Watson-Crick Base-pairing Interactions Research Articles

DNA Logic Gates for Small Molecule Activation Circuits in Cells.

DNA-based devices such as DNA logic gates self-assemble into supramolecular structures, as dictated by the sequences of the constituent oligonucleotides and their predictable Watson-Crick base pairing interactions. The programmable nature of DNA-based devices permits the design and implementation of DNA circuits that interact in a dynamic and sequential manner capable of spatially arranging disparate DNA species. Here, we report the application of an activatable fluorescence reporter based on a proximity-driven inverse electron demand Diels-Alder (IEDDA) reaction and its robust integration with DNA strand displacement circuits. In response to specific DNA input patterns, sequential strand displacement reactions are initiated and culminate in the hybridization of two modified DNA strands carrying probes capable of undergoing an IEDDA reaction between a vinyl-ether-caged fluorophore and its reactive partner tetrazine, leading to the activation of fluorescence. This approach provides a major advantage for DNA computing in mammalian cells since circuit degradation does not induce fluorescence, in contrast to traditional fluorophore-quencher designs. We demonstrate the robustness and sensitivity of the reporter by testing its ability to serve as a readout for DNA logic circuits of varying complexity inside cells.

Short PolyA RNA Homopolymers Undergo Mg2+-Mediated Kinetically Arrested Condensation.

RNA-RNA interactions have increasingly been recognized for their potential to shape the mesoscale properties of biomolecular condensates, influencing morphology, organization, and material state through networking interactions. While most studies have focused on networking via Watson-Crick base pairing interactions, previous work has suggested a potential for noncanonical RNA-RNA interactions to also give rise to condensation and alter overall material state. Here, we test the phase separation of short polyA RNA (polyrA) homopolymers. We discover and characterize the potential for short polyrA sequences to form RNA condensates at lower Mg2+ concentrations than previously observed, which appear as internally arrested droplets with slow polyrA diffusion despite continued fusion. Our work also reveals a negative cooperativity effect between the effects of Mg2+ and Na+ on polyrA condensation. Finally, we observe that polyrA sequences can act as promoters of phase separation in mixed sequences. These results demonstrate the potential for noncanonical interactions to act as networking stickers, leading to specific condensation properties inherent to polyrA composition and structure, with implications for the fundamental physical chemistry of the system and function of polyA RNA in biology.

Reconfigurable Two-Dimensional DNA Lattices: Static and Dynamic Angle Control.

Branched DNA motifs serve as the basic construction elements for all synthetic DNA nanostructures. However, precise control of branching orientation remains a key challenge to further heighten the overall structural order. In this study, we use two strategies to control the branching orientation. The first one is based on immobile Holliday junctions which employ specific nucleotide sequences at the branch points which dictate their orientation. The second strategy is to use angle-enforcing struts to fix the branching orientation with flexible spacers at the branch points. We have also demonstrated that the branching orientation control can be achieved dynamically, either by canonical Watson-Crick base pairing or non-canonical nucleobase interactions (e.g., i-motif and G-quadruplex). With precise angle control and feedback from the chemical environment, these results will enable novel DNA nanomechanical sensing devices, and precisely-ordered three-dimensional architectures.

Programmable and Multifunctional DNA-Based Materials for Biomedical Applications.

DNA encodes the genetic information; recently, it has also become a key player in material science. Given the specific Watson-Crick base-pairing interactions between only four types of nucleotides, well-designed DNA self-assembly can be programmable and predictable. Stem-loops, sticky ends, Holliday junctions, DNA tiles, and lattices are typical motifs for forming DNA-based structures. The oligonucleotides experience thermal annealing in a near-neutral buffer containing a divalent cation (usually Mg2+ ) to produce a variety of DNA nanostructures. These structures not only show beautiful landscape, but can also be endowed with multifaceted functionalities. This Review begins with the fundamental characterization and evolutionary trajectory of DNA-based artificial structures, but concentrates on their biomedical applications. The coverage spans from controlled drug delivery to high therapeutic profile and accurate diagnosis. A variety of DNA-based materials, including aptamers, hydrogels, origamis, and tetrahedrons, are widely utilized in different biomedical fields. In addition, to achieve better performance and functionality, material hybridization is widely witnessed, and DNA nanostructure modification is also discussed. Although there are impressive advances and high expectations, the development of DNA-based structures/technologies is still hindered by several commonly recognized challenges, such as nuclease instability, lack of pharmacokinetics data, and relatively high synthesis cost.

