Profiling chemically modified DNA/RNA units for disease and cancer diagnosis
View Patent ↗The present invention relates to high-throughput methods comprising direct infusion electrospray ionization mass spectrometry (ESI-MS), multistep tandem mass spectrometry (MS n ), consecutive reaction monitoring (CRM), ion mobility spectrometry mass spectrometry (IMS-MS), high-resolution MS, and IMS-MS, for genome-wide (whole cell or tissue) profiling of DNA and RNA nucleotides/nucleosides having a wide variety of variant structural modifications. In particular, these methods are contemplated for providing a specific profile of variant DNA and/or RNA chemically modified nucleic acids (i.e. structures) associated with specific medical conditions. Medical conditions may include, but are not limited to: cancer; including prostate, lung, uterus, larynx, ovary, breast, kidney, and many other types of cancers; specific stages of cancer; bacterial infections; viral infections; genetic and metabolic disorders; and any condition involving changes in DNA and/or RNA structural modifications.
1. A method for identifying a whole cell with a modified nucleic acid structure profile, comprising:
(a) providing,
(i) a biological sample comprising a whole-cell, said whole-cell comprising nucleic acids; and
(ii) a mass spectrometer;
(b) disrupting said whole-cell in a mixture comprising a denaturation solution and an affinity capture media under conditions that form an affinity-captured individual nucleic acid;
(c) eluting said individual nucleic acid from said affinity-captured individual nucleic acid to create a mononucleotide mixture;
(d) infusing said mononucleotide mixture into said mass spectrometer to measure a molecular mass of said individual nucleic acid;
(e) identifying the presence of a modified nucleic acid structure of said nucleic acid based upon said measured molecular weight;
(f) repeating steps c-e so as to identify a genome-wide modified nucleotide structure profile for said whole-cell; and
(g) identifying said whole-cell with said genome-wide modified nucleotide structure profile.
2. The method of claim 1 , wherein said mononucleotide is a ribonucleotide (RNA).
3. The method of claim 1 , wherein said mononucleotide is a deoxyribonucleotide (DNA).
4. The method of claim 1 , wherein said whole-cell is a mammalian cell.
5. The method of claim 1 , wherein said whole-cell is any type of cell or microorganism.
6. The method of claim 1 , wherein said identified whole-cell is selected from the group consisting of a single cell microorganism, a control cell, a healthy cell, a cancer cell, an infected cell and a stressed cell.
7. The method of claim 1 , wherein said mass spectrometer comprises ion mobility spectrometry-mass spectrometry (IMS-MS) and/or high-resolution mass spectrometry.
8. The method of claim 7 , wherein a heat-map plot is derived from said mass spectrometry then used to identify an isobaric modified nucleic acid for including in said profile.
9. The method of claim 1 , wherein said affinity capture media is selected from the group consisting of biotin-streptavidin coupling, iminothiolane coupling, disulfide coupling, poly-A coupling and antisense coupling.
10. The method of claim 1 , wherein said affinity capture media comprises a plurality of beads.
11. The method of claim 10 , wherein said plurality of beads is selected from the group consisting of glass beads, magnetic beads and paramagnetic beads.
12. The method of claim 1 , further comprising contacting said mononucleotide mixture with an immunoassay to identify said modified nucleic acid structure profile.
13. The method of claim 1 , wherein said modified nucleic acid structure comprises a post-transcriptional modification.
14. A method for identifying a whole cell with a modified nucleic acid structure profile, comprising:
(a) disrupting a whole-cell comprising nucleic acids in a mixture comprising a denaturation solution and an affinity capture media under conditions that form an affinity-captured individual nucleic acid;
(b) forming a mononucleotide mixture by eluting an individual nucleic acid from said affinity-captured individual nucleic acid;
(c) infusing said mononucleotide mixture into a mass spectrometer to measure a molecular mass of said individual nucleic acid;
(d) identifying the presence of a modified nucleic acid structure of said nucleic acid based upon said measured molecular weight;
(e) repeating steps b-d so as to identify a genome-wide modified nucleotide structure profile for said whole-cell; and
(f) identifying said whole-cell with said genome-wide modified nucleotide structure profile.
15. The method of claim 14 , wherein said mononucleotide mixture comprises nucleotides comprising ribose sugar.
16. The method of claim 14 , wherein said mononucleotide mixture comprises nucleotides comprising a sugar moiety consisting of ribose sugar.
17. The method of claim 14 , wherein said mononucleotide mixture comprises nucleotides comprising deoxyribose sugar.
18. The method of claim 14 , wherein said mononucleotide mixture comprises nucleotides comprising a sugar moiety consisting of deoxyribose sugar.
19. The method of claim 14 , wherein said whole-cell is a mammalian cell.
20. A method for identifying a whole cell with a modified nucleic acid structure profile, comprising:
(a) disrupting a whole-cell comprising nucleic acids in a mixture comprising a denaturation solution and an affinity capture media comprising a plurality of beads under conditions that form an affinity-captured individual nucleic acid;
(b) forming a mononucleotide mixture by eluting an individual nucleic acid from said affinity-captured individual nucleic acid;
(c) infusing said mononucleotide mixture into a mass spectrometer to measure a molecular mass of said individual nucleic acid, wherein said mass spectrometer comprises ion mobility spectrometry-mass spectrometry (IMS-MS) and/or high-resolution mass spectrometry;
(d) identifying the presence of a modified nucleic acid structure of said nucleic acid based upon said measured molecular weight;
(e) repeating steps b-d so as to identify a genome-wide modified nucleotide structure profile for said whole-cell; and
(f) identifying said whole-cell with said genome-wide modified nucleotide structure profile, wherein said identified whole-cell is selected from the group consisting of a single cell microorganism, a control cell, a healthy cell, a cancer cell, an infected cell, and a stressed cell.