8-Oxo-7,8-dihydroguanosine in aptamer development and its impact on RNA structure

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Project Summary: Aptamers have become important tools with a wide variety of applications, including in diagnostics, as therapeutics, biological reporters, or materials, among others. However, their use has been limited to the four canonical nucleobases (A, G, C, U), with seldom exceptions. The Resendiz lab was the first one to show that 8-oxo-7,8-dihydroguanosine (8-oxoG) can be used as a building block to obtain aptamers of RNA with increased or altered specificity towards their cognate target(s). Thus, effectively increasing the aptamer's chemical space to recognize new targets. The proposed work will exploit this breakthrough to 1) design oxidized RNA conjugate aptamers (ORCAs) bound to a solid support, for their use to select metabolites of potential interest; 2) establish the impact that 8-oxoG has on three structural motifs (hairpins, loop bulges, internal loops) that play important roles in recognition; and 3) introduce chemical modifications that ensure the chemical and enzymatic stability of the ORCAs, while probing that recognition is not diminished or lost. Expanding the aptamer `toolkit' will increase their potential as important biological tools; furthermore, understanding how this modification impacts the structure of RNA has broader biological implications. 8-OxoG is a chemical modification that can be generated, within biological systems, under oxidative stress. The PI's group has shown that its presence within strands of RNA can alter its reactivity towards ribonucleases and other enzymes due, in part, to its ability to undergo conformational changes that expose distinct H-bonding patterns. While this is generally viewed as `unwanted' in nature, the PI's groups uses this property to control the affinity and specificity of aptamers. Aim 1 has the potential to yield technology that can be used in detection/diagnostics and, eventually, in treatment. Aim 2 will yield important information that aids in the rational design of aptamers containing 8-oxoG; while shedding light on the impact that 8-oxoG has on RNA structure and function, a topic of wide interest due to the presence of this oxidative lesion in living organisms, and the lack of knowledge in this regard. Aim 3 will focus on preparing aptamers that are chemically and enzymatically stable; where the synthesis will be developed and expected to be of interest to the scientific community, broadly. The proposed research has the potential to be of high impact, and of interest to various fields. Importantly, its interdisciplinary nature will attract students with various professional interests, and will serve as guide and foundation to ensure a successful transition onto their next academic/professional step. A consequence of these efforts will be the strengthening of the PI's research program, department, and CU Denver as a whole. Shaping/reinforcing the interest of students in the biomedical field, from an early stage, while providing high quality training, will ensure that a knowledgeable pipeline is equipped to tackle the societal challenges of the future.