration of 0.66 pM ISD aptamer strand in binding buffer (20mM Tris-HCl, 100 mM NaCl, 5 mM KC1, 1 mM MgCh, ImM CaCL, pH 7.5); adding various concentrations of glucose (between ~500 pM and 500 mM, as well as 0 M) at a concentration of 10 nM aptamer strand within a 50 uL volume in binding buffer in a black polystyrene 384- well plate in quadruplicate; incubating the sample for 30 minutes and reading the fluorescent signal of the samples with a fluorescence microplate reader; analyzing the background signal at 0 M glucose as well as the dissociation constant of target-induced fluorescent signaling of the aptamer switch by fitting the resulting fluorescence values with a langmuir isotherm model (RFU = TargetConcentration / (KD+TargetConcentration) + background); normalizing each binding curve by dividing by its amplitude; and using the resulting KD from fitting to assess the impact of LNA base addition on aptamer switch function. The aptamer switch containing only natural bases (AS 38) exhibits a relatively low dissociation constant of KD = 19 mM. The addition of 2 LNA bases within AS 66 stabilizes the DS hybridization and decreases binding affinity substantially, leading to an increased dissociation constant of KD = 760 mM. The simultaneous and offsetting addition of both 6 PS modifications and 3 LNA bases in AS 69 leads to a smaller stabilization of DS hybridization, and a smaller increase in the dissociation constant as compared to the natural DNA switch (KD = 143 mM, see Figure 5b and example sequences).

[0058] Example 4: Modulating oligonucleotide switch affinity with non-natural linkers

[0059] A variety of oligonucleotide strands with an ISD aptamer switch architecture that produces a change in fluorescent signal upon binding of an 11 nt DNA oligo were designed by covalently linking a 15nt aptamer sequence to a 15nt displacement strand (DS) through variety of linker designs. In this design, the DS hybridizes to the aptamer in the target-free state, and is displaced upon target oligonucleotide binding, leading to fluorescent signaling of target binding. Linkers were designed to both incorporate natural DNA polyT linkers of various lengths (5, 10, 15, 20, and 25 dT bases denoted as T5, T10, T15, T20, T25 respectively), as well as to incorporate non-natural polymer linkers using a number of hexa-ethyleneglycol repeats (1, 3, or 5 hexa-ethyleneglycol units denoted as Spl8, Spl8x3, SP18x5 respectively). Altering the material of the linker region within the aptamer switch allows for tuning of the aptamer-DS interaction strength, and thus modulation of the overall affinity of the oligonucleotide switch. Increased flexibility of hexa-ethyleneglycol -based linkers as comparedto DNA increases intramolecular hybridization between the DS and aptamer, leading to higher observed dissociation constants given similar lengths of the linker domain (see Figure 6a and example sequences).

[0060] The fluorescent target-binding responses of aptamer switches employing these switching domains are experimentally characterized by: labeling each ISD aptamer switch with a FAM fluorophore at its 5’ end and an Iowa Black FQ quencher at its 3’ end; adding various concentrations of target oligonucleotide (Dl l, between ~2 nM and ~2 pM, as well as 0 M) at a concentration of 10 nM aptamer strand within a 50 uL volume in binding buffer (20mM Tris- HC1, 100 mM NaCl, 5 mM KC1, 1 mM MgCh, ImM CaCh, pH 7.5) in a black polystyrene 384-well plate in quadruplicate; incubating the sample for 30 minutes and reading the fluorescent signal of the samples with a fluorescence microplate reader; fitting the resulting fluorescence values with a langmuir isotherm model (RFU = TargetConcentration / (Ko+TargetConcentration) + background); normalizing each binding curve by dividing by the maximum observed amplitude for all curves; and using the resulting background and binding curve shape to assess the impact of PS bond addition on aptamer switch function. Aptamer switches containing hexa-ethyleneglycol-based linkers exhibit stabilized DS hybridization and higher dissociation constants as compared to aptamers with DNA linkers of similar length. For example, the design with a single hexa-ethyleneglycol linker (Spl8, length ~30 angstroms) has a dissociation constant of KD = 647 nM, while the equivalent DNA linker (T10, length ~34 angstroms) has KD = 470 nM. The design with a three hexaethyleneglycol linker (3xSpl8, length >60 angstroms) has a dissociation constant of KD = 412 nM, while the equivalent DNA linker (T20, length ~68 angstroms) has KD = 226 nM. The design with a five hexa-ethyleneglycol linker (5xSpl8, length >90 angstroms) has a dissociation constant of KD = 369 nM, while the closest equivalent DNA linker (T25, length ~85 angstroms) has KD = 138 nM. These results illustrate that substituting non-natural polymer linkers offer a method to modulate the dissociation constant of an aptamer switch (see Figure 6b and example sequences).

