Attachment of oligonucleotide probes to polymer biochips and its application for the detection of point mutations

This dissertation is on the fabrication of polymer-based microfluidic arrays for the detection of genetic mutations. Poly(methyl methacrylate) was chosen as one of the polymer substrate materials due to its low background noise, low adsorption of biomolecules, and low assembly temperature. The surface modification of polymer substrates for covalent attachment of oligonucleotide probes, the construction of fluidic channels/arrays, and hybridization kinetics will be covered. As an example of the application, point mutation detection using immobilized arrays constructed in microfluidic devices will be demonstrated. The PMMA surface was derivatized with N-lithioethylenediamine solution to introduce amine groups, which were utilized for the covalent immobilization of terminal amino modified oligonucleotide probes via a homo-bi-functional linker molecule. The coupling bonds formed were stable enough to withstand multiple denaturation/rehybridization cycles. To overcome the drawbacks associated with conventional 2-D flat microarrays, such as long hybridization times and large sample consumption, oligonucleotide arrays were constructed into the microfluidic channels hot embossed into PMMA substrate. With the use of these fluidic channels we observed increased hybridization kinetics as compared to that on the flat arrays. Another benefit is that the channel-attached oligonucleotide probes allow the detection of target concentrations down to pM levels. As such, the specially designed oligonucleotide probes, which have similar melting temperatures, were constructed in microfluidic channels. Low-abundance point mutations in K-ras genes were successfully detected by using a ligase detection reaction (LDR) combined with the microfluidic hybridization. Near-IR laser induced fluorescence technique was used for the detection of surface conducted bioanalytical reactions and high detection sensitivity was obtained. In addition, preliminary work was also conducted on direct photo-patterning of deep ultraviolet (UV) light for immobilizing oligonucleotides on poly(methyl methacrylate) and polycarbonate substrates. Deep UV patterning using a through-hole mask indicated that more oligonucleotide molecules were immobilized on the UV-exposed areas than the non-exposed area in the presence of EDC conjugating reagent. However, the proper dose of UV-exposure and the appropriate EDC concentration need to be optimized in future work to increase the contrast on the immobilization efficiency between the exposed regions and un-exposed regions

Microarray analysis of GFP-expressing mouse Dopamine neurons isolated by laser capture microdissection

The Central Nervous System (CNS) contains an enormous variety of cell types which organize in complex networks. The lack of adequate markers to discern unequivocally among this cellular heterogeneity make the task of dissecting out such neural networks and the cells that comprise them very challenging. The present study represents a \u201cbottom-up\u201d approach that entails a description of A9 and A10 nuclei, which are components of the mesencephalic dopaminergic system, and the identification of their molecular make-up through microarray analysis of their gene expression profiles. These mesencephalic dopaminergic nuclei give rise to the mesocortical and mesostriatal projections and are well known for their roles in initiation of movement, reward behaviour and neurobiology of addiction. Moreover, in post mortem brains of Parkinson Disease patients a specific topographic pattern of degeneration of these neurons, also recapitulated in experimental animal models, is noted, with A9 neurons presenting with a higher vulnerability to degeneration with respect to A10 cells among which, neuron loss is almost negligible. Molecular differences may be at the basis of this different susceptibility. In this study we have optimized a protocol for laser-assisted microdissection of fluorescent-expressing cells and have taken advantage of a line of transgenic mice TH-GFP/21-31, which express GFP under the TH promoter in all CA cells, to guide laser capture microdissection of A9 and A10 mDA neurons for differential informative cDNA microarray profiling. Results show that our optimized method retains the GFP-fluorescence of DA cells and achieves good tissue morphology visualization. Moreover, RNA of high quality and good reproducibility of hybridizations support the validity of the protocol. Many of the genes that resulted differentially expressed from this analysis were found to be genes previously known to specifically define the different identities of the two DA neuronal nuclei. Transcripts were verified for expression, in DA neurons, using the collection of in situ hybridization in the Allen Brain Atlas. We have identified 592 differentially expressed transcripts (less than 8%) of which 242 showing higher expression in A9 and 350 showing higher expression in A10. Categorical analysis showed that transcripts associated with mitochondria and energy production were enriched in A9, while transcripts involved in redox homeostasis and stress response resulted enriched in A10. Of all the differentially expressed genes, eight transcripts (Mif, Hnt, Ndufa10, Aurka, Cs, enriched in A9 neurons and Pdia5, Whrn, and Gpx3 enriched in A10 neurons), verified with the Allen Brain Atlas and not noted or confirmed as differentially expressed before, emerged from this analysis. These and other selected genes are discussed

