Self-assembling RNA Aptamer Nanosensors

Aptamers are widely used in biological detection. An aptamer is a single-stranded nucleic acid molecule, either DNA or RNA, folded into a specific three-dimensional structure that binds to its targets with high affinity and specificity. Compared to DNA aptamers, RNA molecules can undergo more complex folding to form a larger number of configurations with target molecules because the RNA structure is more "flexible". This property makes RNA uniquely advantageous for small-molecule detection. However, the biological stability of RNA molecules is poor, and chemically unmodified RNA aptamers are rapidly degraded in biological fluids during sample detection. Thus far, RNA aptamers have not been utilized on a large scale in the biomedical field. In this thesis, three classic RNA aptamers were used as model templates to develop a self-assembling aptamer-based nanosensors, in which the middle sequence of these aptamers that do not interact with target molecules were split off and attached to nanomaterials with unique features (such as gold nanoparticles and graphene oxide). These self-assembling aptamer-based nanosensors were applied in biological fluids (such as serum and milk) and in recognition of chiral molecules. When the target molecules were present in the solution, split aptamers were able to bind to the target molecules, which further reassembled into three-dimensional structures of the aptamer/target molecule. Next, the aptamer/target complex interacted with the nanomaterials to varying degrees to cause fluorescence changes in the solution, which corresponded accurately to the target molecule concentration in the sample. This type of nanosensor takes advantage of the unique features of nanomaterials and shorter split RNA sequences to improve the biological stability of RNA aptamers, further enabling the development of RNA aptamer-based biological detection and biosensing technology. Additionally, the results of this thesis showed that aptamers could function as long as the three-dimensional structure formed by the aptamer/target molecule remained intact. This is a new observation that can be applied in the development of aptamer technology. The main contents are as follows:1. RNA aptamer-based sensor to detect serum theophylline:The theophylline RNA aptamer is an RNA sequence of 33 nucleotides. We split the connecting sequence at rA17 and rG18 into two halves, with one fragment linked to a poly-A tail that was rapidly adsorbed onto the surface of gold nanoparticles (AuNPs), while the end of the other fragment was labeled with a Cy3 fluorophore. When this aptamer-based sensor was used to detect serum theophylline, theophylline drove the rapid self-assembly of these two fragments on the surface of AuNPs to form the three-dimensional structure of the aptamer/theophylline. The Cy3 fluorophore then came into close proximity to the surface of the AuNPs, quenching the fluorescence. Experimental results showed that the concentration of theophylline and the fluorescence-quenching rate were positively correlated. The linear range of the assay was 0-10 μM and the lowest detection limit was 0.05 μM. RNA aptamers of this type of sensor were stable in the presence of RNase I and were not easily degraded. Despite the presence of theophylline-like molecules such as caffeine and theobromine in the serum, their interference with the detection results was less than 9%.2. RNA aptamer-based sensor to detect neomycin B in milk:Based on the interaction between the RNA aptamer of neomycin B and neomycin B in the solution, we split this 23-nucleotide RNA aptamer at positions rA and rG. Similar to the method described above, a fragment was linked to a poly-A tail, which was rapidly adsorbed onto the surface of AuNPs, while the end of the other fragment was labeled with a FAM fluorophore. When neomycin B was present in the milk, neomycin B molecules and these two nucleic acid fragments rapidly self-assembled into a tight three-dimensional structure on the surface of AuNPs, quenching the fluorescence from the FAM fluorophore. At higher neomycin B concentrations in the solution, the quenching rate of the FAM fluorophore was higher and the value of fluorescence was lower. When the concentration of neomycin B ranged from 0 to 10 μM, the decrease in fluorescence showed a linear relationship, and the lowest detection limit was 0.01 μM. Similarly, the sensors increased the biological stability of the connecting RNA sequence and was unaffected by structural analogs of neomycin B such as paromomycin and kanamycin.3. Self-assembling aptamer-based nanosensors for recognition of chiral drugs: Chiral molecules exist for various drug compounds, and they must be identified and detected because chiral molecules exhibit different activities. However, because of their similar structures, the identification and detection of chiral molecules has become a limitation in analytical chemistry. In this study, we adopted the above strategy by splitting the 33-nucleotide L-arginine RNA aptamer at the connecting positions rA2 and rA3 into two fragments. One fragment was linked to a Cy5 fluorophore, which was mixed into graphene oxide solution. Through π-π-stacking interactions, Cy5 was adsorbed on the surface of GO and its fluorescence was quenched by GO. When the other RNA fragment was added to a test solution containing L-arginine, L-arginine caused the added RNA fragment and RNA fragment adsorbed onto the surface of graphene oxide to form a complete RNA aptamer that formed a tight three-dimensional structure. The RNA fragment adsorbed onto GO dissociated from the surface, and the quenched fluorescence from Cy5 was restored. The fluorescence intensity was proportional to the concentration of L-arginine in the solution. The linear detection range was 1-40 mM and the lowest detection limit was 0.09 mM. Similarly, this type of RNA aptamer-based nanosensor resisted degradation by RNaseA and specifically recognized L-arginine; D-arginine and other similar compounds did not show concentration-dependent responses. Based on these results, we also constructed a self-assembling DNA aptamer-based sensor to detect D-vasopressin. As the concentration of D-vasopressin increased, the fluorescence values gradually increased. The detection linear range was 5-100 μg/mL and the lowest detection limit was 1.67 μg/mL. Under the interference of L-vasopressin, this DNA aptamer-based nanosensor was still able to recognize D-vasopressin with high specificity.In summary, we constructed self-assembling RNA aptamer-based nanosensors that could specifically recognize substrates without interference from substrate analogs or enantiomers. These nanosensors were also stable in complex biological fluids and were not affected by nuclease degradation. These advantages allow the nanosensors to be used in the detection of clinical samples. Additionally, the principles determined in this study can be applied in the design of nanosensors for other targets, such as proteins, bacteria, viruses, and cells. Therefore, the RNA aptamer-based sensors constructed using nanomaterials in this thesis are broadly applicable.