Method and composition for measurement of nitric oxide
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.
1 . A nucleic acid complex comprising:
a first single-stranded nucleic acid molecule comprising a fluorophore crosslinked to the first single-stranded nucleic acid molecule, wherein the fluorophore comprises diaminorhodamine-4-methylamine (DAR-4M) conjugated to a dibenzocyclooctyne-polyethylene glycol (DBCO-PEG n ) 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, wherein the first label is capable of producing a signal, and wherein the intensity of the signal is at least dependent on concentration of the nucleic acid complex in the sample.
2 . The nucleic acid complex of claim 1 , wherein n=10.
3 . The nucleic acid complex of claim 1 , wherein the first label is a fluorophore.
4 . The nucleic acid complex of claim 1 , wherein the first label is Alexa488 or Alexa647.
5 . The nucleic acid complex of claim 1 , wherein the first label is crosslinked to the second single-stranded nucleic acid molecule.
6 . The nucleic acid complex of claim 1 , wherein the targeting moiety is crosslinked to the second single-stranded nucleic acid molecule.
7 . The nucleic acid complex of claim 1 , wherein the targeting moiety comprises a cholesterol moiety, a DNA aptamer, oligodeoxynucleotide phosphorothioate (ODP), or oligoribonucleotide phosphorothioate (ORP).
8 . The nucleic acid complex of claim 1 , wherein the first label and the targeting moiety are crosslinked to the second single-stranded nucleic acid molecule.
9 . The nucleic acid complex of claim 1 , wherein the first label is Alexa488 and the targeting moietylabel is a cholesterol moiety.
10 . The nucleic acid complex of claim 1 , wherein the first label is Alexa647 and the targeting moiety is a DNA aptamer.
11 . The nucleic acid complex of claim 1 , wherein the first single-stranded nucleic acid molecule has the following sequence:
(SEQ ID NO: 01)
5′-/5AzideN/ATC AAC ACT GCA CAC CAG ACA GCA-3′.
12 . The nucleic acid complex of claim 1 , wherein the second single-stranded nucleic acid molecule has the following sequence:
(SEQ ID NO: 02)
5′-/Alexa488N/TGC TGT CTG GTG TGC AGT GTT GAT/
3-CholTEG/-3′
or
(SEQ ID NO: 03)
5′-GGC TAT AGC ACA TGG GTA AAA CGA CTT TGC T/
Alexa647N/G TCT GGT GTG CAG TGT TGA T-3′.
13 . The nucleic acid complex of claim 1 , wherein the first label is ATTO647N and the targeting moiety is oligodeoxynucleotide phosphorothioate (ODP), or oligoribonucleotide phosphorothioate (ORP).
14 . The nucleic acid complex of claim 13 , wherein the first single-stranded nucleic acid molecule has the following sequence:
(SEQ ID NO: 04)
5′-/DAR-PEG10/ATC AAC ACT GCA CAC CAG ACA GCA-3′.
15 . The nucleic acid complex of claim 14 , wherein the second single-stranded nucleic acid molecule has the following sequence:
(S2-strand)
(SEQ ID NO: 05)
5′-/ATTO647N/TGC TGT CTG GTG TGC AGT GTT GAT-3′;
(NOckout1826CpG)
(SEQ ID NO: 06)
5′-/ATTO647N/TGC TGT CTG GTG TGC AGT GTT GAT
tttccatgacgttcctgacgtt-3′;
(NOckout1826GC)
(SEQ ID NO: 07)
5′-/ATTO647N/TGC TGT CTG GTG TGC AGT GTT GAT
tttccatgagcttcctgacctt-3′;
(NOckout2007CpG)
(SEQ ID NO: 08)
5′-/ATTO647N/TGC TGT CTG GTG TGC AGT GTT GAT
tttcgtcgttgtcgttttgtcgtt-3′;
(NOckout2007GC)
(SEQ ID NO: 09)
5′-/ATTO647N/TGC TGT CTG GTG TGC AGT GTT GAT
tttgctgctgtgcttttgtgctt-3′;
(NOckoutRNA);
(SEQ ID NO: 10)
5′-/ATTO647N/TGC TGT CTG GTG TGC AGT GTT GAT
tttggacggaaaagaccccgugg-3′
(NOckoutRNA)
(SEQ ID NO: 11)
5′-/ATTO647N/TGC TGT CTG GTG TGC AGT GTT GAT
tttggacgggaagaccccgugg-3′;
or
(pHlicker-SH)
(SEQ ID NO: 12)
5′-HS-ATC AAC ACT GCA CAC CAG ACA GCA-3′.
16 . A method for determining nitric oxide concentration in a sample comprising:
a) contacting the sample with the nucleic acid complex according to claim 13 ;
b) measuring the intensity of the signal; and
c) determining the nitric oxide concentration from the measured signal.
17 . The method of claim 16 , wherein determining nitric oxide concentration is in plasma membrane, trans Golgi network, phagosome, macrophage, or microglia.