[0061] By altering the material of the linker region in an ISD aptamer switch we are able to tune the strength of the of the interaction between the switching strand and the aptamer. To demonstrate this we compared the effect that various natural polyT linkers to that of various lengths (T5, T10, T15, T20, T25) of the non-natural polymer hexa-ethyleneglycol (Spl8, Spl8x3, SP18x5) had on the apparent dissociation constant of an polynucleotide switch towards a short complementary oligonucleotide target molecule (Figure 5a). The polynucleotide switch is in a hairpin loop structure, where the loop is the linker sequence.The constructs were 5’ FAM labeled and 3’ labeled with Iowa Black FQ quencher so that the construct’s fluorescence increased upon binding to the target oligonucleotide strand. 10 nanomolar of the aptamer switch constructs containing different linkers were incubated with various concentrations of the target oligonucleotide molecule in a black fluorescent well plate, incubated for 30 minutes, and then read on a fluorescent microplate reader (Figure 5b). The dissociation constants of each construct, and thus the effect of the linker material on the apparent dissociation constant between the polynucleotide switch and the target molecule could then be determined by fitting the fluorescent data to a Langmuir isotherm model (RFU = TargetConc / (kd+TargetConc) + background). This example demonstrates the ability to tune the dissociation constant of a molecular switch by changing the material and composition of non-natural linkers.

[0062] Example Sequences:

[0063] Switching strand sequences for tuning cortisol-binding anchored displacement strand aptamer switch affinity using phosphorothioate modifications (nucleotides in the sequences that are followed by a * indicate a base with a phosphorothioate modified linkage):

[0064] DS 33: CCGAAGCATAGTGTCAAGCCTTTTTTTTTTTTTTTTTTTTCTACTGCACTGCCAG

[0065] DS 34: CCGAAGCATAGTGTCAAGCCTTTTTTTTTTTTTTTTTTTTCTA*CTGC*ACTG*CC*AG

[0066] DS 35:CCGAAGCATAGTGTCAAGCCTTTTTTTTTTTTTTTTTTTTC*TA*CT*GC*AC*TG*CC*AG*

[0067] DS 36:T*T*T*CCGAAGCATAGTGTCAAGCCTTTTTTTTTTTTTTTTTTTTC*T*A*C*T*G*C*A*C*T*G*C*C*A*G*

[0068] ISD Aptamer switch sequences for decreasing and Increasing the background signal and binding behavior of a glucose-binding ISD aptamer switch with Phosphorothioate (PS) Modifications (nucleotides in the sequences that are followed by a * indicate a base with a phosphorothioate modified linkage):

[0069] AS 38:CTCTCGGGACTACCGTGTGTGTTGCTCTGTAACAGTGTCCTTTGTCGTCCCTTTTTTTTTTTTTTTTTTTTCGGTCGTCC

[0070] AS 62:T*T*T*CTCTCGGGACTACCGTGTGTGTTGCTCTGTAACAGTGTCCTTTGTCGTCCCTTTTTTTTTTTTTTTTTTTTCGGTCGTCCTJi!TJi!T!it

[0071] AS 64:CTCTCGGGACTACCGTGTGTGTTGCTCTGTAACAGTGTCCTTTGTCGTCCCTTTTTTTTTTTTTTTTTTTTCG*G*T*CG*T*C*C

[0072] Increasing apparent dissociation constant of a glucose-binding ISD aptamer switch with Locked Nucleic Acids (LNA) (nucleotides in the sequences that are followed by a + indicate an LNA base, and nucleotides in the sequence that are followed by a *+ indicate a PS modified LNA base):

[0073] AS 69:CTCTCGGGACTACCGTGTGTGTTGCTCTGTAACAGTGTCCTTTGTCGTCCCTTTTTTTTTTTTTTTTTTTTC*G*G*TT*C*TG*T*1’C*C*

[0074] AS 66:CTCTCGGGACTACCGTGTGTGTTGCTCTGTAACAGTGTCCTTTGTCGTCCCTTTTTTTTTTTTTTTTTTTTCGG+ TCGT+ cc

[0075] Modulating oligonucleotide switch affinity with non-natural linkers (where / i Sp 18 / denotes the incorporation of a hexa-ethyleneglycol spacer):

