Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis.

Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin

DNA nanostructures for biotechnological applications

Deoxyribonucleic acid (DNA) is a versatile biomolecule which can be used for the rational design and assembly of nanoscale structures. This thesis explores the use of functional DNA- enzyme nanostructures for applications in biocatalysis and for directed motion on the nanoscale. In the first part of this thesis, a DNA scaffold was outlined for the display and immobilization of enzyme cascades. Confinement or spatial organization of enzyme cascades is adopted in biological systems to prevent loss of reactive intermediates and to facilitate substrate conversion in chemically complex and crowded intracellular environments. We adopt a strategy to create concentrated enzyme assemblies directed by a DNA structure generated by F29 rolling circle amplification (RCA). These DNA assemblies, DNA nanoballs, were investigated for the display of two bi-enzyme systems. Firstly, a horseradish peroxidase and glucose oxidase enzyme pair, and secondly, a transaminase and norcoclaurine synthase bi- enzyme system for the synthesis of biotechnologically relevant benzylisoquinoline (BIA) precursors. The second part of this thesis concerns the use of enzymatic catalysis as a means of affecting the motion of a nanoscale DNA structure. Molecular movement on the micro and nanoscales is a fundamental feature of biological systems, and recreating this functionality represents an important step in the realization of intelligent synthetic devices for directed transport and chemotaxis in response to stimuli. While directed motion has been shown for DNA structures on predefined tracks to which they are hybridized, enzymatic catalysis has not been investigated as an approach to controlling the motion of DNA nanostructures. We show that the motion of a DNA structure tethered to multiple lysine decarboxylase molecules is enhanced by its substrate, L-lysine the ‘fuel’, in a concentration dependent manner, based on nanoparticle tracking analysis (NTA) and DLS analyses

Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 3′-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2′-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2′-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules

BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5′-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm(2) from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studies

BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies

The tricarboxylate reagent benzene-1,3,5-triacetic acid (BTA) was used to attach 5′-aminated DNA primers and templates on an aminosilanized glass surface for subsequent generation of DNA colonies by in situ solid-phase amplification. We have characterized the derivatized surfaces for the chemical attachment of oligonucleotides and evaluate the properties relevant for the amplification process: surface density, thermal stability towards thermocycling, functionalization reproducibility and storage stability. The derivatization process, first developed for glass slides, was then adapted to microfabricated glass channels containing integrated fluidic connections. This implementation resulted in an important reduction of reaction times, consumption of reagents and process automation. Innovative analytical methods for the characterization of attached DNA were developed for assessing the surface immobilized DNA content after amplification. The results obtained showed that the BTA chemistry is compatible and suitable for forming highly dense arrays of DNA colonies with optimal surface coverage of about 10 million colonies/cm2 from the amplification of initial single-template DNA molecules immobilized. We also demonstrate that the dsDNA colonies generated can be quantitatively processed in situ by restriction enzymes digestion. DNA colonies generated using the BTA reagent can be used for further sequence analysis in an unprecedented parallel fashion for low-cost genomic studie

Large-Scale de novo Oligonucleotide Synthesis for Whole-Genome Synthesis and Data Storage: Challenges and Opportunities

Over the past decades, remarkable progress on phosphoramidite chemistry-based large-scale de novo oligonucleotide synthesis has been achieved, enabling numerous novel and exciting applications. Among them, de novo genome synthesis and DNA data storage are striking. However, to make these two applications more practical, the synthesis length, speed, cost, and throughput require vast improvements, which is a challenge to be met by the phosphoramidite chemistry. Harnessing the power of enzymes, the recently emerged enzymatic methods provide a competitive route to overcome this challenge. In this review, we first summarize the status of large-scale oligonucleotide synthesis technologies including the basic methodology and large-scale synthesis approaches, with special focus on the emerging enzymatic methods. Afterward, we discuss the opportunities and challenges of large-scale oligonucleotide synthesis on de novo genome synthesis and DNA data storage respectively

Single-stranded DNA : methods and application in nanotechnology

Basic molecular and cell research, production of recombinant proteins, diagnostic detection of genetic mutations, construction of nanostructures and high-throughput DNA sequencing are only a few examples of the diverse set of applications that are amenable thanks to the availability of synthetic DNA polymers in biomedicine. Strategic investments and technical progress together with the introduction of automation in the synthesis of DNA oligomers enabled to transform, in just a few decades, a process mastered only by a niche of biochemists into an affordable and available custom-made product to every scientific field. Despite such progress, innovation soon reached a plateau due to intrinsic limitations of the synthesis process, putting a barrier at two hundred nucleotides as the maximum length of synthetic DNA molecules. In the meanwhile, molecular biologists closed the gap thanks to a better understanding of polymerases and the mastering of directed evolution protocols making it possible to redesign processes that are more similar to what happens in nature, taking advantage of existing and improved enzymes for the generation of long and high-quality DNA molecules. This enabled to find novel applications for DNA such as gene editing or information storage. In this thesis I focused on the enzymatic production and functionalization of single stranded DNA. More specifically, in paper I we directed our attention to optimize the protocol for the templated enzymatic synthesis of oligonucleotides. We highlighted possible limitations of the technique and proposed a solution in employing a single stranded binding protein greatly decreasing double stranded DNA contaminants. In paper II we further extended the workflow. In here, we focused on continuing the previous protocol to accommodate the production of chimeric DNA-protein molecular tools needed in nanotechnology where DNA is considered more a building material rather than an information rich polymer while the actuation of a particular function is operated by proteins. We worked on a minimal bacteria-derived self-tagging domain that has the capacity to establish a covalent bond with a specific DNA sequence and some applications are suggested. Paper III represents the natural extension of this work even if, in this specific case, the earlier presented rational is reverted. More specifically, a biosensor for the detection of aquatic microorganisms was produced with the characterized bioconjugation technique where the chimeric protein was used as recognition moiety and the oligonucleotide as signal amplification device through its intrinsic DNAzyme activity. Finally, in Paper IV, we decided to use all the previously gathered knowledge – enzymatic DNA production and bioconjugation techniques – to conceive a novel basic biology investigation tool for the study of spatial organization of proteins. Here we took advantage of the possibility to grow a localized and unique DNA polymer with the ability to target proteins with DNA-protein chimeras. The resulting product is then recovered and decoded by next generation sequencing

Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common type of leukemia. The Cancer Genome Atlas Research Network has demonstrated the increasing genomic complexity of acute myeloid leukemia (AML). In addition, the network has facilitated our understanding of the molecular events leading to this deadly form of malignancy for which the prognosis has not improved over past decades. AML is a highly heterogeneous disease, and cytogenetics and molecular analysis of the various chromosome aberrations including deletions, duplications, aneuploidy, balanced reciprocal translocations and fusion of transcription factor genes and tyrosine kinases has led to better understanding and identification of subgroups of AML with different prognoses. Furthermore, molecular classification based on mRNA expression profiling has facilitated identification of novel subclasses and defined high-, poor-risk AML based on specific molecular signatures. However, despite increased understanding of AML genetics, the outcome for AML patients whose number is likely to rise as the population ages, has not changed significantly. Until it does, further investigation of the genomic complexity of the disease and advances in drug development are needed. In this review, leading AML clinicians and research investigators provide an up-to-date understanding of the molecular biology of the disease addressing advances in diagnosis, classification, prognostication and therapeutic strategies that may have significant promise and impact on overall patient survival