Conformationally Constrained Analytical Probes

• US 20080269065A1
• Filed: 08/06/2007
• Published: 10/30/2008
• Est. Priority Date: 04/30/2007
• Status: Abandoned Application

• Current Assignee: Syntrix Biosystems Incorporated
• Original Assignee: Syntrix Biosystems Incorporated
• Inventors: Zebala, John A., Lyon, Robert
• Application Number:
  US11/834,533
• Publication Number:
  US 20080269065A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

There is disclosed probes bearing partial metal chelators that in some embodiments are conformationally constrained. In certain embodiments such probes are useful in methods for the detection of a specific target molecule. These target molecules may include oligonucleotides, peptides, proteins, polysaccharides, or small molecules. There is further disclosed the use of probes with partial metal chelators engaged in a coordination complex with one another that imposes a structural constraint in the probe and increases the specificity factor of the probe.

Claims

1. A probe, comprising a flexible binding element, a first and a second partial metal chelator and a transition metal, wherein the transition metal atom is shared between the first and the second partial metal chelator moieties, and wherein the first and the second partial metal chelators are attached to one another through a portion of the flexible binding element, and wherein a specificity factor of the probe comprises a value selected from the group consisting of K_d, IC₅₀, K_m, k_a, k_d, log₂(PM/MM), T_m, T_d-50 and T_d-w.

2. The probe according to claim 1, wherein the first and second partial metal chelators are independently selected from the group consisting of

wherein R is the location of a covalent attachment to the probe.

3. The probe according to claim 1, further comprising a label, wherein the label is selected from the group consisting of fluorophores and quenchers, radioactive isotopes, antigenic determinants, non-radioactive isotopes, nucleic acids available for hybridization, altered fluorescence-polarization or altered light-scattering, chromogenic, chemiluminescent, electrochemically detectable, and combinations thereof.

4. The probe according to claim 1, wherein the flexible binding element comprises a segment selected from the group consisting of PNA, poly-morphilino, PNAMs, DNA, RNA, siRNA, peptide, oligosaccharide, and combinations thereof.

5. The probe according to claim 1, wherein the first and second partial metal chelators are the same.

6. The probe according to claim 1 wherein the transition metal is selected from the group consisting of zinc, cadmium, copper, nickel, ruthenium, platinum, palladium, cobalt, magnesium, barium, strontium, iron, vanadium, chromium, manganese, rhodium, silver, mercury, molybdenum, tungsten, calcium, lead, cerium, aluminum and thorium.

7. A coordination complex capable of disruption upon binding to a target, comprising a flexible binding element, a first and a second partial metal chelator and a transition metal, wherein the transition metal atom is shared between the first and the second partial metal chelator moieties, and wherein the first and the second partial metal chelators are attached to one another via a covalent bond through a portion of the flexible binding element, and wherein a specificity factor of the probe comprises a value selected from the group consisting of K_d, IC₅₀, K_m, k_a, k_d, log₂(PM/MM), T_m, T_d-50 and T_d-w.

8. The coordination complex according to claim 7, wherein the transition metal is selected from the group consisting of zinc, cadmium, copper, nickel, ruthenium, platinum, palladium, cobalt, magnesium, barium, strontium, iron, vanadium, chromium, manganese, rhodium, silver, mercury, molybdenum, tungsten, calcium, lead, cerium, aluminum and thorium.

9. The coordination complex of claim 7 wherein the first and second partial metal chelators are independently selected from the group consisting of

wherein R is the location of a covalent attachment to the probe.

10. A method for binding a target to a probe comprising:

a) providing a probe in a closed configuration, wherein the probe comprises a flexible binding element, a first and a second partial metal chelator and a transition metal, wherein the transition metal atom is shared between the first and the second partial metal chelator moieties, and wherein the first and the second partial metal chelators are attached to one another through a portion of the flexible binding element, and wherein a specificity factor of the probe comprises a value at least a great as the values selected from the group consisting of K_d, IC₅₀, K_m, k_a, k_d, log₂(PM/MM), T_m, T_d-50 and T_d-w; and

b) opening the closed configuration probe by contacting the probe with the target and the transition metal, wherein the target can only open the closed configuration probe with a high specificity factor.

11. The method according to claim 10, further comprising detecting the target bound to the probe.

12. The method according to claim 10, wherein the step of contacting occurs inside a cell.

13. The method according to claim 12, wherein the amount of an mRNA within the cell is reduced.

14. The method according to claim 12, wherein the amount of a protein within the cell is reduced.

15. The method according to claim 10 wherein the target is selected from the group consisting of DNA, RNA, mRNA, peptide, protein, oligosaccharide, and combinations thereof.

