Nittaya Gale

In the Arabidopsis thaliana shoot apical meristem (SAM) the expression domains of Class III Homeodomain Leucine Zipper (HD-ZIPIII) and KANADI (KAN) genes are separated by a narrow boundary region from which new organs are initiated. Disruption of this boundary through either loss of function or ectopic expression of HD-ZIPIII and KAN causes ectopic or suppression of organ formation respectively, raising the question of how these transcription factors regulate organogenesis at a molecular level. In this study we develop a multi-channel FACS/RNA-seq approach to characterize global patterns of gene expression across the HD-ZIPIII-KAN1 SAM boundary. We then combine FACS, RNA-seq and perturbations of HD-ZIPIII and KAN expression to identify genes that are both responsive to REV and KAN1 and normally expressed in patterns that correlate with REV and KAN1. Our data reveal that a significant number of genes responsive to REV are regulated in opposite ways depending on time after induction, with genes associated with auxin response and synthesis upregulated initially, but later repressed. We also characterize the cell type specific expression patterns of auxin responsive genes and identify a set of genes involved in organogenesis repressed by both REV and KAN1. The plant hormone auxin promotes the formation of lateral organs such as leaves and flowers in a specific region of the shoot called the peripheral zone. Although the restriction of organogenesis to the peripheral zone is known to depend on the Class III Homeodomain Leucine Zipper (HD-ZIPIII) and KANADI1 (KAN1) genes, the transcriptional pathways downstream of these genes have not been studied in the shoot. In this study we investigate regulatory interactions between REVOLUTA (REV), KAN1 and auxin by developing a cell-type specific transcriptomics approach to analyse gene expression patterns and responses to perturbations. Using this approach, we identify cell-type specific genes that respond to changes in REV and KAN1 expression in the shoot. Our data reveal that while REV promotes auxin-related gene expression over the short term, both REV and KAN1 repress auxin induced genes over the long-term, consistent with their influence on organogenesis. We also identify a common set of genes repressed by REV and KAN1 that promote organogenesis.

The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5'-azide and 3'-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.

Controlled covalent attachment of dsDNA horizontally orientated on a gold surface is achieved through the use of a single surface-linker located approximately half way along the attached DNA probe strand.

We show that FRET between Pacific Blue (PB) and Alexa488 (A488) covalently attached to a DNA scaffold can be reversibly controlled by photochromic switching of a spiropyran derivative. With the spiropyran in the closed spiro isomeric form, FRET occurs freely between PB and A488. UV-induced isomerization to the open merocyanine form shuts down the FRET process by efficient quenching of the PB excited state. The process is reversed by exposure to visible light, triggering the isomerization to the spiro isomer.

Strain discrimination within genetically highly similar bacteria is critical for epidemiological studies and forensic applications. An electrochemically driven melting curve analysis monitored by SERS has been utilised to reliably discriminate strains of the bacterial pathogen Yersinia pestis, the causative agent of plague. DNA amplicons containing Variable Number Tandem Repeats (VNTRs) were generated from three strains of Y. pestis: CO92, Harbin 35 and Kim. These amplicons contained a 10 base pair VNTR repeated 6, 5, and 4 times in CO92, Harbin 35 and Kim respectively. The assay also included a blocker oligonucleotide comprising 3 repeats of the 10-mer VNTR sequence. The use of the blocker reduced the effective length of the target sequence available to bind to the surface bound probe and significantly improved the sensitivity of the discrimination. The results were consistent during three replicates that were carried out on different days, using different batches of PCR product and different SERS sphere segment void (SSV) substrate. This methodology which combines low cost, speed and sensitivity is a promising alternative to the time consuming current electrophoretic methods.

The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

Hybridisation assays, which are commonly used to analyse oligonucleotides such as siRNAs and miRNAs, often employ detection probes with fluorescent tags. The signal emitted by a fluorescent tag covers a broad range of wavelengths and this limits the multiplexing potential due to overlapping signals. A novel method of indirect oligonucleotide analysis has been developed which combines a hybridisation assay with cleavable small molecule mass tags using HPLC-ESI MS detection. A self-reporting detection probe has been designed which incorporates a DNA/RNA chimeric oligonucleotide sequence in the reporter region, which generates small nucleotide products upon RNase cleavage of the ribose-phosphate backbone. These small nucleotides can then serve as mass tags for the indirect detection of oligonucleotide analytes. The narrow mass range covered by a small molecule mass tag combined with the wide range of possible mass tags provides a high degree of multiplexing potential. This approach has been demonstrated for the analysis of a synthetic miRNA.

Double-stranded DNA immobilised through a thiol anchor at a gold electrode surface can be unwound and denatured by applying a negative potential. One proposed mechanism for this electrochemical denaturation is that electrostatic field effects are responsible for the destabilisation of the dsDNA through repulsion of the DNA sugar-phosphate backbone away from the electrode surface. Herein, we demonstrate conclusively that electrochemical melting at gold electrodes cannot be explained solely as a simple repulsion mechanism by showing that immobilised DNA denatures at high ionic strengths, where the DNA base-pairs are situated outside of the electrochemical double-layer (and outside the influence of the electric field), and further, that oligomers comprised of the mimic peptide nucleic acid (PNA) can also be denatured at negative potentials, despite the absence of a negatively charged backbone.

The large size of biological molecules such as proteins and oligonucleotides makes them inherently problematic to analyse and quantify directly by mass spectrometry. For these molecules, electrospray ionisation produces multiply charged species and associated alkali metal adducts which can reduce sensitivity and complicate quantification. Whereas time-of-flight mass analysers, often coupled to matrix-assisted laser desorption/ionisation, can have insufficient mass resolution to resolve these large molecules in the higher m/z range. This has led to the development of cleavable small molecule mass tag approaches for the indirect analysis of biomolecules such as proteins and oligonucleotides. Existing methodologies require the design and synthesis of a cleavable linker to join the biomolecule and the mass tag. Here, an alternative approach to small molecule mass tags is presented, which exploits the properties of the RNA molecule to afford self-reporting probes which can be easily synthesised using automated phosphoramidite chemistry. The sugar-phosphate backbone of RNA was used as a built-in enzyme cleavable linker and through the use of RNase digestion of bromine labelled oligonucleotides the observation of a range of small molecule mass tags by mass spectrometry is demonstrated. This study provides a proof-of-concept that RNase digestion can be used to produce labelled small molecule mass tags from oligonucleotide probes, thus eliminating the need for custom design and synthesis of a cleavable linker.