Abstract
Proteins interact with nucleic acids to regulate cellular functions. The study of these regulatory interactions is often hampered by the limited efficiency of current protocols to isolate the relevant nucleic acid-protein complexes. In this report, we describe a rapid and simple procedure to highly enrich cross-linked nucleic acid-bound proteins, referred to as "2C" for "complex capture." This method is based on the observation that silica matrix-based columns used for nucleic acid purification also effectively retain UV cross-linked nucleic acid-protein complexes. As a proof of principle, 2C was used to isolate RNA-bound proteins from yeast and mammalian Huh7 cells. The 2C method makes RNA labelling redundant, and specific RNA-protein interactions can be observed and validated by Western blotting. RNA-protein complexes isolated by 2C can subsequently be immunoprecipitated, showing that 2C is in principle compatible with sensitive downstream applications. We suggest that 2C can dramatically simplify the study of nucleic acid-protein interactions and benefit researchers in the fields of DNA and RNA biology.
Highlights
From storage and transmission of genetic information, in the form of DNA and RNA, to the regulation of gene expression by silencing mechanisms and effector functions like ribozymes, nucleic acids play central roles in cellular life
Because silica columns are used to retain and purify DNA and RNA based on charge, we wondered whether they might retain nucleic acid–binding proteins when covalently cross-linked to nucleic acids
The main finding of this study is that silica matrices are capable of retaining nucleic acids and polypeptides that are cross-linked to them
Summary
Proteins interact with nucleic acids to regulate cellular functions. The study of these regulatory interactions is often hampered by the limited efficiency of current protocols to isolate the relevant nucleic acid–protein complexes. We describe a rapid and simple procedure to highly enrich cross-linked nucleic acid–bound proteins, referred to as “2C” for “complex capture.”. This method is based on the observation that silica matrix–based columns used for nucleic acid purification effectively retain UV cross-linked nucleic acid–protein complexes. We suggest that 2C can dramatically simplify the study of nucleic acid–protein interactions and benefit researchers in the fields of DNA and RNA biology.
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