Characterization of the Secondary Structure and Stability of an RNA Aptamer That Binds Vascular Endothelial Growth Factor

Abstract

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.

Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors

“…As many potential therapeutic applications of chimeras, including cancer therapy, require systemic administration of the therapeutic reagent, it is also necessary to optimize the in vivo kinetics of these chimeras, in addition to enhancing their potency and specificity. Terminal modification of RNAs with PEG increases the half-life and bioavailability of many oligonucleotide-based therapies, including RNA aptamers 28,37,38 . We found that addition of a 20 kDa PEG to the 5′-terminus of the smaller RNA strand promotes increased retention of the chimera in serum (Fig.…”

Current Progress on aptamer-targeted Oligonucleotide Therapeutics

“…Thus, in order to optimize the bioavailability/circulating half-life, the overall size of the aptamer therapeutic must be increased without modifying the length of the aptamer sequence itself. To accomplish this, aptamers have been conjugated to various macromolecules including PEG and cholesterol [53–55]. Similar approaches to improve the pharmacokinetics of AsiCs have been described [34].…”

RNA interference for performance enhancement and detection in doping control

“…Pegaptanib (Macugen ™ ) is a pegylated 2′-fluor-pyrimidine und 2′-O-methyl-purine substituted 27mer (5′-[40kDPEG]-[HN-(CH 2 ) 5 O]CGGArArUCAGUGAAUGCUUAUA-CAUCCG3′-dT, r = ribonucleotides)) to treat macular degeneration by down-regulating the vascular endothelial growth factor (VEGF) 165. [16,17] Most of the ongoing clinical studies investigate medications for different types of cancer such as neoplasms, leukemia, skin melanoma, or lung, bladder, prostate, or breast cancer. Further studies include oligonucleotides designed to treat diabetes mellitus I and II, Crohn's disease, asthma, or Dychenne muscular dystrophy (DMD).…”