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I wish to find an order of magnitude for the binding/unbinding constants of mRNA and RNA-binding proteins.

Suppose molecules A and B reversibly associate to give the complex C. Assuming the diffusion is large enough such that no concentration gradient exist, the reaction equation at equilibrium is:

                                                          π‘˜βž•[A][B] β€” π‘˜βž–[C] = 0

with π‘˜βž• the binding constant (units: concentration-1.time-1), π‘˜βž– the unbinding constant (units: time-1), and [A], [B], [C] the concentrations of the molecules.

I found many papers assessing the equilibrium constant K = π‘˜+ / π‘˜βž– but I am interested in π‘˜+ and π‘˜βž– independently.

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  • $\begingroup$ It's hard to separate the on and off rates, but one approach I've heard of is affinity chromatography. You'd need a column of immobilized protein or RNA, then fluorescently labeled and unlabeled counterpart. You load the column with labeled compound, then pass unlabeled compound through and measure how the labeled compound comes off. $\endgroup$
    – user137
    Jan 6, 2015 at 18:03
  • $\begingroup$ Thanks for your answer. I am not doing experiments so I was searching the literature for some order of magnitudes. $\endgroup$
    – David
    Jan 6, 2015 at 18:07
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    $\begingroup$ The keyword that you're missing is kinetic. You want the kinetic binding constants/rates. And @user137 is right, this type of data is sadly uncommon, but if all you're interested in is OOM it might be possible... maybe. I'll take a look. $\endgroup$
    – tel
    Jan 6, 2015 at 23:07
  • $\begingroup$ @user137 for future reference, there are many experiments that can be used to determine kinetic rates. Surface plasmon resonance (SPR), stop flow spectroscopy, kinetic ELISA, and many others. There's even a lab in my department working on getting binding/unbinding rates from single-molecule microscopy data. $\endgroup$
    – tel
    Jan 6, 2015 at 23:11
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    $\begingroup$ @tel The only reason I was aware of the affinity chromatography method was because my dad did that for his PhD in the 80's. He had to make his own FITC, label his own antibodies, pour his own column, then build his own fluorescence detector for their HPLC using an automobile halogen headlight bulb and then run hundreds of HPLCs at different protein concentrations and salt concentrations and pH. It makes me appreciate working in a lab with funding. $\endgroup$
    – user137
    Jan 6, 2015 at 23:25

2 Answers 2

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Katsamba PS, Park S, Laird-Offringa IA. 2002. Kinetic studies of RNA–protein interactions using surface plasmon resonance. Methods 26(2):95-104

There are many methods for studying kinetics, I only mention this one because the lab I'm in has used it for studying protein-ligand interactions. That article has a good explanation of SPR and how it works, so I won't offer to much detail other than to say it basically measures the change of refractive index as one molecule binds to another, which is immobilized on a chip. This allows direct measurement of on- and off-rates.

The researches studied two RNA binding proteins: HuD and U1A, as well as several mutants. Here are their results (Table 1 in the article): enter image description here

I really couldn't tell you if those rates are generally representative of RNA-protein interactions, but I suppose that's not what you're asking.

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Ridgeway and coworkers designed a really neat microfluidic device for making these kinds of kinetic binding measurements on RNA and protein (Ridgeway et al. 2009). Their device works sort of like a stop-flow, and they used a FRET based scheme to detect binding and unbinding events. They took measurements for one particular rRNA/ribosomal protein pair and got (6 Β± 0.4) Γ— 105 M-1s-1 for the bimolecular on-rate kon and
(5 Β± 0.9) Γ— 10-3 s-1 for the unimolecular off-rate koff.

Ridgeway cites references 23,24,26, and 33 when talking about experiments with "similar constructs and buffer conditions", so those would probably be a good place to start looking if you want to assemble a diverse set of kon and koff  values.

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