16. The method according to claim 10, wherein the probe is attached to a solid support.

17. The method according to claim 16, wherein the solid support is selected from the group consisting of a bead, glass slide, microarray, membrane, microtiter well and dipstick.

18. The method according to claim 10, wherein the transition metal is selected from the group consisting of zinc, cadmium, copper, nickel, ruthenium, platinum, palladium, cobalt, magnesium, barium, strontium, iron, vanadium, chromium, manganese, rhodium, silver, mercury, molybdenum, tungsten, calcium, lead, cerium, aluminum, thorium, and combinations thereof.

19. A method of claim 10, wherein the probe further comprises a label, wherein the label is selected from the group consisting of fluorophores and quenchers, radioactive isotopes, antigenic determinants, non-radioactive isotopes, nucleic acids available for hybridization, altered fluorescence-polarization or altered light-scattering, chromogenic, chemiluminescent, electrochemically detectable, and combinations thereof.

20. The method of claim 12, wherein the probe further comprises a spacer.

21. The method of claim 10, wherein the first and second partial metal chelators are independently selected from the group consisting of

wherein R is an attachment to the probe.

22. The method of claim 10, wherein the portion of the flexible binding element is selected from the group consisting of PNA, poly-morphilino, PNAMS, DNA, RNA, siRNA, peptide, oligosaccharide, and combinations thereof.

23. The method of claim 10, wherein the first and second partial metal chelators are the same.

24. A method of binding a target comprising:

a) providing a probe comprising a first and a second partial metal chelator, wherein the first and second partial metal chelators are covalently attached to one another through a portion of a flexible binding element, wherein the partial metal chelators form a coordination complex with a transition metal, and wherein the coordination complex is disrupted when the binding element contacts a target; and

b) contacting the probe with the target and a transition metal.

25. A partial metal chelator synthon comprising formula (I) or formula (II):

II

wherein

R₁ is a hydroxyl protecting group;

R₂ is selected from the group consisting of a linker;

and salts thereof,

R₃ and R₄ are carboxyl protecting groups;

R₅ and R₆ are independently selected from the group consisting of C_3-10 branched alkyl and C_1-12 unbranched alkyl, and cyclic hydrocarbons;

Y is beta-cyanoethyl;

G is selected from the group consisting of alkyl, heteroalkyl, aryl, aryl(alkylene), heteroaryl, heteroaryl(alkylene), carbocycle, carbocyle(alkylene), heterocycle, heterocycle(alkylene),

wherein n=1 to 10; and

X is from 0 to 10.

26. The partial metal chelator synthon according to claim 25, further comprising glass, wherein the glass is selected from the group consisting of controlled pore glass and flat glass.

27. The partial metal chelator synthon according to claim 26, wherein the partial metal chelator synthon consists of a compound having the structure

28. A microarray device comprising a solid phase substrate having a surface, wherein the solid phase surface comprises a plurality of known locations, and a plurality of probes, wherein each probe is bound to the substrate at a known location, wherein the probe comprises a flexible binding element, a first and a second partial metal chelator and a transition metal, wherein the transition metal atom is shared between the first and the second partial metal chelator moieties, and wherein the first and the second partial metal chelators are attached to one another through a portion of the flexible binding element, and wherein a specificity factor of the probe comprises a value selected from the group consisting of K_d, IC₅₀, K_m, k_a, k_d, log₂(PM/MM), T_m, T_d-50 and T_d-w.

29. The microarray device of claim 28 wherein the first and second partial metal chelators are independently selected from the group consisting of

wherein R is the location of a covalent attachment to the probe.

30. The microarray device of claim 28 wherein the probe further comprises a label, wherein the label is selected from the group consisting of fluorophores and quenchers, radioactive isotopes, antigenic determinants, non-radioactive isotopes, nucleic acids available for hybridization, altered fluorescence-polarization or altered light-scattering, chromogenic, chemiluminescent, electrochemically detectable, and combinations thereof.

31. The microarray device of claim 28 wherein the flexible binding element comprises a segment selected from the group consisting of PNA, poly-morphilino, PNAMs, DNA, RNA, siRNA, peptide, oligosaccharide, and combinations thereof.

32. The microarray device of claim 28 wherein the transition metal is selected from the group consisting of zinc, cadmium, copper, nickel, ruthenium, platinum, palladium, cobalt, magnesium, barium, strontium, iron, vanadium, chromium, manganese, rhodium, silver, mercury, molybdenum, tungsten, calcium, lead, cerium, aluminum and thorium.