[0076] DI 1 : CC TGC CAG TCG

[0077] T5: CGA CTG GCA GGG CAG TTTTT CTG CCC TGT GAA TTC

[0078] T10: CGA CTG GCA GGG CAG TTTTT TTTTT CTG CCC TGT GAA TTC

[0079] T 15 : CGA CTG GCA GGG CAG TTTTT TTTTT TTTTT CTG CCC TGT GAA TTC

[0080] T20: CGA CTG GCA GGG CAG TTTTT TTTTT TTTTT TTTTT CTG CCC TGT GAA TTC

[0081] T25 : CGA CTG GCA GGG CAG TTTTT TTTTT TTTTT TTTTT TTTTT CTG CCC TGT GAA TTC

[0082] Spl8: CGA CTG GCA GGG CAG / iSpl8 / CTG CCC TGT GAA TTC

[0083] 3xSpl8: CGA CTG GCA GGG CAG / iSpl8 / / iSpl8 / / iSpl8 / CTG CCC TGT GAA TTC

[0084] 5xSpl8: CGA CTG GCA GGG CAG / iSp 18 / / iSpl 8 / / iSp 18 / / iSp 18 / / iSp 18 / CTG CCC TGT GAA TTC

[0085] Definitions

[0086] As used herein an “aptamer region” or “aptamer domain” are used interchangeably and refer to a region of a polynucleotide aptamer switch which binds to a target analyte.

[0087] As used herein an “aptamer region” or “aptamer domain” can include a loop region of a beacon aptamer switch, where the loop region binds to a target analyte.

[0088] As used herein a “switching domain” or “displacement strand” are used interchangeably and refer to a region of a polynucleotide aptamer switch which hybridizes or partially hybridizes to a portion of an aptamer region in competition with target binding, such that target binding leads to competitive displacement of the switching domain.

[0089] As used herein a “displacement region” refers to the set of bases within the aptamer switch that undergoes the competitive displacement reaction, both including bases within the aptamer domain and bases within the switching domain. A displacement region can be at least partially complementary to a portion of the aptamer switch. In some embodiments, the region that is at least partially complementary to the portion of the aptamer switch comprises at least 1 base, at least 2 bases, at least 3 bases, at least 4 bases, at least 5 bases, at least 6 bases, at least 7 bases, at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases.

[0090] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWhat is claimed is:

1. A method for sensing an analyte, the method comprising: a. providing an aptamer switch configured to generate a signal upon binding to the analyte, wherein the aptamer switch comprises a displacement region, and wherein the displacement region comprises at least 3 bases; b. modifying the displacement region with one or more non-naturally occurring modifications; and c. contacting the aptamer switch with the analyte to generate a complex comprising the displacement region and a partially complementary strand of the aptamer switch, wherein a dissociation constant of the complex increases or decreases by at least 10% compared to a complex comprising an aptamer switch that does not comprise the one or more non-naturally occurring modifications to the displacement region.

2. A method for sensing an analyte, the method comprising: a. providing an aptamer switch configured to generate a signal upon binding to the analyte, wherein the aptamer switch comprises: i. a sequence that is at least partially complementary to a portion of the aptamer switch, wherein the sequence that is at least partially complementary to the portion of the aptamer switch comprises at least 3 bases; and ii. one or more non-naturally occurring modifications to the sequence that is at least partially complementary to the portion of the aptamer switch; and b. contacting the aptamer switch with the analyte to generate a complex comprising the sequence that is at least partially complementary to the portion of the aptamer switch, wherein a dissociation constant of the complex increases or decreases by at least 10% compared to a complex comprising an aptamer switch that does not comprise the one or more non-naturally occurring modifications to the sequence that is at least partially complementary to the analyte.

3. A method of detecting a target analyte with a polynucleotide aptamer switch comprising: a. providing a polynucleotide aptamer switch configured to undergo a conformation change upon binding to the target analyte, wherein the polynucleotide aptamer switch comprises:i. one or more labels which are configured to produce a change in a signal when the polynucleotide aptamer switch is bound to the target analyte; and ii. one or more modifications which alter a binding affinity between the polynucleotide aptamer switch and the target analyte, the modification comprising at least one of: i. incorporation or substitution of one or more modified nucleobase(s) in an aptamer domain, a switching domain, a linker region, or a stem region of the polynucleotide aptamer switch; ii. incorporation or substitution of one or more sugar modifications in the polynucleotide aptamer switch; iii. incorporation or substitution of one or more modified internucleoside linkage(s) in the polynucleotide aptamer switch; iv. incorporation or substitution of one or more non-natural nucleobase, a non-natural sugar, and a non-natural intemucleoside linkage in a displacement region of the polynucleotide aptamer switch; v. incorporation or substitution of one or more sugar moiety modification(s) in the polynucleotide aptamer switch; or vi. any combination thereof; and ii. contacting the polynucleotide aptamer switch with the target analyte, inducing a conformational change in the polynucleotide aptamer switch and producing the change in signal, thereby detecting the target analyte.

