Photo-induced damage mitigating agents and preparation and methods of use thereof
Compositions, devices, systems and methods for reducing and/or preventing photo-induced damage of one or more reactants in an illuminated analytical reaction by addition of one or more photo-induced damage mitigating agents to the reaction mixture and allowing the reaction to proceed for a period that is less than a photo-induced damage threshold period.
1. A composition comprising:
a first reactant:
a second reactant; and
a photo-induced damage mitigating agent admixture comprising a compound of formula:
wherein R 1 is independently selected from the group consisting of hydrogen, halogen, alkyl, —CH 3 , —(CH 2 ) n R 2 , —(CH 2 ) n R 2 R 3 , —SO 3 H, —NO 2 , —OR 2 , —COR 2 , —COOR 2 , —(CH 2 ) n OR 2 , —CH(OH)R 2 , —S(O) m R 2 , —SO 3 R 2 , —CONH m (R 2 ) 2-m , —SO 2 NH m (R 2 ) 2-m , NH m (R 2 ) 2-m , —CONHSO 3 H, —(CH 2 ) n R 5 , —CH(OR 2 )R 3 , —(CH 2 ) n R 2 R 5 , —R 5 , —OR 5 , —COR 5 , —COOR 5 , —(CH 2 ) n OR 5 , where m is an integer and where n is an integer from 1 to 4;
R 2 is selected from the group consisting of hydrogen, halogen, alkyl, —CH 3 , hydroxyl, —SO 3 H, —NO 2 , —OR 3 , —COR 3 , —COOR 3 , —CH(OR 3 )R 4 , —CH(OH)R 3 , —SO 3 R 3 , —R 5 , —OR 3 R 5 , —COR 5 , —COR 3 R 5 , —COR 3 R 4 R 5 , —CH(OH)R 5 , —S(O) m R 3 , —SO 3 R 3 , —CONH m (R 3 ) 2-m , NH m (R 3 ) —CONHSO 3 H, —SO 2 NH m (R 3 ) 2-m , where m is an integer;
R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, hydroxyl, alkyl, —CH 3 , —SO 2 H, NO 2 , and —COOH;
R 5 is a linker, and Z is a bridging group;
and further wherein interaction of the first reactant with the second reactant under excitation illumination causes photo-induced damage to the first reactant in the absence of the photo-induced damage mitigating agent admixture.
2. The composition of claim 1 , wherein the first reactant is confined.
3. The composition of claim 2 , wherein the first reactant is immobilized on a surface comprising an optical confinement.
4. The composition of claim 3 , wherein the optical confinement comprises a zero mode waveguide.
5. The composition of claim 1 , wherein the first reactant is an enzyme.
6. The composition of claim 1 , wherein the first reactant is a polymerase.
7. The composition of claim 1 , wherein the photo-induced damage mitigating agent admixture further comprises another compound of a formula selected from the group consisting of
8. The composition of claim 7 , wherein the photo-induced damage mitigating agent admixture comprises a compound of the formula
and a compound of the formula
9. The composition of claim 7 , wherein the photo-induced damage mitigating agent admixture comprises multiple racemates and/or diastereomers of the formula
10. The composition of claim 7 , wherein the photo-induced damage mitigating agent admixture further comprises at least 10% of a compound of the formula
and no more than 50% of a compound of formula
11. The composition of claim 1 , wherein the second reactant comprises a fluorescent or fluorogenic molecule.
12. A method of performing an illuminated reaction, comprising:
providing a substrate having a reaction mixture disposed thereon, wherein the reaction mixture comprises the composition of claims 1 , wherein the photo-induced damage mitigating agent admixture reduces an amount of photo-induced damage to the first reactant resulting from interaction of the first reactant with the second reactant under excitation illumination to an amount that is less than that which would occur in the absence of the photo-induced damage mitigating agent admixture; and
illuminating the reaction mixture on the substrate with an excitation illumination.
13. The method of claim 12 , further comprising the step of monitoring a reaction between the first and second reactant while illuminating the reaction mixture.
