**8. Conclusion**

224 Gel Electrophoresis – Advanced Techniques

2006). RIP-chip is an approach that consists on a microarray profiling of RNAs obtained from immunoprecipitated RNA-protein complexes. Genome-wide arrays are used to identify messenger RNAs (mRNAs) that are present in endogenous messenger ribonucleoprotein complexes making it a great tool to identify the physiological substrates of mRNAs. The endogenous complexes are immunoprecipitated from cell lysates which limits this study to kinetically stable interactions. Even though it can identify RNA-protein complexes with heteromultimers, at least one of the proteins has to be previously known to

The RIP-chip method that has just been described is limited to studies of very stable RNAprotein complexes; to remediate this problem another method is available to study RNAbinding proteins. The crosslinking and immunoprecipitation (CLIP) approach uses *in vivo* UV crosslinking prior to the complexes immunoprecipitation to identify less stable interactions (Ule et al., 2003). After immunoprecipitation RNA molecules are separated and cDNA sequencing is carried on. However, this method is not perfect as the commonly used UV 254nm RNA-protein crosslinking has low efficiency and it is difficult to distinguish between crosslinked RNAs from background non-crosslinked fragments that can be

A more recent approach tries to further improve the CLIP method using photoreactive ribonucleoside analogs such as 4-thiouridine or 6-thioguanosine (Hafner et al., 2010). In this photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) protocol the photoreactive nucleosides are incorporated into nascent transcripts within living cells. The irradiation is performed with UV light of 365nm, which induces an efficient crosslink of the labeled cellular RNA to its interacting proteins. The labeled RNAs are isolated after co-immunoprecipitation, and converted into cDNA for deep sequencing. The precise crosslinking position can be identified by mutations in the sequenced cDNA

be the basis of immunoprecipitation and "fish out" the whole complex.

detected in the sample due to the presence of abundant cellular RNAs.

making it possible to distinguish the crosslinked fragments from background.

**(HisT-FLIP)** 

**7.11 High-Throughput Sequencing – Fluorescent Ligand Interaction Profiling** 

Very recently a new method was developed to characterize DNA-protein interactions using second-generation sequencing instruments (Nutiu et al., 2011). This method allows high throughput and quantitative measurement of DNA-protein binding affinity. This High-Throughput Sequencing – Fluorescent Ligand Interaction Profiling (HiTS-FLIP) uses the optics of a high-throughput sequencer to visualize *in vitro* binding of a protein to the sequenced DNA in a flow cell. The new method was initially used on a *Saccharomyces cerevisiae* transcription factor. The fluorescently tagged protein was added at different concentrations to a flow cell containing around 88 million DNA clusters, the equivalent of over 160 yeast genomes. The traditional EMSA was used as an independent validation of the dissociation constants obtained and found a high correlation with values obtained with the new method and those from EMSA as reported in literature. This high-throughput method has an obvious advantage in the fact that it can provide hundreds of millions of measurements but is limited to DNA-protein interactions and requires expensive

**7.10 Crosslinking and Immunoprecicipation (CLIP) and Photoactivable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP)**  Since the first report, 30 years ago, EMSA became one of the most popular methods for detection and characterization of protein-nucleic acid interactions. Hundreds of protocols have been published accommodating modifications in virtually every parameter influencing the experimental outcome. Improvements were made in all EMSA steps including the methods for preparation of protein samples and purification, synthesis and labeling of nucleic acids, and detection. This allowed enlarging and diversifying the applications of EMSA and resulting in a number of variants of the method.

However, despite the large amount of available literature and protocols trial and error will ultimately be the way to optimize the EMSA conditions for the nucleic acid-protein complex to be analyzed. The guidelines discussed above help to provide an initial protocol adjusted to each study but slight changes may be needed to improve binding and detection of the complexes.

In recent years, the use of highthrouhput approaches to detect biologically relevant interactions, including those between proteins and nucleic acids, was reported. Development of these approaches was made possible, at least in part, by the availability of more sensitive and specific equipment and tools. Although EMSA cannot achieve a high throughput level it remains a valuable tool to confirm the detected interactions.
