Characterization of the secondary structure and stability of an RNA aptamer that binds vascular endothelial growth factor.
Thermal denaturation studies and spectroscopic studies were employed to investigate the secondary structure and stability of an RNA-PEG conjugate commercially called Macugen. The RNA aptamer is conjugated to a pegylated moiety, and the majority of its 2'-hydroxyl groups are methylated or otherwise modified. UV optical melting studies and differential scanning calorimetry (DSC) were carried out under different conditions to evaluate the effects of Na+ and oligomer concentrations on the stability of the secondary structure of the RNA oligomer. The results of these studies indicated that the T(m) of the RNA is independent of oligomer concentration but dependent on the salt concentration, in a predictable fashion. Further, the DSC melting profiles obtained under all conditions were highly reversible. Circular dichroism (CD) studies were determined under different salt concentrations, various RNA concentrations, and temperatures as well. Together, the thermal denaturation and CD studies provide evidence that the secondary structure of the RNA oligonucleotide is a stable hairpin at 25 degrees C and that the thermally induced hairpin to single strand transition is highly reversible.
Bozza
Melanie
M
Department of Chemistry and Biochemistry, Seton Hall University, South Orange, New Jersey 07079, USA.
Sheardy
Richard D
RD
Dilone
Enrique
E
Scypinski
Stephen
S
Galazka
Margaret
M
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D052157:Aptamers, Nucleotide; D009841:Oligonucleotides; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C495058:pegaptanib; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D012313:RNA