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

Telomerase Activity Is Required for the Telomere G-Overhang Structure in Trypanosoma brucei

Trypanosoma brucei causes fatal human African trypanosomiasis and evades the host immune response by regularly switching its major surface antigen, VSG, which is expressed exclusively from subtelomeric loci. Telomere length and telomere proteins play important roles in regulating VSG silencing and switching. T. brucei telomerase plays a key role in maintaining telomere length, and T. brucei telomeres terminate in a single-stranded 3′ G-rich overhang. Understanding the detailed structure of the telomere G-overhang and its maintenance will contribute greatly to better understanding telomere maintenance mechanisms. Using an optimized adaptor ligation assay, we found that most T. brucei telomere G-overhangs end in 5′ TTAGGG 3′, while a small portion of G-overhangs end in 5′ TAGGGT 3′. Additionally, the protein and the RNA components of the telomerase (TbTERT and TbTR) and TbKu are required for telomere G-overhangs that end in 5′ TTAGGG 3′ but do not significantly affect the 5′ TAGGGT 3′-ending overhangs, indicating that telomerase-mediated telomere synthesis is important for the telomere G-overhang structure. Furthermore, using telomere oligo ligation-mediated PCR, we showed for the first time that the T. brucei telomere 5′ end sequence- A n important feature of the telomere terminal structure-is not random but preferentially 5′ CCTAAC 3′

Translation of Random Transcripts Generated by TdT: Potential Use in Polysome Peptide Libraries

A thesis presented to the faculty of the College of Science and Technology at Morehead State University in partial fulfillment of the requirements for the Degree of Master of Science in Biology by Michael Lane Spencer on July 22, 1998

Fluorescent labeling of plasmid DNA and mRNA : gains and losses of current labeling strategies

Live-cell imaging has provided the life sciences with insights into the cell biology and dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In this Review, we focus on the current fluorescent labeling strategies for nucleic acids, and in particular mRNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad range of scientific fields. By giving a background of the available techniques and an evaluation of the pros and cons, we try to supply scientists with all the information needed to come to an informed choice of nucleic acid labeling strategy aimed at their particular needs

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

Comparison of two methods of generating random DNA fragments for cloning and sequencing

Thesis (B.S.) in Microbiology -- University of Illinois at Urbana-Champaign, 1990.Includes bibliographical references (leaves 25-26)Microfiche of typescript. [Urbana, Ill.]: Photographic Services, University of Illinois, U of I Library, [1990]. 1 microfiche (33 frames): negative.s 1990 ilu n

Molecular Mechanisms of Flexibility in Nonhomologous End Joining

DNA double strand breaks (DSBs) are highly toxic DNA lesions that play a critical role in human health and disease. The ability to repair these lesions is essential in all kingdoms of life, and in mammals is primarily attributed to the nonhomologous end joining (NHEJ) pathway. NHEJ faces a unique challenge: unlike other forms of DNA damage, DSBs are structurally heterogeneous, varying wildly in end chemistry. To address this problem, NHEJ has evolved uniquely flexible enzymes: DNA polymerases and a DNA ligase that can act on a remarkable variety of substrates, much more so than their counterparts in other pathways. The mechanistic basis of this flexibility, and its significance to biological repair, are unknown. DNA Ligase IV (LIG4) is the only human DNA ligase that participates in NHEJ, and the only one that can efficiently ligate ends across gaps, or with terminal mispairs. We show by single-molecule analysis that terminal mispairs lead NHEJ complexes to mobilize DNA ends and thereby sample more end alignments. This flexibility is what allows LIG4 to join such ends, since pairing flexibility and ligation both require a LIG4-specific structural motif, insert1. Our work showed that pairing flexibility is what enables LIG4 to tolerate a chemically diverse array of substrates, and that this tolerance is essential for cells to survive exogenous DNA damage such as ionizing radiation. NHEJ employs two uniquely flexible polymerases to prepare ends for ligation: DNA polymerase μ (pol µ) and terminal deoxynucleotidyl transferase (TdT). These enzymes act on noncanonical substrates that other polymerases cannot engage. We show these polymerases primarily incorporate ribonucleotides (RNA), not deoxynucleotides (DNA), during NHEJ, both during repair of chromosome breaks made by Cas9 and during V(D)J recombination. These ribonucleotides facilitate NHEJ by enabling ligation of ends with adjacent mispairs, and even single strand ligation. Supplementing cells expressing TdT with deoxynucleotides thus blocks repair of Cas9-induced breaks, while ribonucleotide supplementation can improve Cas9-directed mutagenesis. Our results indicate cellular NHEJ often involves transiently embedded ribonucleotides, which promote flexibility in repair at the cost of more fragile intermediates.Doctor of Philosoph

RNA functionalization strategies and their application to RNA folding dynamics and experimental RNomics