4. A method of tuning an interaction between a target analyte and a polynucleotide aptamer switch comprising: a. modifying the polynucleotide aptamer switch to comprise one or more modifications which alter a binding affinity between the polynucleotide aptamer switch and the target analyte, wherein the one or more modifications comprise at least one of: i. incorporation or substitution of one or more modified nucleobase(s) in an aptamer domain, a switching domain, a linker region, or a stem region of the polynucleotide aptamer switch; ii. incorporation or substitution of one or more sugar modifications in the polynucleotide aptamer switch;iii. incorporation or substitution of one or more modified internucleoside linkage(s) in the polynucleotide aptamer switch; iv. incorporation or substitution of one or more non-natural nucleobase, a non-natural sugar, and a non-natural internucleoside linkage in a displacement region of the polynucleotide aptamer switch; v. incorporation or substitution of one or more sugar moiety modification(s) in the polynucleotide aptamer switch; or vi . any combination thereof.

5. The method of any one of claims 1-4, wherein the one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in a region of the polynucleotide aptamer switch where it hybridizes to itself, and comprise shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch.

6. The method of any one of claims 1-4, wherein one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in an aptamer domain of the polynucleotide aptamer switch, thereby shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch.

7. The method of any one of claims 1-4, wherein the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising a linker domain, wherein the one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in the linker domain, thereby shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch.

8. The method of any one of claims 1-7, wherein the polynucleotide aptamer switch comprises an aptamer domain which binds to the target analyte, wherein the one or more modifications are in the aptamer domain and increase the binding affinity between the aptamer domain and the target analyte relative to an unmodified polynucleotide aptamer switch.

9. The method of any one of the preceding claims, wherein the one or more modifications decrease a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte relative to an unmodified polynucleotide aptamer switch.

10. The method of claim 8 or 9, wherein the one or more modifications increase the binding affinity between the aptamer domain and the target analyte by increasing a number of hydrogen bonds between the target analyte and the aptamer domain, increasing a number of salt bridges between the target analyte and the aptamer domain, increasing a number of hydrophobic interaction sites between the target analyte and the aptamer domain, increasing a number of electrostatic interaction sites between the target analyte and the aptamer domain, or combinations thereof, relative to an unmodified polynucleotide aptamer switch.

11. The method of any one of claims 1-9, wherein the polynucleotide aptamer switch comprises an aptamer domain which binds to the target analyte, wherein the one or more modifications are in the aptamer domain and decrease the binding affinity between the aptamer domain and the target analyte relative to an unmodified polynucleotide aptamer switch.

12. The method of any one of the preceding claims, wherein the one or more modifications increase a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte relative to an unmodified polynucleotide aptamer switch.

13. The method of claim 11 or 12, wherein the one or more modifications decrease the binding affinity between the aptamer domain and the target analyte by decreasing a number of hydrogen bonds between the target analyte and the aptamer domain, decreasing a number of salt bridges between the target analyte and the aptamer domain, decreasing a number of hydrophobic interaction sites between the target analyte and the aptamer domain, decreasing a number of electrostatic interaction sites between the target analyte and the aptamer domain, or combinations thereof, relative to an unmodified polynucleotide aptamer switch.

14. The method of any one of claims 1-7, wherein the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising an aptamer domain covalently attached by a linker region to a switching region, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

15. The method of any one of claims 1-7, wherein the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising an aptamer domain covalently attached by a linker region to a switching region.

16. The method of claim 15, wherein the one or more modifications are in the switching region, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

17. The method of claim 15, wherein the one or more modifications are in the linker region, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

18. The method of claim 15, wherein the one or more modifications are in the linker region, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

19. The method of claim 15, wherein the one or more modifications are in the aptamer domain, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

20. The method of claim 15, wherein the one or more modifications are in the aptamer domain, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

21. The method of any one of claims 1-7, wherein the polynucleotide aptamer switch is a beacon switch comprising an aptamer domain and a stem region, optionally, wherein the aptamer domain is a loop region.

22. The method of claim 21, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, wherein a bound state of the beacon switch to the target analyte occurs upon the hybridization or pairing of the stem region to itself, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

23. The method of claim 21, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, wherein an bound state of the beacon switch to the target analyte occurs upon the hybridization or pairing of the stem region to itself, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

24. The method of claim 21, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, wherein a bound state of the beacon switch to the target analyte occurs upon disruption of the hybridization or pairing of the stem region to itself, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

25. The method of claim 21, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, wherein a bound state of the beacon switch to the target analyte occurs upon disruption of the hybridization or pairing of the stem region to itself, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

26. The method of any one of the preceding claims, wherein the one or more modifications comprising incorporation or substitution of one or more modified nucleobase(s) in the polynucleotide aptamer switch comprise incorporation or substitution of one or more nucleobase(s) which differs from adenine, cytosine, guanine, thymine, or uracil.

