Method and composition for measurement of nitric oxide

Inventors

Krishnan, Yamuna • Veetil, Aneesh T. • Jani, Maulik S.

Assignees

University of Chicago

Abstract

A method for determining nitric oxide concentration in biological samples. The method includes for determining nitric oxide concentration in a sample including: (i) providing a nucleic acid complex comprising a first single-stranded nucleic acid molecule comprising a fluorophore crosslinked to the first strand, the fluorophore comprising diaminorhodamine-4-methylamine (DAR-4M) conjugated, to dibenzocyclooctyne-polyethylene glycol (DBCO-PEGn) linker, wherein n equals 4-12 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 and a targeting moiety conjugated to the first single-stranded nucleic acid molecule or the second single-stranded nucleic acid molecule, the first label is capable of producing a signal, wherein the intensity of the signal is dependent at least on concentration of the nucleic acid complex in the sample; (ii) contacting the sample with the nucleic acid complex; (iii) measuring the intensity of the signal: and (iii) determining the nitric oxide concentration from the measured signal. Compositions for determining nitric oxide concentrations in biological samples are also included.

Core Innovation

The disclosure describes a DNA duplex “NOckout” nucleic-acid complex for determining nitric oxide (NO) concentration in a sample. The complex includes a first single-stranded nucleic acid bearing a fluorophore crosslinked to the strand, where the fluorophore comprises diaminorhodamine-4-methylamine (DAR-4M) conjugated to a dibenzocyclooctyne-polyethylene glycol (DBCO-PEGn) linker with n equal to 4–12, and a second single-stranded nucleic acid that is partially or fully complementary to the first single-stranded nucleic acid.

The nucleic acid complex further includes a first label capable of producing a signal and a targeting moiety conjugated to the first single-stranded nucleic acid molecule or the second single-stranded nucleic acid molecule. The intensity of the signal is at least dependent on the concentration of the nucleic acid complex in the sample, and the disclosure frames the probe as ratiometric by using a normalizing fluorophore such as Alexa488 or Alexa647.

The disclosed method determines nitric oxide concentration in biological samples by contacting the sample with the nucleic acid complex, measuring signal intensity, and determining nitric oxide concentration from the measured signal. Embodiments include targeting particular cellular or compartment locations such as plasma membrane, trans-Golgi network (TGN), phagosome, macrophages, or microglia, including embodiments described as “NOckout PM/TGN.”

The disclosed work enables in-cell calibration of ratiometric NO sensing and supports subcellular, pH-insensitive nitric oxide detection with modular, targetable sensing. Further described applications include mapping NOS3 localization and NOS3-dependent S-nitrosylation, as well as modular trigger and detect concepts for NOS2-derived NO in vivo, including phagosomal NO and TLR-associated activation described for mCpG/zCpG, iRNA, and ssRNA/iRNA-mediated NOS2 activation via zebrafish TLR7.

Claims Coverage

The independent claim coverage includes 3 core features: a nucleic acid complex with specific fluorophore, label, linker, and targeting features; a complementary second strand; and a method for determining nitric oxide concentration from a measured signal after contacting a sample with the nucleic acid complex.

The claims center on a DAR-4M/DBCO-PEGn labeled nucleic acid complex with a complementary strand and a first label whose signal intensity depends on complex concentration in a sample, combined with a targeting moiety on one or both strands. The method claim determines NO concentration by contacting a sample with the complex, measuring signal intensity, and determining nitric oxide concentration from the measured signal.

Stated Advantages

Documented Applications