RNA target enrichment or depletion of biological samples
A method of target enrichment or depletion from a sample with an analyte is described. A probe has one of a left-handed PNA pair and a targeting moiety, in which the left-handed PNA pair are a complementary pair of PNAs that are chiral and have a cyclic backbone modification that induces a left-handed helical structure. A capture surface has the other of the left-handed PNA pair; and the left-handed PNA pair bind to hybridize the probe to the capture surface, which may be a magnetic bead.
1. A method of target enrichment in a sample, the method comprising the steps of:
introducing a probe comprising a left-handed PNA molecule linked to a capture moiety into a sample comprising target analyte;
capturing, with the capture moiety, the target analyte; and
binding the left-handed PNA molecule with a complementary PNA molecule comprising a left-handed chiral structure, the complementary PNA molecule being attached to a surface to thereby enrich for target analyte.
2. The method of claim 1 , wherein the surface comprises a bead.
3. The method of claim 2 , wherein the bead is a magnetic bead.
4. The method of claim 1 , wherein the surface comprises a fixed substrate.
5. The method of claim 1 , wherein the left-handed PNA molecule comprises a linker linking the left-handed PNA molecule to a targeting moiety.
6. The method of claim 1 , wherein the method further includes introducing a second probe into the sample, said second probe comprising a left-handed PNA, a targeting moiety, and a detection moiety.
7. The method of claim 6 , wherein the targeting moiety is a right-handed targeting PNA.
8. A method of claim 1 , wherein the capture moiety comprises an antibody or an antibody fragment.
9. The method of claim 1 , wherein the method is carried out at a temperature in the range of 18° C. to 80° C.
10. The method of claim 9 , wherein the method is carried out at a temperature of about 37° C.
11. The method of claim 1 , wherein the target analyte comprises RNA.
12. The method of claim 11 , wherein the RNA comprises 18s ribosomal RNA.
13. The method of claim 1 , wherein the surface comprises a surface of a bead, and said method further comprises after the binding step, heat the bead to provide a clean capture surface for re-use in a further assay.
14. The method of claim 1 , wherein the capture moiety comprises any one or more of an achiral or chiral PNA, a deoxyribonucleic acid, a ribonucleic acid, a glycol nucleic acid, a threose nucleic acid, a locked nucleic acid, a phosphorothioate oligonucleotide, a phosphorodiamidate morpholino oligomer, a protein, a peptide, a peptide or protein with unnatural amino acids, an enzyme, an antibody, a single domain antibody, an aptamer, a drug molecule, a small molecule, a chemical compound, a cell, or a combination of one or more of the above.
15. The method of claim 1 , wherein a detection moiety is linked with the left-handed PNA molecule, and the detection moiety includes one or more of the following: a fluorescent label, a luminescent label, a chromophore label, a chemiluminescent label, a radioactive label, an enzyme label, a visual label, a metallic label, a microsphere, a nanoparticle, a biotin label, an avidin label, a digoxigenin label, a cholesterol label, a dinitrophenyl label, a Horseradish peroxidase label, or a combination of one or more of the above.
16. The method of claim 1 , wherein the a left-handed PNA molecule comprises a linker, and the linker comprises a length of 1 to 120 atoms, and/or has one or some of the elements: C, N, O, S, P, and Si, and/or is in a chain that contains only one or a combination of the following bonds: a single bond, a double bond, a triple bond, an amide bond, an ester bond, a disulfide bond, an imino group, an ether bond, a thioether bond, and a thioester bond.