| The discovery of small-molecule ligands to proteins and protein receptors of small organic molecules is a central problem in chemistry, biology and medicine. Specifically, small organic compounds that bind to a particular protein with reasonable affinity and specificity offer invaluable probes to perturb the function of the proteins for chemical genetics studies,1and track the location and concentration of the proteins for molecular diagnostics. They are also potential starting points for drug development. Typically, the identification of small-molecule ligands or protein receptors is performed by probing the affinity between the proteins and the small molecules with certain biomolecular interaction assay strategies, including affinity chromatography, kinetic capillary electrophoresis, fluorescence resonant energy transfer and protein-fragment complementation assay. Notwithstanding the importance of these techniques, the need of costly instruments, specific signal reporters or surface-based molecular immobilization can be a detriment. Small molecule-linked DNA is an appealing tool for investigating the interaction between small organic molecules and their protein receptors and offers an alternative possibility avenue for the study of interaction between small molecules and protein receptors due to its versatile signal amplification and various means of detection. Despite of the proliferation of small molecule-linked DNA as a smart module in highly sensitive selection of the synthetic compounds, it has rarely been implemented for the development of biosensor strategies. With these problems mentioned in my heart, a series of novel biosensors strategies were developed based on small molecule-linked DNA and terminal protection assay for interrogating the interaction between small molecules and protein and screening the disease about the SNP and enzyme activity.(1) Small-molecule-linked DNA has emerged as a versatile tool for the interaction assay between small organic molecules and their protein receptors. We reported in Chapter2the proof-of-principle of a terminal protection assay of small-molecule-linked DAN. This assay is based on our new finding that single-stranded DNA (ssDNA) terminally tethered to a small molecule is protected from the degradation by exonuclease I (Exo I) when the small molecule moiety is bound to its protein target. This finding translates the binding of small molecules to proteins into the presence of a specific DNA sequence, which enables us to probe the interaction between small organic molecules and their protein targets using various DNA sequence amplification and detection technologies. On the basis of selective assembly of single-walled carbon nanotubes (SWNTs) with surface-tethered small-molecule-linked ssDNA not protected by protein binding, a novel electrochemical strategy for terminal protection assay has been developed. Through detecting the redox signal mediated by SWNT-s assembly on a16-mercaptohexadecanoic acid-blocked electrode, this strategy is able to ensure substantial signal amplidication and a low background current. This strategy is demonstrated for quantitative analysis of the interaction of folate with a tumor biomarker of folate receptor (FR), and a detection limit of3pM FR is readily achieved with desirable specificity and sensitivity, indicating that the terminal protection assay can offer a promising platform for small molecule-protein interaction studies.(2) Considering the versatile detection technology of DNA, three label-free optical biosensor strategies were developed in Chapter3based on terminal protection assay. Catalytic nucleic acids (DNAzymes) find growing interest as catalytic labels to amplify biosensing events. For example, a single-stranded guanine-rich nucleic acid (aptamer) complexation with hemin revealed peroxidase activity and was used as catalytic label for the colorimetric or chemiluminescence detection of DNA. On the basis of colorimetric of Aptamer3’-terminal tethered to a small molecule is protected from the degradation by Exo I when the small molecule moiety is bound to its protein target. Our strategy relies on the terminal protection assay and HRP-mimicking DANzyme to develop simple, colorimetric, and label-free assays for probing the interaction between small molecules and protein. The folate-FR was used as model system to investigate the interaction of small molecular and proteins. The experiment results indicated that the FR was determined in range of1nM-100nM with a detection limit of0.33nM. The quantitative polymerase chain reaction (QPCR) has been a prevalent technique for nucleic acid analysis, attributed to its powerful capacity in high-sensitivity amplification and high-throughput detection. In parallel, a biosensor for the detection of interaction between interaction