Cross-linking Protein:Nucleic Acid Interactions

Cross-linking Protein:Nucleic Acid Interactions

Heterobifunctional reagents are now available that can be applied to the study of site-specific protein:nucleic acid interactions. These reagents are designed to be deployed in a stepwise manner enabling the capture of a protein:nucleic acid complex. Such conjugations between a protein bait and a nucleic acid prey involve use of cross-linking agents, a subject treated in greater detail as a previous topic in this catalog section. Protein:nucleic acid interactions are most often stabilized by linkage with heterobifunctional cross-linkers that have a photoreactive aryl azide as one of the two reactive groups.

Generally, a purified binding protein is modified by reaction with one of the two reactive groups of the cross-linker. Most reagents target amine functions on the proteins, but other functional groups can be targeted as well. The initial reaction is carried out in the dark since the remaining reactive group of the cross-linker is photoreactive. The photoreactive group of the heterobifunctional reagent will ultimately cross-link the site at which the target protein binds when the resulting complex is exposed to light. Typically, the photoreactive group is an aryl azide-based moiety that can insert nonspecifically upon photolysis.

The modified putative binding protein is incubated with the nucleic acid sample. The complex is captured when exposed to the proper light conditions for the reagent. Band shift analysis can be used to indicate capture of the complex. Alternatively, nucleases can be used to remove those portions of the nucleic acid not protected by the protein binding, thereby isolating the sequence-specific site of interaction. Photoreactive, heterobifunctional reagents with a cleavable disulfide linkage allow reversal of the protein:nucleic acid cross-link and recovery of the components of the interacting pair for further analysis.

Preparation of Protein:Nucleic Acid Conjugates
The ability to conjugate proteins to nucleic acids, including RNA and DNA, is important in a number of life science applications. Perhaps the most common conjugate of these molecules made using cross-linking compounds is the labeling of oligonucleotide probes with enzymes. Conjugating enzymes like horseradish peroxidase (HRP) or alkaline phosphatase (AP) to oligos that can hybridize to specific target sequences is important for detecting and quantifying target DNA or RNA. In this application, the enzyme activity is an indicator of the amount of target present similar to immunoassay detection using ELISA techniques. In this case, the oligo probe takes the place of the antibody, but the enzyme assay is detected by substrate turnover in the same manner.

Conjugation to 5´-Phosphate Groups
Using chemical reagents to effect the conjugation of nucleic acids to enzymes can be done using different strategies. A convenient functional group that can be chemically modified to allow the coupling of protein molecules on oligos is the 5´-phosphate group. Using the 5´ end of the oligo as the conjugation point has an advantage of keeping the rest of the nucleic acid sequence unmodified and free so it can easily hybridize to a complementary target. For oligos that have been synthesized, a 5´-phosphate group may be put on the end of the molecule to facilitate this type of conjugation. The alkyl phosphate is reactive with the water-soluble carbodiimide EDC (Product # 22980, 22981), which forms a phosphate ester similar to the reaction of EDC with a carboxylate group. Subsequent coupling to an amine-containing molecule (i.e., nearly any protein or unmodified peptide) can be done to form a stable phosphoramidate linkage (Figure 1).

If a diamine molecule is used to modify the DNA 5´-phosphate, then the resultant amine-modified oligo can be coupled to enzyme molecules using a heterobifunctional reagent. Using a diamine compound that contains a disulfide (e.g., cystamine) and then reducing the disulfide group results in a sulfhydryl that may be conjugated with proteins rendered sulfhydryl-reactive using the heterobifunctional reagent Sulfo-SMCC (Product # 22322). Pierce offers HRP enzyme that has been made sulfhydryl reactive (i.e., maleimide-activated) by this mechanism (Product # 31485.

Figure 1. Reaction scheme showing typical route to conjugating an amine-containing biomolecule to a 5´ terminal phosphate group.

Conjugation to the 3´ End of RNA
Alternatively, the 3´ end of RNA molecules may be chemically modified to allow coupling with amine-containing molecules or proteins. The diol on the 3´-ribose residue may be oxidized to result in two aldehyde groups using Sodium meta -Periodate (Product # 20504). The aldehydes then can be conjugated to the amine groups on a protein using reductive amination with Sodium Cyanoborohydride (Product # 44892). The aldehyde and the amine first form a Schiff base that is reduced to secondary amine linkage with the cyanoborohydride reductant.

In addition, biotinylation of oligonucleotides can be done using photoreactive reagents. There are two main options commonly used to add one or more biotin residues to nucleic acid probes. Photoactivatable Biotin (Product # 29987) contains a phenyl azide group at the end of a spacer arm with the biotin group at the other end. Photolyzing a solution of the biotin compound together with an oligo in solution results in biotin being non-selectively inserted into the nucleic acid structure. Alternatively, Psoralen-PEO-Biotin (Product # 29986) can be used to label double-stranded DNA or RNA. The psoralen ring structure effectively intercalates into the double-stranded portions, and exposure to UV light causes a cyclo-addition product to be formed with the 5,6-double bond in thymine residues. The poly(ethylene oxide) spacer in Psoralen-PEO-Biotin contributes excellent water solubility, thus assuring that the resultant derivative will have accessibility to streptavidin-containing detection reagents.

Other Nucleic Acid Labeling Methods
Other methods of end-labeling nucleic acids are described fully in the Protein/Gene Expression section.

Biotinylation of Nucleic Acids
Nucleic acid molecules also can be biotinylated by a number of chemical methods. Using the strategies previously described to modify the 5´ or 3´ ends of oligos with a diamine (e.g., Product # 23031 ) will provide a functional group that can be reacted with any amine-reactive biotinylation compound, such as Sulfo-NHS-LC-Biotin (Product # 21335). This modification method would provide a biotin group at the end of an oligo probe, thus allowing streptavidin reagents to be used to detect a hybridization event with a target.

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Recommended Reading

• Simpson, R.J. (2003). The structural organization of protein-DNA and multi-protein- DNA complexes can be analyzed by site-specific protein-DNA photo-cross-linking. In Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press, pp. 685-690. (Product # 20070)

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