Construction of a Bivalent Thrombin Binding Aptamer and Its Antidote with Improved Properties

Aptamers are short synthetic DNA or RNA oligonucleotides that adopt secondary and tertiary conformations based on Watson–Crick base-pairing interactions and can be used to target a range of different molecules. Two aptamers, HD1 and HD22, that bind to exosites I and II of the human thrombin molecule, respectively, have been extensively studied due to their anticoagulant potentials. However, a fundamental issue preventing the clinical translation of many aptamers is degradation by nucleases and reduced pharmacokinetic properties requiring higher dosing regimens more often. In this study, we have chemically modified the design of previously described thrombin binding aptamers targeting exosites I, HD1, and exosite II, HD22. The individual aptamers were first modified with an inverted deoxythymidine nucleotide, and then constructed bivalent aptamers by connecting the HD1 and HD22 aptamers either through a triethylene glycol (TEG) linkage or four consecutive deoxythymidines together with an inverted deoxythymidine nucleotide at the 3′-end. The anticoagulation potential, the reversal of coagulation with different antidote sequences, and the nuclease stability of the aptamers were then investigated. The results showed that a bivalent aptamer RNV220 containing an inverted deoxythymidine and a TEG linkage chemistry significantly enhanced the anticoagulation properties in blood plasma and nuclease stability compared to the existing aptamer designs. Furthermore, a bivalent antidote sequence RNV220AD efficiently reversed the anticoagulation effect of RNV220 in blood plasma. Based on our results, we believe that RNV220 could be developed as a potential anticoagulant therapeutic molecule.

Structures of a DNA Polymerase Inserting Therapeutic Nucleotide Analogues.

Members of the nucleoside analogue class of cancer therapeutics compete with canonical nucleotides to disrupt numerous cellular processes, including nucleotide homeostasis, DNA and RNA synthesis, and nucleotide metabolism. Nucleoside analogues are triphosphorylated and subsequently inserted into genomic DNA, contributing to the efficacy of therapeutic nucleosides in multiple ways. In some cases, the altered base acts as a mutagen, altering the DNA sequence to promote cellular death; in others, insertion of the altered nucleotide triggers DNA repair pathways, which produce lethal levels of cytotoxic intermediates such as single and double stranded DNA breaks. As a prerequisite to many of these biological outcomes, the modified nucleotide must be accommodated in the DNA polymerase active site during nucleotide insertion. Currently, the molecular contacts that mediate DNA polymerase insertion of modified nucleotides remain unknown for multiple therapeutic compounds, despite decades of clinical use. To determine how modified bases are inserted into duplex DNA, we used mammalian DNA polymerase β (pol β) to visualize the structural conformations of four therapeutically relevant modified nucleotides, 6-thio-2'-deoxyguanosine-5'-triphosphate (6-TdGTP), 5-fluoro-2'-deoxyuridine-5'-triphosphate (5-FdUTP), 5-formyl-deoxycytosine-5'-triphosphate (5-FodCTP), and 5-formyl-deoxyuridine-5'-triphosphate (5-FodUTP). Together, the structures reveal a pattern in which the modified nucleotides utilize Watson-Crick base pairing interactions similar to that of unmodified nucleotides. The nucleotide modifications were consistently positioned in the major groove of duplex DNA, accommodated by an open cavity in pol β. These results provide novel information for the rational design of new therapeutic nucleoside analogues and a greater understanding of how modified nucleotides are tolerated by polymerases.

Assembling RNA Nanoparticles.

RNA nanoparticles are designed and self-assembled according to noncanonical interactions of naturally conserved RNA motifs and/or canonical Watson-Crick base-pairing interactions, which have potential applications in gene therapy and nanomedicine. These artificially engineered nanoparticles are mainly synthesized from in vitro transcribed RNAs, purified by denaturing and native polyacrylamide gel electrophoresis (PAGE), and characterized with native PAGE, AFM, and TEM technologies. The protocols of in vitro transcription, denaturing and native PAGE, and RNA nanoparticle self-assembly are described in detail.

DNA Polymerase-Directed Hairpin Assembly for Targeted Drug Delivery and Amplified Biosensing.

