IP Library › Granted Patent US 12,378,612
Granted Patent B2
US 12,378,612 · App. 17/494,626 · Granted Aug 5, 2025

Oligonucleotide-based probes and methods for detection of microbes

Inventors: James O. McNamara (Iowa City, IA); Katie R. Flenker (Iowa City, IA); Hyeon Kim (Iowa City, IA); Alexander R. Horswill (Iowa City, IA); Frank J. Hernandez (Iowa City, IA); Mark Behlke (Coralville, IA); Lingyan Huang (Coralville, IA); Richard Owczarzy (Coralville, IA); Elliot Burghardt (Iowa City, IA); Karen Clark (Iowa City, IA)
Assignee: UNIVERSITY OF IOWA RESEARCH FOUNDATION
C12Q1/689A61K49/0054C07H21/02C07H21/04C12Q1/701G01N21/6428G01N33/581G01N2021/6432
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Quick Facts
Patent No.
US 12,378,612
App. No.
17/494,626
Granted
Aug 5, 2025
Kind
B2
Abstract

The present invention relates to a rapid detection of microbial-associated nuclease activity with chemically modified nuclease (e.g., endonuclease) substrates, and probes and compositions useful in detection assays.

Claims (31)

1. A method of detecting endonuclease activity in a test sample, comprising:

(a) contacting the test sample with a probe for detecting a microbial endonuclease comprising an oligonucleotide, a fluorophore operably linked to the oligonucleotide, and a quencher operably linked to the oligonucleotide, wherein the oligonucleotide comprises one or more modified nucleotides, is capable of being cleaved by a microbial nuclease, and has a DNA TT di-nucleotide, DNA AT di-nucleotide, DNA AA di-nucleotide or DNA TA di-nucleotide to form a digested probe, and

(b) measuring the fluorescence emitted by the digested probe.

2. The method of claim 1 , wherein the at least one fluorophore is selected from the group consisting of the fluorophores Hydroxycoumarin, Alexa fluor, Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer yellow, Alexa fluor 430, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, Cy2, TruRed, FluorX, Fluorescein, FAM, BODIPY-FL, TET, Alexa fluor 532, HEX, TRITC, Cy3, TMR, Alexa fluor 546, Alexa fluor 555, Tamara, X-Rhodamine, Lissamine Rhodamine B, ROX, Alexa fluor 568, Cy3.5 581, Texas Red, Alexa fluor 594, Alexa fluor 633, LC red 640, Allophycocyanin (APC), Alexa fluor 633, APC-Cy7 conjugates, Cy5, Alexa fluor 660, Cy5.5, LC red 705, Alexa fluor 680, Cy7, IRDye 800 CW, JOE, MAX, TAMRA, Licor IRDye 700, Cy7.5, Dy780, Dy781, DyLight 800, Licor, or Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 750, and Alexa Fluor 790.

3. The method of claim 1 , wherein the at least one fluorescence quencher is selected from the group consisting of the fluorescence quenchers DDQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black RQ, QSY-21, BHQ-3, IRDye QC-1, ZEN, IBFQ, BHQ1, BHQ2, IBRQ, ZEN and Licor IRDye QC-1.

4. The method of claim 1 , wherein the DNA di-nucleotide is positioned at nucleotides 2 and 3 as measured from the 5′-end of the oligonucleotide.

5. The method of claim 1 , the DNA TT di-nucleotide is positioned at nucleotides 1 and 2 as measured from the 5′-end of the oligonucleotide.

6. The method of claim 1 , wherein the DNA TT di-nucleotide consists of unmodified deoxythymidines.

7. The method of claim 1 , wherein the oligonucleotide has between 0-50% purines.

8. The method of claim 1 , wherein the oligonucleotide comprises one or more modified pyrimidines, wherein one or more of the pyrimidines other than the DNA TT di-nucleotide, DNA AT di-nucleotide, DNA AA di-nucleotide or DNA TA di-nucleotide are chemically modified.

9. The method of claim 8 , wherein one or more of the pyrimidines are 2′-O-methyl modified or are 2′-fluoro modified.

10. The method of claim 1 , wherein the oligonucleotide comprises one or more purines, and wherein one or more of the purines are chemically modified.

11. The method of claim 10 , wherein one or more of the purines are 2′-O-methyl modified or are 2′-fluoro modified.

12. The method of claim 1 , wherein the oligonucleotide is single-stranded.

13. The method of claim 1 , wherein the oligonucleotide comprises both RNA and DNA.

14. The method of claim 1 , wherein the test sample comprises a biological sample.

15. The method of claim 14 , wherein the biological sample is a blood sample.

16. The method of claim 15 , wherein the blood sample is not subject to a culturing step.

17. The method of claim 1 , wherein the test sample comprises calcium chloride.

18. The method of claim 17 , wherein the calcium chloride is at a concentration of about 5 to 20 mM.

19. The method of claim 1 , wherein the test sample has been heated at 55-100° C. for 10 seconds to 20 hours to form a heat-treated test sample prior to testing.

20. The method of claim 19 , wherein the heat-treated test sample is clarified.

21. The method of claim 1 , wherein the endonuclease present in the test sample has been concentrated prior to testing.

22. The method of claim 21 , wherein the concentration is by means of an aptamer-mediated pull-down.

23. The method of claim 21 , wherein the concentration is by means of immunoprecipitation.

24. The method of claim 23 , wherein the immunoprecipitated endonuclease remains bound to an antibody used in the immunoprecipitation during contact with the probe.

25. The method of claim 23 , wherein the immunoprecipitation is by means of anti-micrococcal nuclease antibody-coupled magnetic beads.

26. The method of claim 25 , wherein the magnetic beads are Protein G-coupled magnetic beads.

27. The method of claim 23 , wherein the immunoprecipitation is specific for a particular microbe.

28. The method of claim 1 , wherein the microbial endonuclease is a Staphylococcus aureus or a Streptococcus pneumoniae endonuclease.

29. A method of detecting a microbial infection of a test sample comprising measuring fluorescence of a sample that has been contacted with a probe for detecting a microbial endonuclease comprising an oligonucleotide, a fluorophore operably linked to the oligonucleotide, and a quencher operably linked to the oligonucleotide, wherein the oligonucleotide is capable of being cleaved by a microbial nuclease, and has a DNA TT di-nucleotide, DNA AT di-nucleotide, DNA AA di-nucleotide or DNA TA di-nucleotide positioned at nucleotides 1 and 2, 2 and 3, or 3 and 4 as measured from the 5′-end of the oligonucleotide, wherein a fluorescence level that is greater than the fluorescence level of an uninfected control indicates that the sample has a microbial infection.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 6, 2021
From: MCNAMARA, JAMES O.; FLENKER, KATIE R.; KIM, HYEON; HORSWILL, ALEXANDER R.; HERNANDEZ, FRANK J.; BEHLKE, MARK; BURGHARDT, ELLIOT; HUANG, LINGYAN; OWCZARZY, RICHARD; CLARK, KAREN
To: UNIVERSITY OF IOWA RESEARCH FOUNDATION
Reel/Frame 057723/0862 →
Continuity (6)
Continuation 16794677 · Feb 19, 2020
Division 15117414
Provisional Application 61937359 · Feb 7, 2014
Provisional Application 61980498 · Apr 16, 2014
Provisional Application 61992034 · May 12, 2014
Related Publication 20220098648A1 · Mar 31, 2022
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