Browsing by Subject "Nucleic acids"

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Design and evolution of functional nucleic acids
(2003) Levy, Matthew; Ellington, Andrew D.
Functional nucleic acids provide insight into the ‘RNA world,’ the proposed period in Earth’s history where RNA served both as catalyst and as the genetic material. Additionally, they are potentially valuable tools for biotechnical applications. Like catalytic RNA, DNA has proven capable of catalyzing chemical reactions. Using a novel ligation chemistry, we have employed in vitro selection to isolate a catalytic DNA molecule (deoxyribozyme) that is capable of forming an unnatural nucleotide linkage. In addition, we have used a combination of rational design and in vitro selection to construct effector-dependent deoxyribozymes that are allosterically activated by the small molecule ATP. These results further strengthen the idea that life arose from a nucleic acid based metabolism and also bode well for the design of more stable nucleic acid based analyte detection systems. In addition to the design of novel DNA catalysts, we have been interested in the origin of the first simple self replicating systems. Simple replicators based on short oligonucleotides as well as peptides have been demonstrated. We have designed a cross-catalytic system based on a well characterized peptide-RNA aptamer interaction, in which the peptide serves as a template for the ligation of RNA aptamer half-molecules. Our results demonstrate that the peptide could specifically enhance the rate of RNA ligation, and suggest the possibility for increased diversity of early replicators. We have also designed an autocatalytic system based on the fast and efficient RNA cleaving 10-23 deoxyribozyme. In this system, two complementary deoxyribozymes have been inactivated by circularization. The circular deoxyribozymes are capable of serving as substrates for the linear enzymes such that linearization results in a cascade of cleaving reactions that exhibits exponential growth. A selection scheme based on this reaction resulted in optimal sequence selection and demonstrates the first in vitro selection experiment conducted in the absence of proteins.
Forces governing nucleic acid compaction investigated by quantitative crosslinking and FRET
(2022-05-06) Hamilton, Ian Scott; Russell, Rick, 1969-; Elber, Ron; Ren, Pengyu; Finkelstein, Ilya
Nucleic acid compaction is ubiquitous in biology, from chromatin and genome packing to RNA folding. Nature achieves these different processes in several different ways, but in all cases nucleic acid helices must be brought into contact or close proximity, often at a substantial free energy penalty. We developed quantitative disulfide crosslinking and tethered FRET techniques to measure the energetics of these fundamental interactions, and to explore the factors influencing them. We used a bimolecular crosslinking assay to measure the electrostatic repulsion governing unmediated encounters between DNA helices. By varying the length of the crosslinker probes, we established the size of the ion atmosphere surrounding the helices under different ionic conditions. We complemented these results with a FRET assay of the relative orientations of two DNA helices tethered by a short PEG linker. In the presence of Na⁺ or Mg²⁺, both bimolecular crosslinking and tethered FRET revealed a transition from a low salt regime dominated by electrostatic repulsion to a high salt electrically screened regime dominated by thermal motion. With Co(NH₃)₆³⁺, both assays revealed a transition to a net attractive regime with dilute phase characteristics consistent with previously observed Co-induced DNA condensates, suggesting that such condensates are the result of pairwise interhelical attraction. We also used an intramolecular crosslinking assay to measure the energetics of sharply bent conformations for a selection of RNA two-helix junctions. For model oligo-U bulges and junctions (Uₓ₌₂₋₇Uᵧ₌₀₋₃), junctions with more unpaired nucleotides on the longer strand (Uₓ) displayed more frequent sharp bending, with little dependence on the length of the shorter strand (Uᵧ). We probed different bent conformations by varying the crosslinker positions, identifying conformational preferences up to ~3 kcal/mol among different bent states. We also examined four naturally-occurring junction sequences (two from ribosomal RNAs and two from group I introns) that are sharply bent in their native folds. We found that these natural junction sequences do not selectively stabilize specific bent conformations corresponding to their native folds, instead exhibiting conformational lability, and behaving much like their oligo-U topological counterparts.
Group II Intron-Based Gene Targeting Reactions in Eukaryotes
(Public Library of Science, 2008-09-01) Mastroianni, Marta; Watanabe, Kazuo; White, Travis B.; Zhuang, Fanglei; Vernon, Jamie; Matsuura, Manabu; Wallingford, John; Lambowitz, Alan M.
Background: Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors (“targetrons”) with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes. Methodology/Principal Findings: By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg2+ concentrations. By supplying additional Mg2+, site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg2+-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization. Conclusions/Significance: Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms.
Nucleic acid based reagentless optical biosensors
(2004-08) Rajendran, Manjula, 1975-; Ellington, Andrew D.