Due to the predictable conformation and programmable Watson-Crick base-pairing interactions, DNA has proven to be an attractive material to construct various nanostructures. Herein, we demonstrate a simple model of DNA polymerase-directed hairpin assembly (PDHA) to construct DNA nanoassemblies for versatile applications in biomedicine and biosensing. The system consists of only two hairpins, an initiator and a DNA polymerase. Upon addition of aptamer-linked initiator, the inert stems of the two hairpins are activated alternately under the direction of DNA polymerase, which thus grows into aptamer-tethered DNA nanoassemblies (AptNAs). Moreover, through incorporating fluorophores and drug-loading sites into the AptNAs, we have constructed multifunctional DNA nanoassemblies for targeted cancer therapy with high drug payloads and good biocompatibility. Interestingly, using the as-prepared AptNAs as building blocks, DNA nanohydrogels are self-assembled after centrifugation driven by liquid crystallization and dense packaging of DNA duplexes. Taking advantage of easy preparation and high loading capacity, the PDHAs are readily extended to the fabrication of a label-free biosensing platform, achieving amplified electrochemical detection of microRNA-21 (miR-21) with a detection limit as low as 0.75 fM and a dynamic range of 8 orders of magnitude. This biosensor also demonstrates excellent specificity to discriminate the target miR-21 from the control microRNAs and even the one-base mismatched one and further performs well in analyzing miR-21 in MCF-7 tumor cells.

Synthesis of alanyl nucleobase amino acids and their incorporation into proteins

Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail.

ChemInform Abstract: Aptamer‐Integrated DNA Nanostructures for Biosensing, Bioimaging and Cancer Therapy

AbstractReview: 150 refs.

Structural DNA nanotechnology: state of the art and future perspective.

Over the past three decades DNA has emerged as an exceptional molecular building block for nanoconstruction due to its predictable conformation and programmable intra- and intermolecular Watson–Crick base-pairing interactions. A variety of convenient design rules and reliable assembly methods have been developed to engineer DNA nanostructures of increasing complexity. The ability to create designer DNA architectures with accurate spatial control has allowed researchers to explore novel applications in many directions, such as directed material assembly, structural biology, biocatalysis, DNA computing, nanorobotics, disease diagnosis, and drug delivery. This Perspective discusses the state of the art in the field of structural DNA nanotechnology and presents some of the challenges and opportunities that exist in DNA-based molecular design and programming.

Thermodynamic Basis for Engineering High-Affinity, High-Specificity Binding-Induced DNA Clamp Nanoswitches

Naturally occurring chemoreceptors almost invariably employ structure-switching mechanisms, an observation that has inspired the use of biomolecular switches in a wide range of artificial technologies in the areas of diagnostics, imaging, and synthetic biology. In one mechanism for generating such behavior, clamp-based switching, binding occurs via the clamplike embrace of two recognition elements onto a single target molecule. In addition to coupling recognition with a large conformational change, this mechanism offers a second advantage: it improves both affinity and specificity simultaneously. To explore the physics of such switches we have dissected here the thermodynamics of a clamp-switch that recognizes a target DNA sequence through both Watson-Crick base pairing and triplex-forming Hoogsteen interactions. When compared to the equivalent linear DNA probe (which relies solely on Watson-Crick interactions), the extra Hoogsteen interactions in the DNA clamp-switch increase the probe's affinity for its target by ∼0.29 ± 0.02 kcal/mol/base. The Hoogsteen interactions of the clamp-switch likewise provide an additional specificity check that increases the discrimination efficiency toward a single-base mismatch by 1.2 ± 0.2 kcal/mol. This, in turn, leads to a 10-fold improvement in the width of the "specificity window" of this probe relative to that of the equivalent linear probe. Given these attributes, clamp-switches should be of utility not only for sensing applications but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines.

Optochemical control of deoxyoligonucleotide function via a nucleobase-caging approach.

Synthetic oligonucleotides have been extensively applied tocontrol a wide range of biological processes such as gene expression, gene repair, DNA replication, and protein activity. Based on well-established sequence design rules that typically rely on Watson-Crick base pairing interactions researchers can readily program the function of these oligonucleotides. Therefore oligonucleotides provide a flexible platform for targeting a wide range of biological molecules, including DNA, RNA, and proteins. In addition, oligonucleotides are commonly used research tools in cell biology and developmental biology. However, a lack of conditional control methods has hampered the precise spatial and temporal regulation of oligonucleotide activity, which limits the application of these reagents to investigate complex biological questions. Nature controls biological function with a high level of spatial and temporal resolution and in order to elucidate the molecular mechanisms of biological processes, researchers need tools that allow for the perturbation of these processes with Nature's precision. Light represents an excellent external regulatory element since irradiation can be easily controlled spatially and temporally. Thus, researchers have developed several different methods to conditionally control oligonucleotide activity with light. One of the most versatile strategies is optochemical regulation through the installation and removal of photolabile caging groups on oligonucleotides. To produce switches that can control nucleic acid function with light, chemists introduce caging groups into the oligomer backbone or on specific nucleobases to block oligonucleotide function until the caging groups are removed by light exposure. In this Account, we focus on the application of caged nucleobases to the photoregulation of DNA function. Using this approach, we have both activated and deactivated gene expression optochemically at the transcriptional and translational level with spatial and temporal control. Specifically, we have used caged triplex-forming oligomers and DNA decoys to regulate transcription, and we have regulated translation with light-activated antisense agents. Moreover, we also discuss strategies that can trigger DNA enzymatic activity, DNA amplification, and DNA mutagenesis by light illumination. More recently, we have developed light-activated DNA logic operations, an advance that may lay the foundation for the optochemical control of complex DNA calculations.

