Hi there, 

For making changes that may result in reduction or enhancement of site-specific DNA binding properties of your protein of interest, you should have a knowledge of its structure, even better - the structure of the binary complex between your protein and DNA. If the structure of a homologous protein exist, you may use that as well if the conservation level is high. The data of previous published  point mutagenesis analysis focused on DNA binding properties of the protein would be also very helpful.

Modeling of the DNA binding properties of a given protein without prior knowledge of its structure is very speculative and not accurate. So, the real question here is what you have got to begin with?  

Dear Dr. Laptenko

Hello,

I would appreciate your immediate attention. Yes, you right. In fact, I am going to work on CRISPR/Cas9 system and its application in genesis.  Crystal structure of Cas9 previously was published (Nishimasu et al 2014). Moreover, applying CRISPR/Cas9 system because of  off-target limitation has a major obstacle to find safe results. Hence, I imagine that if there  is a method for predicting modification's effects on enhancement of site-specific DNA binding properties, we would be able to choose right strategy in genome editing. I was wondering  there is any useful bioinformatical analysis for predicting structural modifications?

Thank you in advance,

Hadi

Hi,

I am sorry for not responding in time.

I am not familiar with any reliable software that would do that. And I do not think that there is an easy solution without investing in a lot of benchwork hours. Here is what I would do. If the structure is available in the complex with the nucleic acid, i would carefully examine the protein interface involved into binary complex formation. The most critical residues responsible for the interaction with nucleic acids and providing the direct contacts  are usually Lysines and Arginines. Usually, site-directed mutagenesis of those residues abrogates binding to a different degree (depending on the importance of a particular residue). In addition, covalent modifications of those Lysines and Arginines (such as Acetylation or Methylation) are known to decrease the affinity of the interactions with nucleic acids. But if you need the opposite - you can perform a saturated mutagenesis of all the NON-criticall residues within the DNA-binding surface of your protein and select for those clones that show enhanced binding characteristics. For simplicity, in the selection experiments one might use only DNA-binding domain of the protein of interest (unless other domains are known to have a significant impact on this process).  Alternatively, one can perform a saturated mutagenesis followed by selection toward specific DNA sequence: kind of reverse SELEX technique with multiple variable ligands being proteins and a fixed DNA sequence.

Dear Dr. Laptenko

I really appreciate your fascinating kindness. Likwise, another big thanks to your comperhensive reply. it would be exteremely useful. It was what I need to know.

Have all the best,

Hadi

Hi again,

Here is an interesting update (a study published just recently by NAR) that may be of use to you. Please, see the attachment.

Good luck.