Granted Patent B2

US 12,436,156 · App. 17/295,704 · Granted Oct 7, 2025

Inventors: Yamuna Krishnan (Chicago, IL); Kasturi Chakraborty (Chicago, IL); Ka Ho Leung (Chicago, IL); Anand Saminathan (Chicago, IL)

Assignee: THE UNIVERSITY OF CHICAGO

G01N33/84C12Q1/6825G01N33/582

Abstract

Claims (37)

1. A method for simultaneously determining 1) pH, and 2) Ca 2+ concentration or Cl − concentration in a sample comprising:

providing a nucleic acid complex comprising

a first single-stranded nucleic acid molecule comprising a Ca 2+ fluorophore or a Cl − fluorophore crosslinked to the first strand; and

a second single-stranded nucleic acid molecule that is partially or fully complementary to the first single-stranded molecule,

wherein the nucleic acid complex further comprises a first label conjugated to the first single-stranded nucleic acid molecule or the second single-stranded nucleic acid molecule and the first label is capable of producing a signal, wherein the intensity of the signal is dependent on change in pH;

measuring the intensity of the signal; and

determining the pH, and Ca 2+ or Cl − concentration from the measured signal by comparing to a reference value.

2. The method of claim 1 , wherein determining is in early endosome, late endosome, plasma membrane, lysosome, autophagolysosome, recycling endosome, cis Golgi network, trans Golgi network, endoplasmic reticulum, peroxisomes, or secretory vesicles.

3. The method of claim 1 , wherein the nucleic acid complex comprises the Cl − fluorophore crosslinked to the first strand.

4. The method of claim 3 , wherein the Cl − fluorophore comprises 10,10′-bis [3-carboxypropyl]-9,9′-biacridinium dinitrate.

5. The method of claim 1 , wherein the nucleic acid complex comprises the Ca 2+ fluorophore crosslinked to the first strand.

6. The method of claim 5 , wherein the Ca 2+ fluorophore is a single wavelength indicator.

7. The method of claim 5 , wherein the crosslinked Ca 2+ fluorophore is:

or

wherein the first single-stranded nucleic acid molecule comprising a Ca 2+ fluorophore is of formula:

wherein R is a linker.

8. The method of claim 1 , wherein the intensity of the signal dependent on change in pH varies as a function of the conformation of the nucleic acid complex.

9. The method of claim 1 , wherein the nucleic acid further comprises a second label conjugated to the first single-stranded nucleic acid or the second single-stranded nucleic acid.

10. The method of claim 9 , wherein the intensity of the signal varies as a function of at least one of the distance between the first label and second label and the relative orientation of the first label and second label.

11. The method of claim 1 , wherein the first single-stranded nucleic acid molecule and second single-stranded nucleic acid form an i-motif under acidic conditions.

12. The method of claim 1 , wherein the second single-stranded nucleic acid is capable of forming an intramolecular complex comprising two parallel-stranded C—HC+ base paired duplexes that are intercalated in an anti-parallel orientation at acidic conditions.

13. The method of claim 1 , wherein the nucleic acid complex further comprises a third single-stranded nucleic acid molecule that is partially complementary to the first single-stranded molecule.

14. The method of claim 1 , wherein the nucleic acid complex further comprises a targeting moiety.

15. The method of claim 1 , wherein the first and/or second single-stranded nucleic acid molecule is less than 200 nucleotides; or less than 100 nucleotides; or less than 50 nucleotides.

16. The method of claim 1 , wherein the determined Ca 2+ concentration is in a range of 10 nM to 10 mM; or in a range of 10 nM to 1 μM; or in a range of 1 μM to 10 mM; or wherein the determined Cl − concentration is in a range of 10 nM to 10 mM; or in a range of 10 nM to 1 μM; or in a range of 1 μM to 10 mM.

17. The method of claim 1 , wherein the determined pH is less than pH 5.5; or wherein the determined pH is more than pH 7.0.

18. The method of claim 1 , wherein

(a) the first strain has the sequence 5′-the Ca 2+ fluorophore-GAC TCA CTG TTT GTC TGT CGT TCT AGG ATA/the second label/AT ATT TTG TTA TGT GTT ATG TGT TAT-3′ (SEQ ID NO: 07); and

the second strain has the sequence 5′-the first label-CCC CTA ACC CCT AAC CCC TAA CCC CAT ATA TAT CCT AGA ACG ACA GAC AAA CAG TGA GTC-3′ (SEQ ID NO:08);

(b) the first strain has the sequence 5′-TTA TAG GAT CCT GCG GTC GG/the Ca2+fluorophore/GGC ACC AGG CGT AAA ATG TA-3′ (SEQ ID NO:09);

the second strain has the sequence: 5′-the first label-CCC CAA CCC CAA TAC ATT TTA CGC CTG GTG CC-3′ (SEQ ID NO: 10); and

the third strain has the sequence: 5′-CCG ACC GCA GGA TCC TAT AAA ACC CCA ACC CC-the second label-3 (SEQ ID NO:11);

(c) the first strain has the sequence 5′-the Ca2+fluorophore-GAC TCA CTG TTT GTC TGT CGT TCT AGG ATA/the second label/AT ATT TTG TTA TGT GTT ATG TGT TAT-3′ (SEQ ID NO: 07); and

the second strain has the sequence 5′-the first label-AT AAC ACA TAA CAC ATAACAAAA TAT ATA TCC TAG AAC GAC AGA CAA ACA GTG AGT C-3′ (SEQ ID NO:12); or

(d) the first strain has the sequence 5′-the Cl − fluorophore-ATC AAC ACT GCA-Lys-COOH (SEQ ID NO:22);

the second strain has the sequence 5′-TAT TGT GTA TTG TGT ATT GTT TTA TAT AT/the first label/A TAG GAT CTT GCT GTC TGG TGT GCA GTG TTG AT-3′ (SEQ ID NO:23); and

the third strain has the sequence: 5′-CAC CAG ACA GCA AGA TCC TAT ATA TAT ACC CCA ATC CCC AAT CCC CAA TCC CC-the second label-3′ (SEQ ID NO:24).

Assignments (2)

Continuity (2)

References Cited (258)