NASCENT DNA PROTEOMICS ANALYSIS UNCOVERS DNA REPLICATION DYNAMICS IN THE HUMAN PATHOGEN TRYPANOSOMA BRUCEI

DNA is the substrate of many cellular processes including DNA replication, transcription and chromatin remodeling. These processes are coordinated to maintain genome integrity and ensure accurate duplication of genetic and epigenetic information. Genome-wide studies have provided evidence of the relationship between transcription and DNA replication timing. A global analysis of DNA replication initiation in T. brucei showed that TbORC1 (subunit of the origin recognition complex, ORC) binding sites are located at the boundaries of transcription units. Although recent studies in T. brucei indicate functional links among DNA replication and transcription, the underlying mechanisms remain unknown. In this study, we adapted an unbiased technology for the identification of replication fork proteins called iPOND (isolation of proteins on nascent DNA) to T. brucei, its first application to a parasite system.The iPOND approach relies on labeling newly replicated DNA with the thymidine analog EdU (5-ethynyl-2′-deoxyuridine), which contains an alkyne functional group that enables the cycloaddition of a biotin azide. This click chemistry reaction yields a stable covalent linkage, facilitating streptavidin capture of cross-linked biotinylated DNA-protein complexes. First, we described how we adapted the iPOND protocol in T. brucei cells to generate a suitable sample for mass spectrometry analysis (Chapter 2). We performed MS label-free quantification to determine which proteins are enriched in an active replication fork in T. brucei (Chapter 3). We identified 410 proteins, including key DNA replication factors and proteins associated with transcription, chromatin organization, DNA repair and mRNA splicing. Around 25% of the proteins identified were of unknown function that might have the potential to be essential trypanosome-specific replication proteins. Additionally, we characterized two proteins from our iPOND-derived protein list (Chapter 3). These are a protein annotated as a Replication Factor C subunit (Tb927.10.7990), and a protein of unknown function (Tb927.3.5370). Both revealed nuclear localization. Tb927.10.7990 proved to be essential since its silencing caused a growth defect and impaired DNA replication. However,Tb927.3.5370 appeared to be a dispensable gene. We propose nucleosomes are assembled close to the replication fork followed by RNA pol II recruitment, transcription, and co-transcriptional RNA splicing. Further studies are needed to determine how these processes are linked and co-regulated, and how rapidly they are initiated during DNA replication

Development and Application of Ultrastructural in Situ Hybridization to Visualize the Spatial Organization of mRNA: a Dissertation

It has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this thesis was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems and structures involved. This work required the development of novel in situ hybridization methods for use with electron microscopy. This allowed resolution to visualize single mRNA molecules and individual filaments. The development of a silver enhancement methodology for both the light and electron microscopic detection of biotinated oligo-dT probes permitted a synoptic view of the intracellular distribution of poly(A) mRNA. At the light microscope, the distribution of poly(A) mRNA did not resemble the individual distribution patterns of microfilaments, intermediate filaments or microtubules. Ultrastructural examination revealed that poly(A) mRNA was not uniformly distributed along cytoskeletal filaments, but clustered at their intersections. The composition of these mRNA containing structures was investigated by both morphologic and in situ hybridization analysis using antibodies to cytoskeletal proteins. In thin sections, polysomes were observed attached to both microfilaments and intermediate filaments. To permit the simultaneous detection of oligo-dT hybridization and specific cytoskeletal proteins, a double labelling method using colloidal gold conjugated antibodies was developed. The majority of poly(A) mRNA was associated with the actin cytoskeleton, with 72% of the hybridization localized within 5nm of a labelled microfilament. Within the actin cytoskeleton, poly(A) mRNA was localized to intersections of orthogonal networks. Greater than 50% of poly(A) colocalized with the actin crosslinking proteins, filamin and α-actinin, but not vinculin. A significant amount of poly(A) mRNA was found to be associated with intermediate filaments. The double label gold analysis demonstrated that 33% of the hybridization signal localized within 5nm of labelled vimentin filaments. Prior disorganization of the actin cytoskeleton using cytochalasin did not disrupt the association of mRNA with vimentin. These observations are consistent with our morphologic results of polysome-intermediate filament associations, and indicate that microfilaments are not the only filament system to which mRNA is bound. Furthermore, a small amount of hybridization signal (12%) consistently was observed along microtubules, providing an additional cytoskeletal network to distribute mRNA. To further characterize the spatial organization of mRNA within the cytoskeleton, ultrastructural methods were developed to directly visualize individual mRNA molecules. First, oligonucleotide probes chemically modified with a single hapten and directly conjugated primary reagents were used to permit detection of an individual hybridized probe molecule by a single gold particle. Second, biotin and digoxigenin labelled oligonucleotide probes were used to simultaneously visualize the intermolecular and intramolecular relationships of two nucleic acid sequences. Third, reverse transcriptase was used to extend hybridized primers in situ which permitted visualization of the poly(A) sequence concomittant with the conformation of an mRNA molecule. These methods have permitted analysis of how single mRNA molecules may be positioned with respect to each other within the cytoskeleton. The ultrastructural visualization of mRNA within its structural environment has demonstrated heterogeneous interactions with the cytoskeleton. Future work will be needed to further characterize the mechanism of mRNA attachment. The proteins which bridge nucleic acid sequences to specific intersections can be identified. It will be interesting to learn how the identified mRNA-cytoskeletal interactions might be involved in the regulation of both mRNA translation and intracellular location. Lastly, and perhaps the most challenging goal, is to investigate whether the identified mRNA-cytoskeletal interactions are used by the cell to influence its own shape, polarity and architecture