27. The method of any one of the preceding claims, wherein the one or more modified nucleobase(s) comprise one or more of: Iso-dG-iso deoxyguanosine, 2-aminopyrimidine, 2-MethoxyEthoxy A, 2-MethoxyEthoxy MeC, 2-MethoxyEthoxy G, 2-MethoxyEthoxy T, 2'-O-Methyl RNA Bases (G,C,U,A), Fluoro C, Fluoro U, Fluoro A, Fluoro G, 2- Aminopurine, 5-Bromo dU, deoxyUridine, 2,6-Diaminopurine (2-Amino-dA), Dideoxy- C, deoxyinosine, Hydroxymethyl dC, Inverted dT, Iso-dGm, Iso-dC, Inverted Dideoxy-T, 5-Methyl dC, 5-Nitroindole , 5-hydroxybutynl-2’-deoxyuridine, 8-aza-7- deazaguanosine, deoxyinosine, or combinations thereof.

28. The method of any one of the preceding claims, wherein the polynucleotide aptamer switch is an aptamer beacon switch and the incorporation or substitution of one or more modified nucleobase(s) are in a stem region of the aptamer beacon switch, or wherein the polynucleotide aptamer switch is intramolecular strand displacement switch and the incorporation or substitution of one or more one or more modified nucleobase(s) are in a switching domain or an aptamer domain of the polynucleotide aptamer switch.

29. The method of any one of the preceding claims, wherein the one or more modifications further comprise increasing or decreasing a number of nucleobases in a region of the polynucleotide aptamer switch where it hybridizes to itself.

30. The method of any one of the preceding claims, wherein the one or more modifications comprising incorporation or substitution of one or more sugar modifications in the polynucleotide aptamer switch comprise incorporation or substitution of one or more different furanose(s) into the polynucleotide aptamer switch.

31. The method of any one of the preceding claims, wherein the different furanose(s) comprises one or more of a: LNA-locked nucleic acid, TNA-threose nucleic acid, CeNA- cyclohexane nucleic acid, FANA-2’-fluoro-aribinonucleic acid, HNA-hexitol nucleic acid, GNA-glycol nucleic acid, L-DNA, or combinations thereof.

32. The method of any one of the preceding claims, wherein the polynucleotide aptamer switch is an aptamer beacon switch and the incorporation or substitution of one or more LNA-locked nucleic acid(s) are in a stem region of the aptamer beacon switch.

33. The method of claim 31, wherein the polynucleotide aptamer switch is intramolecular strand displacement switch and the incorporation or substitution of one or more LNA- locked nucleic acid(s) are in a switching domain or an aptamer domain of the polynucleotide aptamer switch.

34. The method of any one of the preceding claims, wherein the one or more modifications comprising incorporation or substitution of one or more modified intemucleosidelinkage(s) in the polynucleotide aptamer switch comprise incorporation or substitution of one or more internucleoside linkage(s) different from a phosphate linkage.

35. The method of any one of the preceding claims, wherein the internucleoside linkage different from a phosphate linkage comprises: phosphorothioate, morpholino, PNA- peptide nucleic acids, glycol linkers, PEG (polyethylene glycol)-linkers, and hexaethyleneglycol linkers, or combinations thereof.

36. The method of any one of the preceding claims, wherein the one or more modifications comprising incorporation or substitution of one or more modifications are in a displacement region of the polynucleotide aptamer switch and comprise incorporation or substitution of one or more of: a non-natural nucleobase, a non-natural sugar, a nonnatural intemucleoside linkage, or combinations thereof.

37. The method of claim 36, wherein the non-natural intemucleoside linkage comprises at least 1, at least 4, at least 8, at least 10, at least 15, at least 20 phosphorothioate intemucleoside linkages, or a combination thereof.

38. The method of any one of the preceding claims, wherein the one or more modifications in the displacement region of the polynucleotide aptamer switch comprise: Hoogsteen and Wobble base-pairing, non-Watson and Crick base pairing, Z / P base pairing, incorporation of 2-aminopurine, substitution of adenine with 2-aminopurine, a duplex stabilizing modification, a duplex de-stabilizing modification, incorporation or substitution of PNA, incorporation or substitution of 5-hydroxybutynl-2’ -deoxyuridine, or combinations thereof.

39. The method of any one of the preceding claims, wherein the one or more modifications comprising incorporation or substitution of one or more sugar moiety modification(s) in the polynucleotide aptamer switch comprise: incorporation or substitution of a non- naturally occurring sugar or non- naturally occurring polymer into the polynucleotide aptamer switch, incorporation or substitution of four-carbon threose sugars, incorporation or substitution of peptide nucleic acids (PNAs), or combinations thereof.

