|dc.description.abstract||Purine nucleosides, such as adenosine and guanosine, are important biomolecules to regulate physiological functions, including maintain heart and brain health, exert inflammatory responses, and take part in metabolisms. Abnormal levels of purine nucleosides can lead to serious problems. Therefore, monitoring their concentrations is critical for understanding their biological roles and performing related disease diagnoses. Compared with conventional methods to detect them, such as high-performance liquid chromatography (HPLC) and mass spectrometry, DNA aptamer-based strategies are highly attractive due to their high specificity, binding affinity, low cost, and in situ detection ability. Ideally, the aptamers with any desired specific binding abilities could be isolated by systematic evolution of ligands by exponential enrichment (SELEX); however, currently, some selected aptamers cannot distinguish closely related molecules. For example, the widely used adenosine aptamer is effective in distinguishing it from other nucleosides (G, C, T and U) but not the adenine monophosphate (AMP) and adenine triphosphate (ATP), consequently aptamer-sensing platforms built by this sequence are also limited in molecular recognition. In addition, the DNA aptamers for guanosine are not isolated yet. On the other hand, to further graft aptamers on hydrogel matrix for applications, chemical modifications are also inevitable, leading to the high cost of aptamer engineering. To solve these problems, the primary focus of this thesis is to improve the specificity of aptamer-based sensors for detecting purine nucleosides, and develop a modification-free method for preparing DNA-hydrogels at reduced costs.
Targeting the problem of some SELEX-derived aptamers with intrinsically limited specificity, a novel method is developed in chapter 2 to achieve highly specific recognition of adenosine. Typically, an entire adenine nucleotide was excised from the backbone of the existing adenosine aptamer (mentioned above), in which the resulting vacancy on DNA scaffold allowed highly specific re-binding of free adenosine, this way realized its molecular recognition and other cognate analytes including AMP, ATP, guanosine, cytidine, uridine, and theophylline are distinguished. This method is termed “base-excision”. To characterize the adenosine recognition, SYBR Green I (SGI) fluorescence spectroscopy and isothermal titration calorimetry (ITC) were used. The ITC demonstrated that one A-excised aptamer strand can bind to two adenosine molecules, with a Kd of 17.0 ± 1.9 µM at 10 degree, and entropy-driven binding. Since the wide-type aptamer cannot discriminate adenosine from AMP and ATP, we attribute this improved specificity to the excised site. Finally, the A-excised aptamer was tested in diluted fetal bovine serum (FBS) and showed a limit of detection of 46.7 µM adenosine. This work provides a facile, cost-effective, and non-SELEX method to engineer existing aptamers for new features and better applications.
In chapter 3, the aptamer engineering strategy described in chapter 2 is further used to generate new DNA aptamers for specific recognition of guanosine. Both the Na+-binding aptamer and classical adenosine aptamer were manipulated as the base-excising scaffold. A total of seven guanosine aptamers were designed, in which a guanine-excised Na+-aptamer showed the highest binding specificity and affinity for guanosine, with an apparent Kd of 0.78 mM. Both the aptamer scaffold generality and excised-site generality were systematically studied. This work provides a few guanosine binding aptamers by non-SELEX method. It also provides deeper insights into engineering aptamers for molecular recognition.
On the other hand, since the adenosine only differs deoxyadenosine by a 2´-OH and the specific recognition of adenosine from their mixture have not been realized by current methods, in chapter 4, molecularly imprinted polymers (MIPs) and aptamers as two different recognition strategies are combined. A boronic acid-containing monomer, 3-acrylamidophenylboronic acid (AAPBA), was incorporated into the MIPs to specifically target cis-diol moiety in the ribose of adenosine. ITC and SYBR Green I staining were used to measure the binding. The AAPBA-containing aptamer-MIP exhibited a 115-fold high selectivity for adenosine against deoxyadenosine at pH 6.4. The ribose in adenosine may interact with the boronic acid unit and decrease its inhibition effect to the aptamer in the MIP. Whereas for deoxyadenosine, it does not bear a cis-diol, and thus cannot rescue the aptamer. This work provides insights into the combination of aptamers with other functional groups in MIPs, which may further broaden applications in ways that free aptamers cannot achieve alone.
From an application perspective, since the current preparations of DNA-hydrogels are heavily relying on acrydite-modified DNA, lowering the cost of grafting DNA on hydrogels is another issue. To this end, a modification-free method is studied in chapter 5. We show that unmodified penta-adenine (A5) can reach up to 75% conjugation efficiency in 8 h under a freezing polymerization condition in polyacrylamide hydrogels. DNA incorporation efficiency was reduced by forming duplex or other folded structures and by removing the freezing condition. By designing diblock DNA containing an A5 block, various functional DNA sequences were attached. Such hydrogels were designed for ultrasensitive DNA hybridization and Hg2+ detection, with detection limits of 50 pM and 10 nM, respectively, demonstrating the feasibility of using unmodified DNA to replace acrydite-DNA. The same method works for both gel nanoparticles and monoliths. This work reveals interesting reaction products by exploiting freezing and has provided a cost-effective way to attach DNA to hydrogels.
Overall, improved molecular recognition of adenosine and guanosine has been achieved, through engineering existing aptamers for new functions or combining aptamers with other functional molecules in MIPs. To further facilitate the incorporation of DNA aptamers in hydrogel systems for various applications, the modification-free method is also developed. This thesis deepens our understandings in DNA aptamer-based molecular recognition and in nucleic acid chemistry, as well as provides opportunities for researchers to achieve more specific adenosine and guanosine recognitions in real applications for disease monitoring and diagnosis purpose.||en