Targets for the identification of antibiotics that are not susceptible to antibiotic resistance
View Patent ↗To identify conserved and variable regions of the 16 S rRNA, an instant evolution experiment was performed on the entire 16 S rRNA. Analysis of these mutants identified regions that are required for function. These conserved sequences may be used as targets for pharmaceuticals that are taxonomically specific and which are refractory to the development of drug resistance.
1. A nucleic acid represented by formula V:
Z 1 -[L] m -[T 1 ]-[L] n -[T 2 ]-[L] p -[T 3 ]-[L] q -[T 4 ]-[L] r -[T 5 ]-[L] s -Z 2 V (5′→3′)
wherein, independently for each occurrence,
T 1 , T 2 , T 3 , T 4 and T 5 are selected from the group consisting of SEQ ID NOs: 4, 8, 9, 10, 11, 43, 44, 45, 46, 47, 48, 49, 52, 53, 54, 55, 56, 57, 58 and 59;
L is a nucleic acid selected from the group consisting of adenosine, cytidine, guanosine and uridine; optionally substituted with one substituent selected from the group consisting of biotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3 amidite, Cy5 amidite, digoxigenin, TEXAS RED® maleimide and tetramethylrhodamine;
Z 1 and Z 2 are selected from the group consisting of biotin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3 amidite, Cy5 amidite, digoxigenin and resin beads; and
m, n, p, q, r and s are independently an integer from 0 to 40.
2. The nucleic acid of claim 1 , wherein T 1 is SEQ ID NO 4; T 2 is SEQ ID NO 8; T 3 is SEQ ID NO 9; T 4 is SEQ ID NO 10; and T 5 is SEQ ID NO 11.
3. The nucleic acid of claim 2 , wherein p is 2, q is 17, r is 15, Z 1 is fluorescein, Z 2 is biotin, and each L is, independently for each occurrence, selected from the group consisting of unsubstituted adenosine, unsubstituted cytidine, unsubstituted guanosine and unsubstituted uridine.
4. The nucleic acid of claim 1 , wherein T 1 is SEQ ID NO 43; T 2 is SEQ ID NO 44; T 3 is SEQ ID NO 55; T 4 is SEQ ID NO 56; and T 5 is SEQ ID NO 57.
5. The nucleic acid of claim 4 , wherein n is 2, r is 3, Z 1 is fluorescein, Z 2 is biotin, or each L is, independently for each occurrence, selected from the group consisting of unsubstituted adenosine, unsubstituted cytidine, unsubstituted guanosine and unsubstituted uridine.
6. The nucleic acid of claim 4 , wherein n is 2, r is 3, Z 1 is fluorescein, Z 2 is biotin, and each L is, independently for each occurrence, selected from the group consisting of unsubstituted adenosine, unsubstituted cytidine, unsubstituted guanosine and unsubstituted uridine.
7. The nucleic acid of claim 1 , wherein T 1 is SEQ ID NO 45; T 2 is SEQ ID NO 46; T 3 is SEQ ID NO 54; T 4 is SEQ ID NO 58; and T 5 is SEQ ID NO 59.
8. The nucleic acid of claim 7 , wherein n is 16, r is 6, Z 1 is fluorescein, Z 2 is biotin, or each L is, independently for each occurrence, selected from the group consisting of unsubstituted adenosine, unsubstituted cytidine, unsubstituted guanosine and unsubstituted uridine.
9. The nucleic acid of claim 7 , wherein n is 16, r is 6, Z 1 is fluorescein, Z 2 is biotin, and each L is, independently for each occurrence, selected from the group consisting of unsubstituted adenosine, unsubstituted cytidine, unsubstituted guanosine and unsubstituted uridine.
10. The nucleic acid of claim 1 , wherein T 1 is SEQ ID NO 47; T 2 is SEQ ID NO 48; T 3 is SEQ ID NO 49; T 4 is SEQ ID NO 52; and T 5 is SEQ ID NO 53.
11. The nucleic acid of claim 10 , wherein n is 7, p is 37, r is 7, Z 1 is fluorescein, Z 2 is biotin, or each L is, independently for each occurrence, selected from the group consisting of unsubstituted adenosine, unsubstituted cytidine, unsubstituted guanosine and unsubstituted uridine.
12. The nucleic acid of claim 10 , wherein n is 7, p is 37, r is 7, Z 1 is fluorescein, Z 2 is biotin, and each L is, independently for each occurrence, selected from the group consisting of unsubstituted adenosine, unsubstituted cytidine, unsubstituted guanosine and unsubstituted uridine.
13. The nucleic acid of claim 1 , wherein Z 1 is fluorescein.
14. The nucleic acid of claim 1 , wherein Z 2 is biotin.
15. The nucleic acid of claim 1 , wherein each L is, independently for each occurrence, selected from the group consisting of unsubstituted adenosine, unsubstituted cytidine, unsubstituted guanosine and unsubstituted uridine.
16. The nucleic acid of claim 1 , wherein m and s are integers between 0-25.
17. The nucleic acid of claim 1 , wherein m and s are integers between 5-15.
18. A method of identifying an inhibitor of protein synthesis comprising
measuring the fluorescence of the nucleic acid of claim 1 , thereby establishing a first fluorescence reading;
contacting a test compound with said nucleic acid, and measuring the resulting fluorescence, thereby establishing a second fluorescence reading;
determining the difference between said first fluorescence reading and said second fluorescence reading;
selecting the compound wherein the difference between said first fluorescence reading and said second fluorescence reading is non-zero, thereby identifying said agent;
assaying the inhibitory properties of the agent by administering it to a cell, a cell extract or purified ribosomes; and
detecting protein synthesis; wherein a decrease in protein synthesis indicates that the agent is an inhibitor of protein synthesis.
19. The method of claim 18 , wherein assaying the inhibitory properties of the agent comprises detecting protein synthesis.
20. The method of claim 18 , wherein assaying the inhibitory properties of the agent comprises determining the inhibitor constant for inhibiting mRNA translation.
21. The method of identifying an agent which binds to the nucleic acid of claim 1 , comprising the steps of:
measuring the fluorescence of said nucleic acid, thereby establishing a first fluorescence reading;
contacting a test compound with said nucleic acid, and measuring the resulting fluorescence, thereby establishing a second fluorescence reading;
determining the difference between said first fluorescence reading and said second fluorescence reading; and
selecting the compound wherein the difference between said first fluorescence reading and said second fluorescence reading is non-zero, thereby identifying said agent.