40. The method of any one of the preceding claims, wherein the polynucleotide aptamer switch is an aptamer beacon switch, or wherein the polynucleotide aptamer switch is intramolecular strand displacement switch, or wherein the polynucleotide aptamer switch is an anchored displacement strand switch.

41. The method of any one of the preceding claims, wherein the one or more labels comprise a fluorophore and quencher, a redox reporter, or an electrochemical label.

42. The method of any one of the preceding claims, wherein the fluorophore and quencher comprises an acceptor fluorophore and donor fluorophore, wherein contacting the polynucleotide aptamer switch with the target analyte comprises inducing a ratiometric FRET readout.

43. The method of any one of the preceding claims, wherein the polynucleotide aptamer switch is comprised in a device comprising one or more members which are configured to penetrate the skin of a subject, wherein contacting the polynucleotide aptamer switch with the target analyte at least partially occurs by contacting an interstitial fluid of the subject.

44. The method of any one of the preceding claims, wherein the partially complementary strand comprises a linker.

45. The method of claim 44, wherein the linker is a poly-dT linker.

46. The method of claim 45, wherein the poly-dT linker comprises at least 5, at least 10, at least 15, at least 20, or at least 25 nucleotides.

47. The method of any one of claim 44-46, wherein the linker comprises a non-natural polymer linker.

48. The method of claim 47, wherein the non-natural polymer linker comprises at least 1, at least 2, at least 5, or at least 10 hexa-ethyleneglycol repeats.

49. A polynucleotide aptamer switch configured to undergo a conformation change upon binding to the target analyte, wherein the polynucleotide aptamer switch comprises: i. one or more labels which are configured to produce a change in a signal when the polynucleotide aptamer switch is bound to the target analyte; and ii. one or more modifications which alter a binding affinity between the polynucleotide aptamer switch and the target analyte, the modification comprising at least one of: i. incorporation or substitution of one or more modified nucleobase(s) in an aptamer domain, a switching domain, a linker region, or a stem region of the polynucleotide aptamer switch; ii. incorporation or substitution of one or more sugar modifications in the polynucleotide aptamer switch; iii. incorporation or substitution of one or more modified internucleoside linkage(s) in the polynucleotide aptamer switch; iv. incorporation or substitution of one or more non-natural nucleobase, a non-natural sugar, and a non-natural intemucleosidelinkage in a displacement region of the polynucleotide aptamer switch; v. incorporation or substitution of one or more sugar moiety modification(s) in the polynucleotide aptamer switch; or vi. any combination thereof.

50. The polynucleotide of claim 49, wherein the one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in a region of the polynucleotide aptamer switch where it hybridizes to itself, thereby shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch.

51. The polynucleotide of claim 49, wherein one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in an aptamer domain of the polynucleotide aptamer switch, thereby shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch.

52. The polynucleotide of claim 49, wherein the polynucleotide aptamer switch an intramolecular strand displacement (ISD) switch comprising a linker domain, wherein the one or more modifications which alter the binding affinity between the polynucleotide aptamer switch and the target analyte are in the linker domain, thereby shifting a relative stability and / or binding kinetics of a bound state or an unbound state of the aptamer switch polynucleotide relative to an unmodified polynucleotide aptamer switch.

53. The polynucleotide of any one of claims 49-52, wherein the polynucleotide aptamer switch comprises an aptamer domain which binds to the target analyte, wherein the one or more modifications are in the aptamer domain and increase the binding affinity between the aptamer domain and the target analyte relative to an unmodified polynucleotide aptamer switch.

54. The polynucleotide of any one of the preceding claims, wherein the one or more modifications decrease a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte relative to an unmodified polynucleotide aptamer switch.

55. The polynucleotide of claim 53 or 54 wherein the one or more modifications increase the binding affinity between the aptamer domain and the target analyte by increasing a number of hydrogen bonds between the target analyte and the aptamer domain, increasing a number of salt bridges between the target analyte and the aptamer domain, increasing anumber of hydrophobic interaction sites between the target analyte and the aptamer domain, increasing a number of electrostatic interaction sites between the target analyte and the aptamer domain, or combinations thereof, relative to an unmodified polynucleotide aptamer switch.

56. The polynucleotide of any one of claims 49-52, wherein the polynucleotide aptamer switch comprises an aptamer domain which binds to the target analyte, wherein the one or more modifications are in the aptamer domain and decrease the binding affinity between the aptamer domain and the target analyte relative to an unmodified polynucleotide aptamer switch.

57. The polynucleotide of any one of the preceding claims, wherein the one or more modifications increase a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte relative to an unmodified polynucleotide aptamer switch.

