Resonance Energy Transfer System and Method

1 . A donor-insert-acceptor resonance energy transfer system comprising:

(i) a donor molecule;

(ii) an insert molecule attached to said donor molecule, said insert molecule being a fragment of an actual or putative substrate of an enzyme that post-translationally modifies said substrate or a variant of said substrate; and

(iii) an acceptor molecule attached to said insert molecule;

wherein said insert molecule maintains a predetermined spacing, r, between said donor molecule and said acceptor molecule within the range of 0.7R 0 to 1.1R 0 , and wherein said donor molecule emits energy in the presence of a donor activator and said acceptor receives energy from said donor, resulting in a ratio of detectable signal of acceptor divided by detectable signal of said donor, and wherein the co-occurrence of an event modifying said insert molecule to alter said spacing, r, causes a measurable change in the ratio of detectable signals.

2 . The donor-insert-acceptor system of claim 1 , wherein the donor energy is in the form of fluorescence and the acceptor detectable signal is in the form of fluorescence.

3 . (canceled)

4 . The donor-insert-acceptor system of claim 1 , wherein the insert is between 10 and 27 amino acid residues in length.

5 . The donor-insert-acceptor system of claim 1 , wherein the variant has up to 12 amino acid residues replaced.

6 . The donor-insert-acceptor system of claim 1 , wherein the donor and acceptor are polypeptides.

7 . The donor-insert-acceptor system of claim 6 , wherein the acceptor polypeptide is an autofluorescent polypeptide and the donor protein is a luciferase.

8 . The donor-insert-acceptor system of claim 7 , wherein the autofluorescent polypeptide is green fluorescent protein 2, the luciferase is Renilla luciferase, and the donor activator molecule is coelenterazine.

9 . The donor-insert-acceptor system of claim 8 , wherein the insert is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

10 . The donor-insert-acceptor system of claim 1 , wherein the insert selected from the group consisting of a MAPK pathway recognition site and a Src pathway recognition site.

11 . (canceled)

12 . A donor-insert-acceptor resonance energy transfer system comprising:

a nucleic acid molecule encoding

(i) a donor molecule;

(ii) an insert molecule attached to said donor molecule, said insert molecule being a fragment of an actual or putative substrate of a tyrosine kinase or a serine/threonine kinase or a variant of said substrate; and

(iii) an acceptor molecule attached to said insert molecule;

wherein said insert molecule maintains a predetermined spacing, r, between said donor molecule and said acceptor molecule within the range of 0.7R 0 to 1.1R 0 , and wherein said donor molecule emits energy in the presence of a donor activator and said acceptor molecule displays a detectable signal in response to the emission of energy by said donor molecule and upon the co-occurrence of an event modifying the insert molecule to alter said spacing, r, resulting in a measurable change in said detectable signal, wherein said change is indicative of said event.

13 . The donor-insert-acceptor system of claim 12 , wherein the donor energy is in the form of fluorescence and the acceptor detectable signal is in the form of fluorescence.

14 . (canceled)

15 . The donor-insert-acceptor system of claim 12 , wherein the nucleic acid molecule is a plasmid.

16 . The donor-insert-acceptor system of claim 12 , wherein the insert is between 12 and 27 amino acid residues in length.

17 . The donor-insert-acceptor system of claim 12 , wherein the variant has up to 12 amino acid residues replaced.

18 . The donor-insert-acceptor system of claim 12 , wherein the acceptor polypeptide is an autofluorescent polypeptide and the donor polypeptide is a luciferase.

19 . The donor-insert-acceptor system of claim 18 , wherein the autofluorescent polypeptide is green fluorescent protein 2, the luciferase is Renilla luciferase, and the donor activator molecule is coelenterazine.

20 . The donor-insert-acceptor system of claim 19 , wherein the insert is a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

21 . The donor-insert-acceptor system of claim 12 , wherein the insert is selected from the group consisting of a MAPK pathway recognition site and a Src pathway recognition site.

22 . (canceled)

23 . A method of detecting an event associated with an insert in a donor-insert-acceptor resonance energy transfer system comprising the steps:

(i) measuring a first ratio of detectable signal of a first donor-insert-acceptor system according to claim 1 within a first test sample and a second ratio of detectable signal of a second donor-insert-acceptor system according to claim 1 within a second control sample; and

(ii) determining a signal ratio for the first and second samples;

wherein a difference in the signal ratios indicates that an event associated with the insert has occurred.

24 - 34 . (canceled)

35 . A donor-insert-acceptor resonance energy transfer kit comprising:

(i) a first component comprising a donor-insert-acceptor resonance energy transfer system of claim 1 ; and

(ii) a second component comprising a donor-acceptor resonance energy transfer system which comprises

(a) a second donor molecule; and

(b) a second acceptor molecule attached to the second donor molecule;

wherein the second donor molecule emits energy in the presence of a second donor activator and the second acceptor molecule displays a detectable signal in response to the emission of energy by the donor molecule.