of small molecules and protein was demonstrated combining QPCR with the terminal protection assay. We designed a DNA sequence with small molecule linked to the3’-terminal. In the presence of target protein, small molecule-linked DNA was protected from the degradation of Exo I, which can then be quantitatively detected using QPCR. In the current study, SYBR Green I was chosen as the fluorescence reporter in QPCR. This affords a low-cost, label-free strategy for investigating the interaction of small molecule and proteins. We used the biosensor developed to detect the FR with a linear range from100fM to1nM. Otherwise, a novel Exo I protection-based colorimetric biosensing strategy was developed for rapid, sensitive, and visual detection of small molecule-binding protein. This strategy relied on the protection of small molecule-linked DNA crosslinked AuNP from Exo I-mediated digestion by specific binding of target proteins with small molecule. The Exo I protection-based colorimetric biosensor was demonstrated using a model molecule pairs of biotin and streptavidin (SA). The results revealed that the method allowed a specific, simple and quantitative assay of the target protein with a linear response range from1to35nM.(3) In order to further extend the appolication range of the Exo I terminal protection assay, Chapter4reports a new finding of generalized terminal protection that small molecule-DNA chimeras are protected from degradation by various DNA exonucleases, when the small molecule moieties are bound to their protein targets. This generalization converts small molecule-protein interaction assays into the detection of DNA of various structures, affording a useful mechanism for the analytics of small molecules. Based on this mechanism, a label-free biosensor strategy has been developed for homogeneous assay of protein-small molecule interactions based on fluorescence staining detection. Also, a label-free SNP genotyping technique is proposed based on polymerase extension of a single nucleotide with small molecule label. The developed techniques are demonstrated using a model proteinsmall molecule system of biotin/streptavidin and a model SNP system of human β-globin gene around codon39positions. The results revealed that the protein small molecule interaction assay strategy shows dynamic responses in the concentration range from0.5nM to100nM with a detection limit of0.1nM, and the SNP typing technique gives dynamic responses in the concentration range from0.1nM to200nM with a detection limit of0.02nM. Besides desirable sensitivity, the developed strategies also offer high selectivity, excellent reproducibility, low cost and simplified operations, implying that these techniques may hold considerable potential for molecular diagnostics and genomic researches.(4) Note that small-molecule and protein interactions we investigated above typically displayed dissociation constants in the nanomolar range. For instance, the dissociation constant was-0.1nM for folate and its receptor FR and was-10nM for FITC and its antibody. Therefore, terminal protection of small molecule-linked DNA could be a general mechanism for interrogating the interactions of protein and small-molecule pairs with nanomolar dissociation constants, and it is applicable to common double-strand or single-strand DNA exonucleases. Despite the limit, terminal protection may still hold great potential for the analysis of small moleculeprotein interactions, because there have been lots of small-molecule antibodies or receptors as well as potent drugs with nanomolar dissociation constants. Moreover, by utilizing the based activity probe, photo cross-linking and covalent capture technology that allows covalent conjugation of small molecule-linked DNA to the interacting proteins, terminal protection can be further extended beyond the assays of proteinsmall molecule pairs with varying dissociation constants (nanomolar to micromolar). In Chapter5, we present a novel colorimetric biosensing strategy, which combines the covalent capture technology with terminal protection assay to develop simple, colirimetric, and label-free assays for detection of Dnmt1and hOGG1activity. The DNA substrates of enzymes were tethere on AuNPs by5’-terminal thiol anchor and covalently bound to the enayme active site. The enzyme covalently captured prevents the Exo I and Exo III from digesting the enzymatic substrate, so the formation of the active enzyme can be detected directly. The terminal protection colorimetric biosensor based on covalent captur was demonstrated using two model enzymes of Dnmt land hOGG1. The results revealed that the method allowed quantitative assay of enzymatic activity of Dnmt land hOGG1with a dynamic response range from2U/mL to104U/mL and1.6U/mL to256U/mL respectively. |
PDF Full Text Request