14. The method of claim 12 , wherein said illuminated reaction is a base extension reaction.
15. The method of claim 12 , wherein said first reactant is a polymerase enzyme.
16. The method of claim 12 , wherein said second reactant comprises fluorescent or fluorogenic molecule.
17. The method of claim 12 , wherein the photo-induced damage mitigating agent admixture further comprises another compound of a formula selected from the group consisting of
18. The method of claim 17 , wherein the photo-induced damage mitigating agent admixture comprises both a compound of the formula
and a compound of the formula
19. The method of claim 18 , wherein the photo-induced damage mitigating agent admixture comprises multiple racemates and/or diastereomers of the formula
20. A device comprising:
a substrate having an observation region;
a first reactant immobilized within the observation region;
a second reactant disposed within the observation region, wherein interaction between the first and second reactants under excitation illumination causes photo-induced damage to the first reactant; and
at least one photo-induced damage mitigating agent admixture comprising a compound of formula
disposed within the observation region,
wherein R 1 is independently selected from the group consisting of hydrogen, halogen, alkyl, —CH 3 , —(CH 2 ) n R 2 , —(CH 2 ) n R 2 R 3 , —SO 3 H, —NO 2 , —OR 2 , —COR 2 , —COOR 2 , —(CH 2 ) n OR 2 , —CH(OH)R 2 , —S(O) m R 2 , —SO 3 R 2 , —CONH(R 2 ) 2-m , —SO 2 NH m (R 2 ) 2-m , NH m (R 2 ) 2-m , —CONHSO 3 H, —(CH 2 ) n R 5 , —CH(OR 2 )R 3 , —(CH 2 ) n R 2 R 5 , —R 5 , —OR 5 , —COR 5 , —COOR 5 , —(CH 2 ) n OR 5 , where m is an integer and where n is an integer from 1 to 4;
R 2 is selected from the group consisting of hydrogen, halogen, alkyl, —CH 3 , hydroxyl, —SO 3 H, —NO 2 , OR 3 , —COR 3 , —COOR 3 , —CH(OR 3 )R 4 , —C(OH)R 3 , —SO 3 R 3 , —R 5 , —OR 3 R 5 , COR 5 , —COR 3 R 5 , —COR 3 R 4 R 5 , —CH(OH)R 5 , —S(O) m R 3 , —SO 3 R 3 , —CONH m (R 3 ) 2-m , NH m (R 3 ) 2-m , —CONHSO 3 H, —SO 2 NH m (R 3 ) 2-m , where m is an integer;
R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, hydroxyl, alkyl, —CH 3 , —SO 3 H, NO 2 , and —COOH;
R 5 is a linker, and Z is a bridging group.
21. The device of claim 20 , wherein the admixture further comprises at least one compound of formula
22. The device of claim 20 , wherein the admixture comprises multiple racemates and/or diastereormers of the formula
23. The device of claim 20 , wherein the admixture comprises at least 10% of the compound having the formula
and no more than 50% of a compound of formula
24. A photo-induced damage mitigating agent admixture comprising at least about 10% of a compound of formula,
and no more than about 20% of a compound of formula
wherein R 1 is independently selected from the group consisting of hydrogen, halogen, alkyl, —CH 3 , —(CH 2 ) n R 2 , —(CH 2 ) n R 2 R 3 , —SO 3 H, —NO 2 , —OR 2 , —COR 2 , —COOR 2 , —(CH 2 ) n OR 2 , —CH(OH)R 2 , —S(O) m R 2 , —SO 3 R 2 , —CONH m (R 2 ) 2-m , SO 2 NH m (R 2 ) 2-m , NH m (R 2 ) 2-m , —CONHSO 3 H, —(CH 2 ) R 5 , —CH(OR 2 )R 3 , (CH 2 ) n R 2 R 5 , —R 5 , —OR 5 , —COR 5 , —COOR 5 , —(CH 2 ) n OR 5 , where m is an integer and where n is an integer from 1 to 4;
R 2 is selected from the group consisting of hydrogen, halogen, alkyl, —CH 3 , hydroxyl, —SO 3 H, —NO 2 , OR 3 , —COR 3 , —COOR 3 , —CH(OR 3 )R 4 , —C(OH)R 3 , —SO 3 R 3 , —R 5 , —OR 3 R 5 , —COR 5 , —COR 3 R 5 , —COR 3 R 4 R 5 , —CH(OH)R 5 , —S(O) m R 3 , —SO 3 R 3 , —CONH m (R 3 ) 2-m , NH m (R 3 ) 2-m , CONHSO 3 , —SO 2 NH m (R 3 ) 2-m , where m is an integer;
R 3 and R 4 are independently selected from the group consisting of hydrogen, halogen, hydroxyl alkyl, —CH 3 , SO 3 H, NO 2 , and —COOH;
R 5 is a linker, and Z is a bridging group.
25. The method of claim 18 , wherein the photo-induced damage mitigating agent admixture further comprises at least 10% of the compound of the formula
and no more than 50% of a compound of formula