Solid-phase DNA sequencing reactions performed in micro-capillary reactors and solid-phase reversible immobilization in microfluidic chips for purification of dye-labeled DNA sequencing fragments

The research presented in this dissertation involves micro-capillary reactors for solid phase DNA sequencing, the identification of dye terminator sequencing fragments with time-resolved methods, and purification of dye-labeled DNA fragments using solid- phase reversible immobilization in microfluidic chips. Solid-phase micro-reactors have been prepared for DNA sequencing applications using slab gel electrophoresis. A PCR product was immobilized to the interior wall of a fused-silica capillary tube via a biotin-streptavidin linkage. Solid-phase sequencing was carried out in micro-capillary reactors using a four-lane, single color dye primer chemistry strategy. The read length was found to be 589 bases. The complementary DNA fragments generated in the small volume (~62 nL) reactor were directly injected into the gel-filled capillary for size separation with detection accomplished using near-IR laser-induced fluorescence. A set of terminators labeled with near-IR heavy-atom modified tricarbocyanine dyes were investigated for a terminator sequencing protocol in conjunction with slab gel electrophoresis. This protocol gave 605 bp read lengths. A one color, two lifetime format of DNA sequencing was implemented. A pixel-by-pixel analysis was employed to identify each of the bases in the run. The resulting read accuracy for the two-dye capillary run was 90.6%. The use of photoactivated polycarbonate (PC) for purification of dye-labeled terminator sequencing fragments using solid-phase reversible immobilization (SPRI) was investigated. SPRI cleanup of dye-terminator sequencing fragments using a photoactivated PC microchannel and slab gel electrophoresis produce a read length of 620 bases with a calling accuracy of 98.9%. The PC-SPRI cleanup format was also integrated to a capillary gel electrophoresis system. In this case, the immobilization microchannel contained microposts to increase the loading level of DNAs to improve signal intensity without the need for pre-concentration

Solid-state nanopores: a new platform for DNA biomarker discovery

Solid-state (SS) nanopores emerged as a molecular detection platform in 2001, offering many advantages over their biological counterparts, α-hemolysin nanopores (α-HL). These advantages include better chemical, electrical, mechanical, and thermal stability, as well as size tunability and device integration. In addition, the size of α-HL restricts its application to translocations of single-stranded polynucleotides (ssDNA and ssRNA). This research project focused on developing a SS-nanopore platform for biomarker detection, based on differentiating ssDNA and double-stranded DNA (dsDNA) at the single-molecule scale. Reported dsDNA translocation measurements result in an average residence time of ~ 30 ns/bp, so the temporal resolution required for detection of small DNA duplexes can exceed available bandwidth limitations. To address this issue, several system parameters were explored in order to slow down translocation speed, thereby increasing temporal resolution and signal-to-noise ratio. These parameters included: applied voltage, pH, pore geometry, DNA binding agents, salt composition and concentration, and temperature. Experimental findings showed that SS-nanopores can be precisely fabricated using a controlled helium ion milling technique, acidic conditions cause DNA depurination that results in slower translocation durations, and single-stranded binding proteins (SSBs) bind preferentially to ssDNA, forming complexes with distinct translocation characteristics that permit large (> 7 kb) ds- and ssDNA to be effectively distinguished. Together, these data show that SS-nanopores can serve as a tool to electronically detect the presence and relative concentration of target DNA molecules with ultrahigh sensitivity, thus demonstrating their potential utility as a biomarker discovery platform in both biomedical and environmental applications

