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Using modified aptamers for site specific protein-aptamer conjugations.

ABSTRACT: Conjugation of DNAs to defined locations on a protein surface will offer powerful tools for positioning functional groups and molecules in biological and biomedical studies. However, tagging protein with DNA is challenging in physiological environments, which requires a bioorthogonal approach. Here we report a chemical solution to selectively conjugate DNA aptamer with a protein by protein-aptamer template (PAT)-directed reactions. Since protein-aptamer interactions are bioorthogonal, we exploit PAT as a unique platform for specific DNA-protein cross-linking. We develop a series of modified oligonucleotides for PAT-directed reactions and screen out F-carboxyl as a suitable functionality for selective and site-specific conjugation. The functionality is incorporated into aptamers by our F-carboxyl phosphoramidite with easy synthesis. We also demonstrate the necessity of a linker between the reactive functionality and the aptamer sequences.

SUBMITTER: Wang R

PROVIDER: S-EPMC5201207 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was possible to create protein tail-to-tail dimers in a one-pot procedure through the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. Overall, with the flexibility of using different isoprenoid analogues, this system greatly extends the utility of protein labeling using prenyltransferases.

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Using modified aptamers for site specific protein-aptamer conjugations.

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