Investigation into the Chiral Selectivity of DNA Aptamers for Essential Biomolecule Targets
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Date
2024-08-30
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University of Waterloo
Abstract
In nature, chiral molecules are typically represented as a single enantiomer for most biomolecules. This aspect of homochirality is said to be connected to prehistoric RNA which led to the dominance of sugar and amino acids to exist exclusively as one chiral form. With the advent of geomatics technology becoming more prominent in therapeutics like biosensing and drug delivery, understanding a deeper aspect on nucleic acid chemistry can help with both improving efficiency and expanding applications. In this thesis, the focus will be on DNA, specifically aptamer technology and how their affinity to ligands can be influenced by chirality. The chiral biomolecules of both lactate and tryptophan are explored by conducting various selections for the different isomers. Both targets are important in clinical applications. Tryptophan is an essential biomolecule responsible for the production of neurological hormones in the body, while lactate is an unique biomolecule in that both of its enantiomers have distinct role in the body.
In our lactate selection, even using only D-lactate as a target, high specificity aptamers for L-lactate were obtained. The aptamers showed capabilities of reaching KD of 0.23 mM and a limit of detection (LOD) of 0.21 mM in blood serum. These concentrations nicely cover the physiological range of lactate (1-20 mM), which demonstrates its potential for therapeutics applications. Additionally, the aptamer also demonstrates a 5-fold enantioselectivity for L-lactate compared to D-lactate. From the evidence present of this experiment, it is likely that DNA aptamer exhibit a preference towards L-chirality for lactate.
For the selection with tryptophan, two separate experiments were conducted using racemic and homochiral solution of tryptophan as the selection targets. The obtained aptamers from these selections demonstrated high enantioselectivity for both L-tryptophan and D-tryptophan. One of the D-tryptophan aptamer exhibited a KD of 11 μM and a 7-fold greater affinity compared to L-tryptophan. We can compare this affinity to that of aptamers specific to L-tryptophan reported in other studies, which displayed similar affinity and selectivity for the opposite enantiomer. Due to this result, we proposed that DNA’s affinity for both enantiomers stems from the greater complexity and binding features presented in tryptophan’s molecular structure.
By studying the sequences that were obtained from the selections, we observed two distinct cases of chiral bias in DNA for different biomolecules. We demonstrated how using a homochiral target solution can be applied to improve the selection of high affinity aptamers, as seen by the lactate study. Additionally, we demonstrated that highly selective DNA aptamers can also be obtained for both enantiomer of a target, as seen by the tryptophan study. Although the exact reason for the chiral preference in some targets remains uncertain, our findings suggests that variance in size may be a plausible reason to explain this phenomenon. Future studies should be taken to explore this case further by selecting other essential biomolecules that are similar in size to the two targets used. Exploring how DNA interacts with targets that have varying functional groups would help provide some more insight on the underlying mechanism of DNA’s chiral binding.
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Keywords
DNA, aptamers, lactate, tryptophan, chirality