Nucleic acid binding species called aptamers are intrinsically well suited for biosensing applications. Not only do they have universal target recognition abilities, but they are also specific, exhibit high binding affinities, and can be easily engineered. Aptamers that contain fluorophores in conformationally labile positions and that signal the presence of their cognate ligands in solution have been termed `signaling aptamers´. An advantage with these optical nucleic acid based sensors is that they are “reagentless biosensors” and can enable real time, continuous, reagentless detection of targets in solution. Although several different strategies have been used to generate signaling aptamers, these methods are not general and have to be adapted to address the individual needs of different aptamers. We have developed a direct selection method that couples ligand binding to signaling, such that all selected aptamers can also of-necessity signal the presence of target analyte. Selection is based on analyte binding mediated conformational changes, vii and consequent release of a complementary quencher oligonucleotide by a fluorescently labeled aptamer beacon, thus generating a fluorescent signal. The selection method was initially proofed using oligonucleotides as target analytes. A novel type of molecular beacons were thus selected that exhibited performance characteristics comparable to those of designed molecular beacons. The selection method could also be successfully adapted to isolate aptamer beacons against small metal ions as analytes. The principal advantage of this method is that it is based on in vitro selection, and can potentially be applied to a wide variety of target molecules, thereby allowing the development of numerous reagentless biosensors. We also employed in vitro selection to isolate RNA aptamers against MEK-1, a protein kinase known to have diagnostic importance for cancer. In addition, signaling aptamers were also designed against various medically relevant proteins which can be used for diagnostic applications.
Nucleic acid localization in diagnostics and therapeutics
(2010-05) Pai, Supriya Sudhakar; Ellington, Andrew D.; Georgiou, George; Williams, Robert O.; Tian, Ming; Sullivan, Chris
Aptamers are short nucleic acid ligands generated by the process of iterative selection. Nucleic acid counterparts to protein antibodies, aptamers bind their targets with relatively high affinities by assuming characteristic shapes. Highly thermostable, open to manipulations and non-immunogenic, these olignucleotides can be readily adapted to a variety of diagnostic assays and harvested for their therapeutic potential. We have particularly focused on the unique prospects that stem from their localization patterns both in vitro and in vivo. While several assays exist for protein diagnostics, many of these are limited by the amount of target they can detect. To overcome these limitations it might prove effective to couple protein detection with nucleic acid based amplification. The Proximity Ligation Assay (PLA) is an innovative technique that facilitates protein detection on a zeptomolar range by amplifying a tiny signal via the polymerase chain reaction. PLA is based on the concept that two DNA tags when co-localized adjacent to one another on a protein surface and ligated via a connector nucleotide will form a unique amplicon that can detected using real-time PCR and in turn detect the protein. We have adapted PLA to the peptide based detection of Bacillus spores as well as the RNA aptamer based detection of cancer cells. Highly sensitive and specific, nucleic acid based PLA could serve as a promising tool in diagnostics. Aptamers have also been analyzed for their localization patterns in vivo. Using two anti-prostate specific membrane antigen RNA aptamers, we have demonstrated that there is an inherent bias for some circulating oligonucleotides over others based solely on their sequence. This phenomenon has also been explored in cancer models of mice for persistence of specific aptamers over others in tumors for therapy. An in vivo “Stealth” selection scheme has also been designed and executed to hunt for stable and robust aptamer species that are naturally chosen for survival within a mouse system. Generation of such ligands could benefit several therapeutic ventures such as targeted drug delivery past complex vasculature as in the case of the blood:brain barrier.
Probing HIV-1 NC-induced nucleic acid structural rearrangement by single-molecule fluorescence spectroscopy
(2007-12) Zeng, Yining, 1976-; Barbara, P. F. (Paul F.), 1953-
Reverse transcription of HIV-1 genome involves multiple nucleic acids structural rearrangements chaperoned by nucleocapsid protein (NC). One such critical step is that the DNA transactivation response element (TAR) anneals to it complementary RNA region on the genome. It has been challenging to investigate mechanistic details on the annealing process because of the involvement of heterogeneous sets of protein/nucleic acid complexes. Here, we use single-molecule spectroscopy to study the NC induced melting of nucleic acid structure and the annealing activity of different regions along TAR structure. We find that NC induced secondary fluctuations are limited to the terminal stems, and the mechanism for the fluctuations is complex. By employing complementary targeted oligomers, we kinetically trap and investigate stable states of the putative nucleation complexes for the annealing process. This single molecule spectroscopy method directly probes kinetic reversibility and the chaperone role of NC at various stages along the reaction sequence. The new results lead to detailed understanding of NC chaperoned reverse-annealing and the partially annealed conformational sub-states. Argininamide, because of its specific binding to the loop regions of TAR, was studied on its specific inhibition to strand transfer. The loopmediated annealing is found to be more potentially inhibited than stem-mediated one.
