Modified nucleotides methods and kits
Modified nucleotides, and methods to modify nucleotides with a moiety or label, such as biotin, that permits their detection and results in a modified nucleotide, and methods of use of the modified nucleotide in quantitative and qualitative assays.
1. A compound having the structure (II):
or a salt, conjugate base, tautomer, or ionized form thereof, where
Base* is a purine or pyrimidine base;
R is H, OH, CH 3 , or a hydroxyl protecting group;
Alk is a connecting group having the structure -//—(CH 2 ) m —Y—//- wherein m is an integer ranging from 3 to 6 inclusive, Y is a bond or bond forming group selected from
and
the leftmost bond is to Base* and the rightmost bond is to Lnk;
Lnk is a linking group having the structure
where n is an integer ranging from 2 to 48 inclusive,
A 1 is a bond forming group selected from
A 2 is a bond forming group selected from
A 3 when present is a bond forming group selected from
X is a cleavable group that can undergo silicon-carbon cleavage, nucleophilic cleavage, redox cleavage, photochemical cleavage, enzymatic cleavage, or exchange-based cleavage;
Z is a branching group that contains a modifying molecule (Mod); and
the leftmost bond is to Alk and the rightmost bond is to Obs; and
Obs is an observable label moiety.
2. The compound of claim 1 where the sugar is ribose; the purine or pyrimidine base is cytosine (C); m is 3; Lnk is
where
n is 4, A 1 is
A 2 is
when present Z is a branching group that contains a modifying molecule (Mod), and when present A 3 is
Mod is a modifier or modifying molecule, and
Obs is selected from the group consisting of biotin, a fluorophore, and an azide.
3. The compound of claim 1 where the sugar is ribose, and the purine or pyrimidine base is selected from cytosine (C), uracil (U), adenine (A), guanine (G), or inosine (I).
4. The compound of claim 3 where the purine or pyridine base is selected from 1-methyladenine, N 6 -methyladenine, N 6 -isopentyladenine, N,N-dimethyladenine, 7-deazaadenine, 2-thiocytosine, 3-methylcytosine, N 4 -acetylcytosine, 2-thiocytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, N 2 ,N 2 -dimethylguanine, 7-deazaguanine, 2-thiouracil, 6-thiopurine, or 2,6-diaminopurine.
5. The compound of claim 1 where the observable label is a chromogen, a fluorophore, a mass label, a spin label, a streptavidin-binding label, or a secondary detection label.
6. The compound of claim 1 where n is an integer selected from 2 to 24 inclusive.
7. The compound of claim 1 where the sugar is ribose; the purine or pyrimidine base is selected from adenine (A), cytosine (C), guanine (G), uracil U), or inosine (I); m is 3; n is 4; and the observable label is a streptavidin-binding compound selected from biotin, desthiobiotin, or iminobiotin.
8. The compound of claim 1 where the sugar is ribose; the purine or pyrimidine base is cytosine (C); m is 3; Lnk is
where
n is 4, A 1 is
A 2 is
and when present, A 3 is
and
Z is a branching group that contains a modifying molecule (Mod), and Obs is selected from the group consisting of biotin, a fluorophore, and an azide.
9. The compound of claim 1 including a salt, conjugate base, tautomer, and/or ionized form of the compound.
10. A method for enriching for an RNA-binding protein or RNA binding complex, the method comprising
labeling an RNA with the compound of claim 1 by incubating under suitable conditions the RNA and the compound of claim 1 with an enzyme capable of ligating the compound of claim 1 to the RNA molecule to result in a labeled RNA,
contacting a protein source that contains at least one RNA-binding protein or RNA binding complex with the labeled RNA under conditions suitable for forming a complex between the labeled RNA and the RNA-binding protein, and
enriching for the RNA-binding protein or RNA binding complex utilizing the labeled RNA as bait.
11. The method of claim 10 where the RNA is labeled by contacting the RNA with an excess of the compound.
12. The method of claim 11 further comprising a step of including a crosslinking agent to the compound.
13. The method of claim 11 where the affinity handle is selected from at least one of biotin-PEG 4 -alkane-3′5′-bisphosphate cytidine; desbiotin-PEG 4 -alkane-3′5′-bisphosphate cytidine, biotin-PEG 12 -alkane-3′5′-bisphosphate cytidine; desbiotin-PEG 12 -alkane-3′5′-bisphosphate cytidine; azido-PEG 4 -alkane-3′5′-bisphosphate cytidine; azido-PEG 12 -alkane-3′5′-bisphosphate cytidine; TMT-PEG 4 -alkane-3′5′-bisphosphate cytidine; and/or TMT-PEG 12 -alkane-3′5′-bisphosphate cytidine.
14. The method of claim 10 where the protein source is at least one of cell lysate, tissue lysate, exogenously expressed protein in cell or tissue lysates, protein expressed in an in vitro transcription and/or translation system, and/or protein purified form bacteria, yeast, fungi, tissue, or mammalian cells.
15. The method of claim 14 where the labeled RNA is contacted with the protein lysate and affinity resin or matrix in an optimized binding buffer.
16. The method of claim 15 further comprising washing, eluting, and detecting the RNA protein complex.
17. The method of claim 16 where detection is by Western blot, array, and/or mass spectrometry.
18. The method of claim 10 wherein the labeled RNA contains a crosslinker within the ligated compound, the compound attached to the RNA using T4 RNA ligase.
19. The method of claim 10 wherein the labeled RNA contains a crosslinker cleavable within the ligated compound that is attached to the RNA using T4 RNA ligase.
20. The method of claim 10 further comprising a step utilizing a crosslinking reagent.
21. The method of claim 10 using an affinity matrix to enrich for the RNA-binding protein or RNA binding complex, where the affinity matrix is streptavidin agarose resin, streptavidin magnetic breads, hydrazide resin, alkoxyamine resin, modified agarose resin, soft-release streptavidin magnetic beads, soft-release agarose resin, and/or resin coupled with at least one anti-tandem mass tag (TMT) antibody.
22. A kit for enriching an RNA binding protein or RNA binding protein complex, the kit comprising the compound of claim 1 and reagents for labeling an RNA with the compound of claim 1 .
23. The kit of claim 22 further comprising at least one control comprising labeled RNA and at least one control comprising unlabeled RNA to assess RNA ligation and/or binding affinity.
24. The kit of claim 22 further comprising the compound of claim 7 , and instructions for labeling the RNA, resulting in a labeled RNA with a crosslinker, such that the labeled RNA contains an affinity handle and crosslinking reagent.
25. The kit of claim 22 further comprising reagents for capture and detection of RNA-protein interactions.
26. A compound selected from the group consisting of