58. The polynucleotide of claim 56 or 57, wherein the one or more modifications decrease the binding affinity between the aptamer domain and the target analyte by decreasing a number of hydrogen bonds between the target analyte and the aptamer domain, decreasing a number of salt bridges between the target analyte and the aptamer domain, decreasing a number of hydrophobic interaction sites between the target analyte and the aptamer domain, decreasing a number of electrostatic interaction sites between the target analyte and the aptamer domain, or combinations thereof, relative to an unmodified polynucleotide aptamer switch.

59. The polynucleotide of any one of claims 49-52, wherein the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising an aptamer domain covalently attached by a linker region to a switching region, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

60. The polynucleotide of any one of claims 49-52, wherein the polynucleotide aptamer switch is an intramolecular strand displacement (ISD) switch comprising an aptamer domain covalently attached by a linker region to a switching region.

61. The polynucleotide of claim 60, wherein the one or more modifications are in the switching region, wherein the one or more modifications alter the binding affinitybetween the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

62. The polynucleotide of claim 60, wherein the one or more modifications are in the linker region, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

63. The polynucleotide of claim 60, wherein the one or more modifications are in the linker region, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

64. The polynucleotide of claim 60, wherein the one or more modifications are in the linker region, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

65. The polynucleotide of claim 60, wherein the one or more modifications are in the aptamer domain, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

66. The polynucleotide of claim 60, wherein the one or more modifications are in the aptamer domain, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the switching domain and the aptamer domain, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

67. The polynucleotide of any one of claims 49-52, wherein the polynucleotide aptamer switch is a beacon switch comprising an aptamer domain covalently attached to a stem region, optionally, wherein the aptamer domain is a loop region.

68. The polynucleotide of claim 67, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, wherein a bound state of the beacon switch to the target analyte occurs upon the hybridization or pairing of the stem region to itself, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

69. The polynucleotide of claim 67, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, wherein an bound state of the beacon switch to the target analyte occurs upon the hybridization or pairing of the stem region to itself, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

70. The polynucleotide of claim 67, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by decreasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, wherein a bound state of the beacon switch to the target analyte occurs upon disruption of the hybridization or pairing of the stem region to itself, thereby decreasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

71. The polynucleotide of claim 67, wherein the one or more modifications alter the binding affinity between the polynucleotide aptamer switch and the target analyte by increasing a stability of a DNA duplex defined by hybridization or pairing of the stem region to itself, wherein a bound state of the beacon switch to the target analyte occurs upon disruption of the hybridization or pairing of the stem region to itself, thereby increasing a dissociation constant measuring the binding affinity between the polynucleotide aptamer switch and the target analyte.

72. The polynucleotide of any one of the preceding claims, wherein the one or more modifications comprising incorporation or substitution of one or more modified nucleobase(s) in the polynucleotide aptamer switch comprise incorporation or substitutionof one or more nucleobase(s) which differs from adenine, cytosine, guanine, thymine, or uracil.

73. The polynucleotide of any one of the preceding claims, wherein the one or more modified nucleobase(s) comprise one or more of: Iso-dG-iso deoxyguanosine, 2-aminopyrimidine, 2-MethoxyEthoxy A, 2-MethoxyEthoxy MeC, 2-MethoxyEthoxy G, 2-MethoxyEthoxy T, 2'-O-Methyl RNA Bases (G,C,U,A), Fluoro C, Fluoro U, Fluoro A, Fluoro G, 2- Aminopurine, 5-Bromo dU, deoxyUridine, 2,6-Diaminopurine (2-Amino-dA), Dideoxy- C, deoxyinosine, Hydroxymethyl dC, Inverted dT, Iso-dGm, Iso-dC, Inverted Dideoxy-T, 5-Methyl dC, 5-Nitroindole, 5-hydroxybutynl -2’ -deoxyuridine, 8-aza-7-deazaguanosine, deoxyinosine, or combinations thereof.

74. The polynucleotide of any one of the preceding claims, wherein the polynucleotide aptamer switch is an aptamer beacon switch and the incorporation or substitution of one or more modified nucleobase(s) are in a stem region of the aptamer beacon switch, or wherein the polynucleotide aptamer switch is intramolecular strand displacement switch and the incorporation or substitution of one or more one or more modified nucleobase(s) are in a switching domain or an aptamer domain of the polynucleotide aptamer switch.

75. The polynucleotide of any one of the preceding claims, wherein the one or more modifications further comprise increasing or decreasing a number of nucleobases in a region of the polynucleotide aptamer switch where it hybridizes to itself.