The oversimplified notion of RNA being a mere carrier of sequence information from gene to protein has been repeatedly undermined over the decades by yet another newly discovered function performed by certain RNA species. These new species include in particular RNAs which regulate gene expression in response to a metabolite sensing event. These RNAs — known as riboswitches — elegantly couple metabolite recognition with gene regulation in the apparent absence of protein helpers. Here we first sought to investigate the folding dynamics of the S-adenosyl-L-methionine responsive riboswitch by FRET spectroscopy. This requires the synthesis of full-length riboswitch constructs site-specifically modified with multiple fluorophores. For this challenging task we have established a 5-way splinted-ligation strategy to prepare dual-fluorophore labelled full-length riboswitch constructs in an unprecedented overall yield of 10 %. These constructs have further been subjected to bulk and single molecule FRET spectroscopy for ligand induced folding analysis. We confirmed similar folding dynamics for the aptamer of the complete riboswitch (aptamer + expression platform) as reported earlier for constructs containing the aptamer alone. However, we also observed a few other folding phenomena induced by a chemically slightly different, yet non-cognate metabolite, which cannot be explained by any facts known about this riboswitch to date and require further experiments to reach a final conclusion. During the course of the aforesaid work, we realized the limitations of existing nucleic acid functionalization strategies. Therefore we decided to use bioorthogonal click reactions as part of our labelling strategy. Among various different click reactions, we first had to find the one which best suits our purpose and to optimize its conditions. Having the optimized click reaction conditions at hand, we developed enzymatic strategies to site-specifically functionalize long RNAs with clickable residues. In our nucleic acid labelling strategy a diverse array of different chemical functionalities can be introduced exploiting the modular nature of click chemistry. This does not demand either de novo synthesis or optimizations of enzymatic reaction conditions for each new single compound. Furthermore, we developed a chemical approach using two different mutually orthogonal click reactions for concurrent, site-specific labelling of DNA molecules with multiple fluorophores. Moreover, we sought to extend this strategy of enzymatic, site-specific transfer of clickable residues to long RNAs towards photochemical transfer of clickable moieties to a target RNA in a mixture of many unrelated sequences. This technique, which we call Affinity-based Chemical RNomics is a chemical approach in experimental RNomics whereby RNA sequences which bind to a given small-molecule metabolite are to be isolated from a total RNA isolate of any organism just by the virtue of its tight binding to its cognate metabolite and without any prior knowledge of its sequence. This method would therefore allow for the discovery of previously unknown riboswitches, currently the only known kind of natural RNA that binds small-molecule metabolites. Since all currently known riboswitches have been discovered by rational approaches, this will considerably extend the chances of discovering new riboswitches

Investigation of messenger RNA stability in the yeast Saccharomyces cerevisiae

Factors which influence mRNA stability in the yeast, Saccharomvces cerevisiae have been investigated, in particular the influence of mRNA length and translation. The properties of 13 mRNAs of unknown function have been analysed using yeast cDNA clones as molecular probes. The pyruvate kinase mRNA was also analysed using a genomic clone. Preliminary characterization of each cDNA clone by northern blotting, Southern blotting and cross hybridization experiments demonstrated that each was derived from a different, single copy gene (with the possible exception of cDNA10). A new and convenient technique has been developed for the measurement of mRNA half-life in yeast. The method involves the quantitation of specific mRNAs by dot-blotting after inhibition of transcription using phenanthroline. The chemical half-lives for the 14 mRNAs ranged from 6.6 +/- 0.67 to over 100 minutes, relative to the half-life of the 18S rRNA control. Comparison of mRNA length and half-life revealed that two populations of mRNAs containing relatively stable or unstable mRNAs were present. Within each population there was an inverse relationship between mRNA length and half-life. The distribution of each mRNA across sucrose density gradients of yeast polysomes was analysed. There was no obvious correlation between ribosome loading' and mRNA half-life. Therefore mRNA length, but not ribosome loading, would appear to be an important factor in the determination of the stability of an mRNA in S. cerevisiae. To account for the division of yeast mRNAs into two clear populations on the basis of mRNA length and half-life, it is suggested that at least one other factor must have an influence

Assessment of DNA structure and integrity in the human spermatozoon

Male fertility is routinely assessed by basic semen analysis, but this is poorly predictive of fertility outcome. One promising advanced diagnostic is assessing sperm DNA damage, but there is a lack of method standardisation, clinical thresholds and comparison data for different assays. The objective of this study was to validate standardised methods for the assessment of sperm DNA quality using TUNEL, Acridine orange (AO) and Chromomycin A3 (CMA3) in slide-based assays; and assess prognostic value for fertility and miscarriage. The data obtained reveals that using differing optimal mounting solution for different assays is key to reliable results. The use of DNA fragmentation inductors such as DNase and hydrogen peroxide in donor samples confirmed and validated the use of AO and TUNEL for detecting DNA damaged cells. A novel semi-automated computer-based scoring system for fluorescence microscopy images was devised and compared with visual operator results for intra-assay variability of AO and TUNEL assays. This system allowed objective and consistent results free of operator subjectivity. The assessment of TUNEL, AO and CMA3 values in a subset of patients from the HABSelect trial showed no correlation between the assays corroborating that different assays measure different aspects of DNA quality. The number of patient samples assessed were insufficiently powered to draw firm conclusions related to clinical outcome, but we believe they are useful in making a case for further investigations in the field