Genetic Targeting of Adult Renshaw Cells Using a Calbindin 1 Destabilized Cre Allele for Intersection With Parvalbumin or Engrailed1

Renshaw cells (RCs) are one of the most studied spinal interneurons; however, their roles in motor control remain enigmatic in part due to the lack of experimental models to interfere with RC function, specifically in adults. To overcome this limitation, we leveraged the distinct temporal regulation of Calbindin (Calb1) expression in RCs to create genetic models for timed RC manipulation. We used a Calb1 allele expressing a destabilized Cre (dgCre) theoretically active only upon trimethoprim (TMP) administration. TMP timing and dose influenced RC targeting efficiency, which was highest within the first three postnatal weeks, but specificity was low with many other spinal neurons also targeted. In addition, dgCre showed TMP-independent activity resulting in spontaneous recombination events that accumulated with age. Combining Calb1-dgCre with Parvalbumin (Pvalb) or Engrailed1 (En1) Flpo alleles in dual conditional systems increased cellular and timing specificity. Under optimal conditions, Calb1-dgCre/Pvalb-Flpo mice targeted 90% of RCs and few dorsal horn neurons; Calb1-dgCre/En1-Flpo mice showed higher specificity, but only a maximum of 70% of RCs targeted. Both models targeted neurons throughout the brain. Restricted spinal expression was obtained by injecting intraspinally AAVs carrying dual conditional genes. These results describe the first models to genetically target RCs bypassing development

The Role of Initiation Factor Dynamics in Translation Initiation

Like most biological polymerization reactions, ribosome-catalyzed protein synthesis, or translation, can be divided into initiation, elongation, and termination stages. Initiation is the rate-limiting stage of translation and a critical site for translational control of gene expression. Throughout all stages of protein synthesis, the ribosome is aided by essential protein co-factors known as translation factors. I have studied the role that two translation initiation factors, IF1 and IF3, play in the mechanism and regulation of translation initiation in Escherichia coli. Specifically, I have used single-molecule fluorescence resonance energy transfer (smFRET) as a primary tool for investigating how the dynamics of IF1 and IF3 regulate the accuracy with which the translational machinery selects an initiator transfer RNA (tRNA) and the correct messenger RNA (mRNA) start codon during the initiation stage of protein synthesis

High-throughput Automated Muropeptide Analysis (HAMA) Reveals Peptidoglycan Composition of Gut Microbial Cell Walls

Peptidoglycan (PGN), a net-like polymer constituted by muropeptides, provides protection for microorganisms and has been a major target for antibiotics for decades. Researchers have explored host-microbiome interactions through PGN recognition systems and discovered key muropeptides modulating host responses. However, most common characterization techniques for muropeptides are labor-intensive and require manual analysis of mass spectra due to the complex cross-linked PGN structures. Each species has unique moiety modifications and inter-/intra-bridges, which further complicates the structural analysis of PGN. Here, we developed a high-throughput automated muropeptide analysis (HAMA) platform leveraging tandem mass spectrometry and in silico muropeptide MS/MS fragmentation matching to comprehensively identify muropeptide structures, quantify their abundance, and infer PGN cross-linking types. We demonstrated the effectiveness of HAMA platform using well-characterized PGNs from E. coli and S. aureus and further applied it to common gut bacteria including Bifidobacterium, Bacteroides, Lactobacillus, Enterococcus, and Akkermansia muciiniphila. Specifically, we found that the stiffness and strength of the cell envelopes may correspond to the lengths and compositions of interpeptide bridges within Bifidobacterium species. In summary, the HAMA framework exhibits an automated, intuitive, and accurate analysis of PGN compositions, which may serve as a potential tool to investigate the post-synthetic modifications of saccharides, the variation in interpeptide bridges, and the types of cross-linking within bacterial PGNs.</p