Scaling up DNA computation with next-generation sequencing and modified nucleic acids
(2022-05-05) Wang, Siyuan Stella; Ellington, Andrew D.; Soloveichik, David; Finkelstein, Ilya; Marcotte, Edward; Anslyn, Eric
A central goal of biomolecular engineering is the construction of tools to manipulate nanoscale processes. DNA has proved to be a programmable material suited for this task. DNA strand displacement reactions can be designed to process chemical information in the form of concentrations and sequences. DNA nanotechnology has thus far produced devices for the detection of disease biomarkers, performed computation on chemical inputs, powered mechanical action at both the nanoscale and the macroscale, and assembled precise sub-micron structures from the bottom up. This dissertation addresses three main topics. First, we develop predictive models for non-canonical nucleic acid hybridization that enable rational design. Second, we show how rationally designed DNA strand displacement reactions can be used to perform computations on information stored in DNA. Third, we present nucleic acid computation with both strand displacement and transcription and discuss strategies for facilitating the scale up of networks. Finally, we discuss data storage in nucleic acid variants in the appendix. Rational design of DNA circuits and structures is possible because the thermodynamics of DNA and RNA hybridization can be approximated using a nearest-neighbor model. The parameters of this model are typically experimentally determined through the hyperchromism of denatured nucleic acids. This is measured through low-throughput UV-Vis spectrophotometry melting experiments that require a sizable amount of duplexes for a large set of sequences. For non-canonical nucleic acids or non-standard interactions, this characterization can be prohibitively costly and time consuming. Initially, we considered repurposing a next-generation sequencing (NGS) platform for high-throughput mapping of nucleic acid hybridization across a large sequence space; however, we found that the platform is suitable for mapping protein-nucleic acid interactions but not nucleic acid-nucleic acid hybridization due to its dynamic range. We then assessed whether high-resolution melting (HRM) can be used as a rapid method for determining approximate model parameters and found that HRM models can predict relative stabilities between duplexes of different sequences. Using this method, we developed a predictive model for phosphorothioate DNA which we then applied to the design of a phosphorothioate-modified catalytic hairpin assembly circuit. DNA strand displacement reactions can be used not only to manipulate chemical information in the form of concentration, but also to read and write to more permanent forms of information, such as sequence and secondary structure. We developed and demonstrated a DNA data storage scheme that enables in-memory computation. DNA is a promising data storage medium for meeting today's rapidly growing data storage needs; however, because computation on the stored data is usually performed in silico, strands must be sequenced and re-synthesized at every read-write cycle. Our scheme circumvents the bottleneck of de novo oligonucleotide synthesis by updating information using strand displacement cascades that result in sequence changes readable by NGS. We experimentally demonstrated two algorithms - binary counting and cellular automaton Rule 110 - and additionally showed that biologically-occurring DNA sequences without sequence design can be repurposed for storage and computation. Our scheme is capable of computation on multiple data in parallel, as well as random access and sequential computation, allowing for scaled up storage. Programmable chemical computation is also possible with enzymatic reactions such as transcription. Catalytic activity from enzymes has the potential to simplify circuit design and produce biologically potent signals. Practical concerns to expanding chemical computation circuits such as transcription networks include limited readout of signals and time-consuming purification. We addressed these concerns by expanding on previous efforts to build scalable in vitro transcription networks. We updated a single-stranded inhibitory transcription switch design for compatibility with multiplexed NGS readout and developed an analogous single-stranded switch that is activated by nucleic acid signals.
SELEX: a tool to study the sequence specific molecular recognition of single stranded nucleic acids
(2004) Manimala, Joseph Chacko; Anslyn, Eric V.; Ellington, Andrew D.
Self-assembly of functionalized synthetic nucleic acid bases
(2005) Jayawickramarajah, Janarthanan; Sessler, Jonathan L.
Inspired by the significance and utility of hydrogen bond driven self-assembly, as exemplified through base-pairing interactions of double helical DNA, this dissertation discusses the synthetic preparation and structural analysis of a number of functionalized supramolecular ensembles derived from nucleic acid base-pairing. Specifically, three projects are detailed. Each of these projects, although under the general purview of basepairing, addresses a distinct issue in the field of molecular recognition. The first chapter will serve to introduce the general modalities of base-pairing as well as provide an overview of the diverse synthetic supramolecular ensembles that have been prepared by exploiting such interactions. Chapter 2 will detail the development and self-assembly properties of a novel guanosine-cytidine dinucleoside, which is capable of forming a cyclic ensemble through Watson-Crick hydrogen bonding. Chapter 3 will focus on the development of a porphyrin-fullerene derived photoinduced electron transfer model system, which self-assembles via Watson-Crick base-pairing interactions. Chapter 4 will introduce a new pyrrole-tethered purine nucleoside, which is capable of forming an extended three-point Hoogsteen-type base-pairing interaction with guanosine. In addition, this chapter will also investigate the capability of this purine derivative in disrupting guanosine-derived self-association. At the end of each of these chapters, a section pertaining to ongoing efforts and proposed future research is included. Chapter 5 provides experimental methods and characterization data.