8-Oxo-7,8-dihydroguanosine in aptamer development and its impact on RNA structure Supplement - Project Summary:
Aptamers are biomolecular entities that can bind to a target molecule with high selectivity, which makes
them attractive constructs with potential applications as therapeutic tools, in biosensing, or as materials,
among others. The proposal associated with this supplement submission focuses on the use of a chemical
modification, 8-oxo-7,8-dihydroguanosine (8-oxoG), as a building block for the discovery of aptamers of RNA
with enhanced specificities and unique structural motifs. Therefore, the use of instrumentation/methodology
that characterizes interactions between the designed aptamers and their potential targets, is of utmost
importance. Some of the common techniques, that are used by the scientific community, include isothermal
titration calorimetry (ITC), microscale thermophoresis (MST), surface plasmon resonance (SPR), and
electrophoretic mobility shift assays (EMSA). Amongst these, the PI opted for ITC as the instrumentation of
choice. Advantage of this technique, over the other, include that 1) there is no need for chemical labeling of
the RNA construct, e.g., with fluorophores, radiolabels, or biotin; 2) the potential to recover the aptamer of
interest; 3) the possibility to extract thermodynamic data, stoichiometry, and affinity strength; and 4) price
accessibility, compared to MST and SPR. It is worth noting that this is a well-accepted technique, in the
community, and that the data that can be extrapolated is of value, to learn about the novel RNA constructs that
will be obtained.
The addition of this instrument, to the PI's research facilities, will expedite the research progress/activities and
ensure that the proposed goals are met in a timely and efficient manner. Furthermore, improving the PI's
laboratory infrastructure will provide hands-on experience, and high-quality training to all students involved in
this research enterprise.
As mentioned, the instrument will be used to characterize RNA-metabolite interactions. In addition,
structural information, will be obtained by recording the thermodynamic parameters on suitable RNA
constructs, that will provide valuable information about the impact of the oxidative modifications of interest.
Locating the ITC within the PI's facility will significantly increase the rate at which experiments are being
carried out, currently (the closest ITC instrument, for research purposes, is located at the biophysics core of
the Medical campus, ~ 10 miles away). Importantly, the student population at CU Denver will have access to
this technique, while improving the research infrastructure within the PI's department. Student exposure to this
technique will enhance the training experience, and prepare them better for their next academic/professional
step.