Structural, energetic and dynamic properties of guanine(C8)–thymine(N3) cross-links in DNA provide insights on susceptibility to nucleotide excision repair

The one-electron oxidation of guanine in DNA by carbonate radical anions, a decomposition product of peroxynitrosocarbonate which is associated with the inflammatory response, can lead to the formation of intrastrand cross-links between guanine and thymine bases [Crean et al. (Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines. Nucleic Acids Res. 2008; 36: 742–755.)]. These involve covalent bonds between the C8 positions of guanine (G*) and N3 of thymine (T*) in 5′-d(…G*pT*…) and 5′-d(…G*pCpT*…) sequence contexts. We have performed nucleotide excision repair (NER) experiments in human HeLa cell extracts which show that the G*CT* intrastrand cross-link is excised with approximately four times greater efficiency than the G*T* cross-link embedded in 135-mer DNA duplexes. In addition, thermal melting studies reveal that both lesions significantly destabilize duplex DNA, and that the destabilization induced by the G*CT* cross-link is considerably greater. Consistent with this difference in NER, our computations show that both lesions dynamically distort and destabilize duplex DNA. They disturb Watson–Crick base-pairing and base-stacking interactions, and cause untwisting and minor groove opening. These structural perturbations are much more pronounced in the G*CT* than in the G*T* cross-link. Our combined experimental and computational studies provide structural and thermodynamic understanding of the features of the damaged duplexes that produce the most robust NER response.

Reversibly switchable DNA nanocompartment on surfaces: experiments, applications, and theory

This paper summarizes our studies of DNA nano-compartement in recent years. Biological macromolecules have been used to fabricate many nanostructures, bio-devices, and biomimetics because of their physical and chemical properties. But dynamic nanostructure and bio-machinery that depend on collective behavior of biomolecules have not been demonstrated. Here, we report the design of DNA nanocompartment on surfaces that exhibit reversible changes in molecular mechanical properties. Such molecular nanocompartment is served to encage molecules, switched by the collective effect of Watson-Crick base-pairing interactions. This effect is used to investigate the dynamic process of nanocompartment switching and molecular thermosensing, as well as perform molecular recognition. Further, we found that ‘fuel’ strands with single-base variation cannot afford an efficient closing of nanocompartment, which allows highly sensitive label-free DNA array detection. Theoretical analysis and computer simulations confirm our experimental observations, which are discussed in this review paper. Our results suggest that DNA nanocompartment can be used as building blocks for complex biomaterials, because its core functions are independent of substrates and mediators.

Self-association of adenine-dependent hairpin ribozymes

Hairpin ribozymes are flexible molecules that catalyse reversible self-cleavage after the docking of two independently folded internal loops, A and B. The activities, self-association and structures in solution of two 85 base adenine-dependent hairpin ribozymes (ADHR1 and ADHR2) were studied by native gel electrophoresis, analytical centrifugation, and small angle neutron scattering. Bi-molecular RNA interactions such as linear-linear, loop-loop, loop-linear or kissing interactions have been found to be important in the control of various biological functions, and hairpin loops present rich potential for establishing both intra- and intermolecular interactions through standard Watson-Crick base pairing or non-canonical interactions. Similar results were obtained for ADHR1 and ADHR2. At room temperature, they indicated end-to-end self-association of the ribozymes in rod-like structures with a cross-section corresponding to two double strands side-by-side. Dimers, which predominate at low concentration ( approximately 0.1 mg/ml), associate into longer rods, with increasing concentration ( approximately 1 mg/ml). Above 65 degrees C, the dimers and rods dissociated into compact monomers, with a radius of gyration similar to that of tRNA (about 70 bases). The dimers were non-active for catalysis, which suggests that dimer formation, probably by preventing the correct docking of loops A and B, could act as an inhibition mechanism for the regulation of hairpin ribozyme catalysis.

