Study of specific interaction between nucleotides and dye support by nuclear magnetic resonance.
The binding between four matrices (beaded cellulose, cellulose acetate, cellulose triacetate and Sepharose CL-6B) and beaded cellulose derivatized with a thiacarbocyanine dye with 5'-mononucleotides is investigated by Saturation Transfer Difference Nuclear Magnetic Resonance (STD-NMR) technique. This procedure intends to identify unspecific interactions between 5'-mononucleotides and matrices commonly used in affinity chromatography systems and also clarify the contribution of a thiacarbocyanine dye immobilized onto cellulose beads in a biorecognition process. The differences between non-derivatized and derivatized beaded cellulose evidence the contribution of thiacarbocyanine dye in the observed interaction. STD-NMR experiments show that Sepharose CL- 6B interact less with the 5'-mononucleotides comparatively with beaded cellulose. Indeed, beaded cellulose shows nonspecific interactions with almost all 5'-mononucleotides that compromises the specificity of the interaction between the thiacarbocyanine dye immobilized with the 5'-mononucleotides. The cellulose matrices where the hydroxyl groups are replaced by acetate and triacetate groups do not exhibit binding response to the 5'- mononucleotides, whereas the thiacarbocyanine dye contribution is evidenced by the reinforcement of the interactions with the sugar moiety of 5'-GMP and 5'-UMP and with base of 5'-AMP, 5'-CMP and 5'-TMP. This screening of the nucleotide atoms involved in the binding to the supports can be very useful in chromatography evaluations in which dye-affinity chromatography supports may be used, such as purification of nucleic acids.
CICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506, Covilhã, Portugal. carlacruz@fcsaude.ubi.pt
Carla
C
Cruz
Renato E F
RE
Boto
Paulo
P
Almeida
João A
JA
Queiroz
D002482:Cellulose / Q000031:analogs & derivatives / Q000737:chemistry*; D002846:Chromatography, Affinity; D004396:Coloring Agents / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009696:Nucleic Acids / Q000302:isolation & purification; D009711:Nucleotides / Q000737:chemistry*; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry
D004396:Coloring Agents; D009696:Nucleic Acids; D009711:Nucleotides; C047264:sepharose CL 6B; D002482:Cellulose; D012685:Sepharose