Oligonucleotide-based probes for detection of bacterial nucleases
The present invention relates to a rapid detection of microbial-associated nuclease activity with chemically modified nuclease (e.g., ribonuclease) substrates, and probes and compositions useful in detection assays.
1. A probe for detecting a microbial endonuclease comprising an oligonucleotide of 2-30 nucleotides in length, a fluorophore operably linked to the oligonucleotide, and a quencher operably linked to the oligonucleotide, wherein the oligonucleotide comprises a chemically modified purine comprising a 2′ substituted sugar and is capable of being cleaved by a microbial nuclease but not by a mammalian nuclease.
2. The probe of claim 1 , wherein the oligonucleotide is 10-15 nucleotides in length.
3. The probe of claim 1 , wherein one or more of the chemically modified purines comprises a sugar selected from the group consisting of 2′-O-methyl-ribose, 2′-O-alkyl-ribose, 2′-O-allyl-ribose, 2′-S-alkyl-ribose, 2′-S-allyl-ribose, 2′-fluoro-ribose, 2′-halo-ribose, 2′-azido-ribose, carbocyclic sugar analogue, a-anomeric sugar, epimeric sugar, arabinose, xylose, lyxose, pyranose sugar, furanose sugar, and sedoheptulose, or is a 2′-O-methyl ribonucleotide, a 2′-methoxyethoxy ribonucleotide, a 2′-O-allyl ribonucleotide, a2′-O-pentyl ribonucleotide, a 2′-O-butyl ribonucleotide, 2′-fluoro-β-D-arabinonucleotide (FANA), Locked Nucleic Acid (LNA), or Unlocked Nucleic Acid (UNA).
4. The probe of claim 1 , wherein the fluorophore is selected from the group consisting of 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, and IRDye 800 CW.
5. The probe of claim 1 , wherein the quencher is selected from the group consisting of DDQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2, and DDQ-II.
6. The probe of claim 1 , wherein the oligonucleotide is single-stranded.
7. The probe of claim 1 , wherein the oligonucleotide comprises both RNA and DNA.
8. The probe of claim 7 , wherein the oligonucleotide comprises a DNA di-nucleotide.
9. A method of detecting a microbial infection of a sample comprising measuring fluorescence of a sample that has been contacted with a probe of claim 1 , wherein a fluorescence level that is greater than the fluorescence level of an uninfected control indicates that the sample has a microbial infection.
10. The method of claim 9 , wherein the method is performed in vivo for the detection of a microbial infection in a mammal, wherein a test fluorescence of potentially infected tissue that is greater than the fluorescence level of control tissue indicates that the sample has a microbial infection.
11. The method of claim 9 , wherein the test fluorescence level is at least 1-100% greater than the control level.
12. The method of claim 9 , wherein the fluorophore is detectable at a depth of 7-14 cm in the mammal.