Small molecule-RNA aptamer SPR

Hello all, I have a couple of questions that have been bothered me for months. I hope to get some insights from you guys. I am trying to study interactions between RNA aptamers and small molecules. In particular, I am hoping to develop a secondary screening strategy to isolate my SELEX pool. 1). I have been trying to validate my idea by coupling dopamine on CM5 via the amine group and inject RNA as analyte. Dopamine is a very small molecule (~150 Da) so no matter what buffers I use (Borate pH 8.0, NaOAc pH 4.0) or how long I activate the chip and immobilize dopamine ligand, I could only get negative response (negative means RU lower than EDC/NHS activation). I went ahead and inject my RNA over the chip. I do not see any binding and for some reason the background-subtracted sensorgram will show negative binding, and this phenomenon is consistent on Biacore 3000 and T100 and also in some other people's hand. 2). After having all the troubles described above, I decide to try using a SA chip. I immobilized a biotinylated DNA linker that has a linker region that can anneal to the spacer in my RNA aptamer. I can see the hybridization between my DNA linker and RNA aptamer and the chip is quite stable. However, when I inject my small molecule analyte, I always get a negative response (background subtracted). It seems like it may be a bulk solvent effect but I am not really sure what is going on. I would appreciate any comment and if you would like to see my data, I can email them to you. Thanks! Joe

Hi Joe,

1) In priciple your set-up should work, but propably with very low responses. Dopamine (150 Da) is about the same size as the NHS (115 Da) which is bound to the surface during activation so only a very low reponse to be expected. Very thorough equilibration before immobilization can help you. In addition inject some of the buffer of dopamine (without dopamine) to observe the effect of the buffer to the sensor surface.

2) Did you already try to match the flowbuffer to yor RNA-buffer. This will make it easier to observe binding. In addition inject the RNA-buffer over your dopanine and reference channel. When subtracting the reference, the dopamine channel should give almost a flat line. If not you can use 'double referencing' of make a calibration to compensate.

I don't have a solution for you but found two articles which may be of use for you.

Arnoud

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Nucleic Acids Res. 2006 November; 34(19) 5670-5682.

Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay

Maung Nyan Win, Joshua S. Klein, and Christina D. Smolke*

DOI: 10.1177/1087057106288491

J Biomol Screen 2006; 11; 599 originally published online Jun 7, 2006;

Joonsung Hwang and Satoshi Nishikawa

Aptamers

Novel Approach to Analyzing RNA Aptamer-Protein Interactions: Toward Further Applications of

jbx.sagepub.com/cgi/content/abstract/11/6/599