COMPOSITIONS AND METHODS INVOLVING APTAMER SWITCH POLYNUCLEOTIDES
The disclosure provides aptamer switch polynucleotides whose kinetics and effective binding affinity to a target analyte can be independently tuned. The aptamer switch polynucleotides comprise an aptamer, an intramolecular linker, and a displacement strand.
1 . An aptamer switch polynucleotide comprising:
an aptamer, wherein the aptamer is linked to a first label at a first terminus of the aptamer;
a polynucleotide linker, wherein a first terminus of the polynucleotide linker is linked to a second terminus of the aptamer; and
a displacement strand, wherein a first terminus of the displacement strand is linked to a second terminus of the polynucleotide linker, wherein a second terminus of the displacement strand is linked to a second label, and wherein the displacement strand is at least partially complementary to a portion of the aptamer,
wherein in the concentration of a target analyte causes a shift in equilibrium between a reporting state and a non-reporting state of the aptamer switch polynucleotide, and
wherein one of the first and second labels produces a detectable readout in the reporting state of the aptamer switch polynucleotide.
2 . The aptamer switch polynucleotide of claim 1 , wherein in the absence of the target analyte, the displacement strand hybridizes to the portion of the aptamer.
3 . The aptamer switch polynucleotide of claim 1 , wherein in the presence of the target analyte, the aptamer binds to the target analyte, the displacement strand does not hybridize to the portion of the aptamer.
4 . The aptamer switch polynucleotide of claim 1 , wherein one of the first and second labels is a fluorophore and the other of the first and second labels is a quencher.
5 The aptamer switch polynucleotide of claim 4 , wherein the first label is a fluorophore and the second label is a quencher.
6 . The aptamer switch polynucleotide of claim 4 , wherein the first label is a quencher and the second label is a fluorophore.
7 . The aptamer switch polynucleotide of claim 4 , wherein the quencher quenches the fluorescence from the fluorophore in the non-reporting state of the aptamer switch polynucleotide.
8 . The aptamer switch polynucleotide of claim 4 , wherein the fluorophore produces fluorescence as a detectable readout in the reporting state of the aptamer switch polynucleotide.
9 . The aptamer switch polynucleotide of claim 1 , wherein the polynucleotide linker is a homopolymeric polynucleotide.
10 . The aptamer switch polynucleotide of claim 9 , wherein the polynucleotide linker is a poly-thymine polynucleotide.
11 . The aptamer switch polynucleotide of claim 1 , wherein the displacement strand is between 60% and 100% complementary to the portion of the aptamer.
12 . The aptamer switch polynucleotide of claim 11 , wherein the displacement strand is between 80% and 100% complementary to the portion of the aptamer.
13 . The aptamer switch polynucleotide of claim 12 , wherein the displacement strand is between 95% and 100% complementary to the portion of the aptamer.
14 . The aptamer switch polynucleotide of claim 1 , wherein the displacement strand comprises 1 or 2 mismatched nucleotides.
15 . The aptamer switch polynucleotide of claim 1 , wherein the displacement strand is between 3 and 15 nucleotides long.
16 . The aptamer switch polynucleotide of claim 1 , wherein the displacement strand is at least partially complementary to an end or internal sequence of the aptamer.
17 . The aptamer switch polynucleotide of claim 1 , wherein the polynucleotide linker is between 10 and 100 nucleotides long.
18 . The aptamer switch polynucleotide of claim 1 , wherein the aptamer switch polynucleotide comprises natural and/or non-natural nucleotides.
19 . The aptamer switch polynucleotide of claim 18 , wherein the natural and/or non-natural nucleotides are natural and/or non-natural DNA and/or RNA nucleotides.