76. The polynucleotide of any one of the preceding claims, wherein the one or more modifications comprising incorporation or substitution of one or more sugar modifications in the polynucleotide aptamer switch comprise incorporation or substitution of one or more different furanose(s) into the polynucleotide aptamer switch.

77. The polynucleotide of any one of the preceding claims, wherein the different furanose(s) comprises one or more of a: LNA-locked nucleic acid, TNA-threose nucleic acid, CeNA- cyclohexane nucleic acid, FANA-2’-fluoro-aribinonucleic acid, HNA-hexitol nucleic acid, GNA-glycol nucleic acid, L-DNA, or combinations thereof.

78. The polynucleotide of any one of the preceding claims, wherein the polynucleotide aptamer switch is an aptamer beacon switch and the incorporation or substitution of one or more LNA-locked nucleic acid(s) are in a stem region of the aptamer beacon switch.

79. The polynucleotide of any one of the preceding claims, wherein the polynucleotide aptamer switch is intramolecular strand displacement switch and the incorporation or substitution of one or more LNA-locked nucleic acid(s) are in a switching domain or an aptamer domain of the polynucleotide aptamer switch.

80. The polynucleotide of any one of the preceding claims, wherein the one or more modifications comprising incorporation or substitution of one or more modified internucleoside linkage(s) in the polynucleotide aptamer switch comprise incorporation or substitution of one or more intemucleoside linkage(s) different from a phosphate linkage.

81. The polynucleotide of any one of the preceding claims, wherein the internucleoside linkage different from a phosphate linkage comprises: phosphorothioate, morpholino, PNA-peptide nucleic acids, glycol linkers, PEG (polyethylene glycol)-linkers, and hexaethyleneglycol linkers, or combinations thereof.

82. The polynucleotide of any one of the preceding claims, wherein the one or more modifications comprising incorporation or substitution of one or more modifications are in a displacement region of the polynucleotide aptamer switch and comprise incorporation or substitution of one or more of: a non-natural nucleobase, a non-natural sugar, a non-natural internucleoside linkage, or combinations thereof.

83. The polynucleotide of claim 82, wherein the non-natural internucleoside linkage comprises at least 1, at least 4, at least 8, at least 10, at least 15, at least 20 phosphorothioate intemucleoside linkages, or a combination thereof.

84. The polynucleotide of any one of the preceding claims, wherein the one or more modifications in the displacement region of the polynucleotide aptamer switch comprise: Hoogsteen and Wobble base-pairing, non-Watson and Crick base pairing, Z / P base pairing, incorporation of 2-aminopurine, substitution of adenine with 2-aminopurine, a duplex stabilizing modification, a duplex de-stabilizing modification, incorporation or substitution of PNA, incorporation or substitution of 5-hydroxybutynl-2’-deoxyuridine, or combinations thereof.

85. The polynucleotide of any one of the preceding claims, wherein the one or more modifications comprising incorporation or substitution of one or more sugar moiety modification(s) in the polynucleotide aptamer switch comprise: incorporation or substitution of a non-naturally occurring sugar or non- naturally occurring polymer into the polynucleotide aptamer switch, incorporation or substitution of four-carbon threose sugars, incorporation or substitution of peptide nucleic acids (PNAs), or combinations thereof.

86. The polynucleotide of any one of the preceding claims, wherein the polynucleotide aptamer switch is an aptamer beacon switch, or wherein the polynucleotide aptamer switch is intramolecular strand displacement, or wherein the polynucleotide aptamer switch is an anchored displacement strand switch.

87. The polynucleotide of any one of the preceding claims, wherein the one or more labels comprise a fluorophore and quencher, a redox reporter, or an electrochemical label.

88. The polynucleotide of any one of the preceding claims, wherein the fluorophore and quencher comprises an acceptor fluorophore and donor fluorophore, wherein contacting the polynucleotide aptamer switch with the target analyte comprises inducing a ratiometric FRET readout.

89. The polynucleotide of any one of the preceding claims, wherein the polynucleotide aptamer switch is comprised in a device comprising one or more members which are configured to penetrate the skin of a subject, wherein contacting the polynucleotide aptamer switch with the target analyte at least partially occurs by contacting an interstitial fluid of the subject.

90. The polynucleotide of any one of the preceding claims, wherein the partially complementary strand comprises a linker.

91. The polynucleotide of claim 90, wherein the linker is a poly-dT linker.

92. The polynucleotide of claim 91, wherein the poly-dT linker comprises at least 5, at least 10, at least 15, at least 20, or at least 25 nucleotides.

93. The polynucleotide of any one of claims 90-92, wherein the linker comprises a nonnatural polymer linker.

94. The polynucleotide of claim 93, wherein the non-natural polymer linker comprises at least 1, at least 2, at least 5, or at least 10 hexa-ethyleneglycol repeats.

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