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DNA/RNA as analyte in SPR

  • cmondiel
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1 year 8 months ago #1 by cmondiel
DNA/RNA as analyte in SPR was created by cmondiel
Hello everyone,

I have some questions about how to handle oligonucleotides as analytes in SPR; I know that it is preferred to immobilise them due to their negative charge, but I have one case where we need to inject them as analytes over an immobilized protein. We use NTA sensorchips

Immobilized ligand : His tag in C-ter / 23kDa
Immobilization type : NTA capture coupling
Immobilization buffer: PBS-P
Running buffer : Tris pH 7,5 NaCl 150mM, 0,05% tween, 2%DMSO


Would you have some recommendations to increase chances of success? I thought about:
- variation of pH
- Increasing NaCl conc.
- Work with DNAse free water

Best regards,

Clémence
  • Arnoud
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1 year 8 months ago #2 by Arnoud
Replied by Arnoud on topic DNA/RNA as analyte in SPR
Hi Clémence,
I don't have personal experience with oligonlucleotides binding to immobilized proteins. Quick search showed that this is relative uncommonly done. Besides possible problems with the negative charge I would try the standard assay conditions first. You can try injecting the oligonucleotides first over a surface without ligand to assess possible non-specific interaction.

One extended protocol which may be of help and two older publications.
protocols.scienceexchange.com/protocols/...ce-plasmon-resonance


1. Gambari, R., Feriotto, G., Rutigliano, C., et al.; Biospecific interaction analysis (BIA) of low-molecular weight DNA-binding drugs. J.Pharmacol.Exp.Ther. (294) 1: 370-377; 2000.
2. Palau, W. and Di Primo, C.; Single-cycle kinetic analysis of ternary DNA complexes by surface plasmon resonance on a decaying surface. Biochimie (94) 9: 1891-1899; 2012.

Kind regards
Arnoud
  • mat25077
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6 months 1 day ago #3 by mat25077
Replied by mat25077 on topic DNA/RNA as analyte in SPR
Hi Arnoud,
thank you for these really helpful pages! The question at hand is whether it is possible to immobilize a protein and have DNA as the analyte. The literature I can find refers to immobilization of the DNA.
Perhaps you have some additional thoughts? 
Thank you,
Best,
Till
  • Arnoud
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5 months 3 weeks ago - 5 months 3 weeks ago #4 by Arnoud
Replied by Arnoud on topic DNA/RNA as analyte in SPR
Hi,
Indeed it is difficult to find articles about binding of DNA to immobilized proteins. My thoughts about it is that it should be possible with 'short' DNA-fragements. I can imaging that longer DNA fragments (no idea where the cut-off length is) will give more non-specific interaction and possible secundairy structures in the DNA interfering with binding.
DNA-DNA hybridisation is more often done with short DNA fragments.

I have three publications which may be of help. Furhtermore you can look at the literature pages[url] www.sprpages.nl/sensor-chips-menu/dna [/url] and [url] www.sprpages.nl/sensor-chips-menu/aptamers [/url] or do a serach on Pubmed with:"DNA/analysis"[MeSH] AND surface plasmon resonance.

Calcagno, M., et al. (2022). "Nanoparticle-Enhanced Surface Plasmon Resonance Imaging Enables the Ultrasensitive Detection of Non-Amplified Cell-Free Fetal DNA for Non-Invasive Prenatal Testing." Analytical Chemistry 94(2): 1118-1125.

Jiang, T., et al. (2005). "Detection of TP53 mutation using a portable surface plasmon resonance DNA-based biosensor." Biosens Bioelectron 20(10): 1939-1945.

Seefeld, T. H., et al. (2011). "Rapid microarray detection of DNA and proteins in microliter volumes with surface plasmon resonance imaging measurements." Langmuir : the ACS journal of surfaces and colloids 27(10): 6534-6540.

kind regards
Arnoud
 
Last edit: 5 months 3 weeks ago by Arnoud.
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