Reversibly switchable DNA nanocompartment on surfaces

Biological macromolecules have been used to fabricate many nanostructures, biodevices and biomimetics because of their physical and chemical properties. But dynamic nanostructure and biomachinery that depend on collective behavior of biomolecules have not been demonstrated. Here, we report the design of DNA nanocompartments on surfaces that exhibit reversible changes in molecular mechanical properties. Such molecular nanocompartments are used to encage molecules, switched by the collective effect of Watson-Crick base-pairing interactions. This effect is used to perform molecular recognition. Furthermore, we found that 'fuel' strands with single-base variation cannot afford an efficient closing of nanocompartments, which allows highly sensitive label-free DNA array detection. Our results suggest that DNA nanocompartments can be used as building blocks for complex biomaterials because its core functions are independent of substrates and mediators.

Using Double-stranded RNA to Prevent in Vitro and in Vivo Viral Infections by Recombinant Baculovirus

Introduction of double-stranded RNA (dsRNA) into a wide variety of cells and organisms results in post-transcriptional depletion of the homologue endogenous mRNA. This well-preserved phenomenon known as RNA interference (RNAi) is present in evolutionarily diverse organisms such as plants, fungi, insects, metazoans, and mammals. Because the identification of the targeted mRNA by the RNAi machinery depends upon Watson-Crick base-pairing interactions, RNAi can be exquisitely specific. We took advantage of this powerful and flexible technique to demonstrate that selective silencing of genes essential for viral propagation prevents in vitro and in vivo viral infection. Using the baculovirus Autographa californica, a rapidly replicating and highly cytolytic double-stranded DNA virus that infects many different insect species, we show for the first time that introduction of dsRNA from gp64 and ie1, two genes essential for baculovirus propagation, results in prevention of viral infection in vitro and in vivo. This is the first report demonstrating the use of RNAi to inhibit a viral infection in animals. This inhibition was specific, because dsRNA from the polyhedrin promoter (used as control) or unrelated dsRNAs did not affect the time course of viral infection. The most relevant consequences from the present study are: 1) RNAi offers a rapid and efficient way to interfere with viral genes to assess the role of specific proteins in viral function and 2) using RNAi to interfere with viral genes essential for cell infection may provide a powerful therapeutic tool for the treatment of viral infections.

Synthesis and spectroscopic properties of the first phthalocyanine–nucleobase conjugates

The adenine-containing zinc(II) phthalocyanines 3 , 4a , and 4b have been prepared by standard O-alkylation of the tetrahydroxy analogue 1 . These macrocycles exhibit rather strong intermolecular interactions resulting in a poor solubility in organic solvents and unusual spectral properties that are highly solvent-dependent. Self-assembly of the tetra-adenine phthalocyanine 3 and the thymine-containing 9,10-anthraquinone 6 through the Watson–Crick base-pairing interactions has also been demonstrated by fluorescence quenching experiments.

The Donor Substrate Site within the Peptidyl Transferase Loop of 23 S rRNA and its Putative Interactions with the CCA-end of N-blocked Aminoacyl-tRNA Phe

An RNA region associated with the donor substrate site, located at the base of the peptidyl transferase loop of 23 S rRNA, was subjected to a comprehensive single-site mutational study. Growth phenotypes of Escherichia colicells were characterized on induction of synthesis of the mutated rRNAs and the mutated ribosomes were tested, selectively, for their capacity to generate peptide bonds under the conditions of the “fragment” assay. Most of the mutants exhibited dominant or recessive lethal growth phenotypes and, in general, defective growth correlated with low activities in peptide bond formation, although exceptions were observed with normal growth and low activities, and vice versa. All these phenotypes are consistent with defects occurring in the structure of the ribosomal donor site and/or the capacity of the donor substrate to enter or leave this site. A compensating base change approach was employed to test for Watson-Crick base-pairing interactions between the -CCA end of the P-site bound tRNA Pheand this region of the peptidyl-transferase loop. Single nucleotide substitutions were introduced into the -CCA end of tRNA Pheand the ability of the 3′-terminal pentanucleotide fragments to act as donor substrates was examined for ribosomes carrying the different mutated 23 S rRNAs. No evidence was found for the occurrence of Watson-Crick base-pairing interactions. However, the data are consistent with the formation of a Hoogsteen pair between the 3′-terminal adenosine base of the donor substrate and U2585 of the 23 S rRNA.