Watson-Crick Pairing Research Articles

A Dynamic Control Center Based on a DNA Reaction Network for Programmable Building of DNA Nanostructures.

DNA-based nanostructures allow for complex self-assembly with nanometer precision through the specificity of Watson-Crick base pairing, but network behavior-directed control of the kinetic process is less studied. Here we show how the DNA reaction network (DRN), which has emerged as a reliable and programmable way to implement artificial network dynamics, can be built as the control center of programmable nanostructures, allowing spatiotemporal control over the dynamic behavior of DNA nanotubes. We chose a common network motif in biological control systems, the feed-forward loop, as the model network and demonstrated that dynamic behaviors, such as self-tuning control and multilayer hierarchical assembly, could be programmed by constructing an inhibition network and an excitation network, separately, in buffer solution and inside protocells.

Self-transfecting GMO-PMO chimera targeting Nanog enable gene silencing in vitro and suppresses tumor growth in 4T1 allografts in mouse

Phosphorodiamidate morpholino oligonucleotide (PMO)-based antisense reagents cannot enter cells without the help of a delivery technique, which limits their clinical applications. To overcome this problem, self-transfecting guanidinium-linked morpholino (GMO)-PMO or PMO-GMO chimeras have been explored as antisense agents. GMO facilitates cellular internalization and participates in Watson-Crick base pairing. Targeting NANOG in MCF7 cells resulted in decline of the whole epithelial to mesenchymal transition (EMT) and stemness pathway, evident through its phenotypic manifestations, all of which were promulgated in combination with Taxol due to downregulation of MDR1 and ABCG2. GMO-PMO-mediated knockdown of no tail gene resulted in desired phenotypes in zebrafish even upon delivery after 16-cell stages. In BALB/c mice, 4T1 allografts were found to regress via intra-tumoral administration of NANOG GMO-PMO antisense oligonucleotides (ASOs), which was associated with occurrence of necrotic regions. GMO-PMO-mediated tumor regression restored histopathological damage in liver, kidney, and spleen caused by 4T1 mammary carcinoma. Serum parameters of systemic toxicity indicated that GMO-PMO chimeras are safe. To the best of our knowledge, self-transfecting antisense reagent is the first report since the discovery of guanidinium-linked DNA (DNG), which could be useful as a combination cancer therapy and, in principle, can render inhibition of any target gene without using any delivery vehicle.

Ratiometric fluorescence biosensing of silver NanoCluster Beacons for ATP detection based on ligation- triggered rolling cycle amplification

NanoCluster Beacon (NCB) provided a label-free ratiometric fluorescence method with obvious dual-color signal output. In this work, a novel method was developed for ATP detection by coupling NCB and rolling cycle amplification (RCA). NCB was composed of the silver clusters (P2-AgNCs) and the converter (P3), and the linker sequence (P1) kept them in close proximity by Watson-Crick base pairing. The fluorescence performance of NCB was correlated with the status of the AgNCs. Controllable color switching from green to red was thus achieved. ATP served as cofactor of T4 DNA ligase to catalyze the ligation reaction, then the subsequent RCA reaction produced a long-stranded linear DNA with a lot of P1 sequences that assembled with NCBs. So that the green fluorescence of AgNCs (λem = 542 nm) was transformed to bright red fluorescence (λem = 630 nm), lighting up the ratiometric fluorescence of NCBs. Under the optimized conditions, the value of F630 nm/F542 nm (F630/542) was linearly correlated with the logarithmic ATP concentration with a detection limit of 122 pM. Furthermore, the proposed method showed good performance in human serums, indicating its potential to be applied in clinical diagnosis.

Synergistic binding of actinomycin D and echinomycin to DNA mismatch sites and their combined anti-tumour effects.

Combination cancer chemotherapy is one of the most useful treatment methods to achieve a synergistic effect and reduce the toxicity of dosing with a single drug. Here, we use a combination of two well-established anticancer DNA intercalators, actinomycin D (ActD) and echinomycin (Echi), to screen their binding capabilities with DNA duplexes containing different mismatches embedded within Watson-Crick base-pairs. We have found that combining ActD and Echi preferentially stabilised thymine-related T:T mismatches. The enhanced stability of the DNA duplex-drug complexes is mainly due to the cooperative binding of the two drugs to the mismatch duplex, with many stacking interactions between the two different drug molecules. Since the repair of thymine-related mismatches is less efficient in mismatch repair (MMR)-deficient cancer cells, we have also demonstrated that the combination of ActD and Echi exhibits enhanced synergistic effects against MMR-deficient HCT116 cells and synergy is maintained in a MMR-related MLH1 gene knockdown in SW620 cells. We further accessed the clinical potential of the two-drug combination approach with a xenograft mouse model of a colorectal MMR-deficient cancer, which has resulted in a significant synergistic anti-tumour effect. The current study provides a novel approach for the development of combination chemotherapy for the treatment of cancers related to DNA-mismatches.

Systematic Investigation of Tether Length and Phosphorus Configuration in Backbone Constrained Macrocyclic Nucleic Acids to Modulate Binding Kinetics for RNA.

We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson-Crick base pairing for modulating the binding kinetics of single-stranded oligonucleotides (ONs) using bis-phosphonate esters bridging hydrocarbon tethers to provide 11- and 15-membered macrocyclic analogues. In this manuscript, we describe the synthesis of all eight P-stereoisomers of macrocyclic 12-, 13-, 14-, and 16-membered hydrocarbon-bridged nucleotide trimers, their incorporation into ONs, and biophysical characterization of the modified ONs. The size of the macrocyclic tether and configuration at phosphorus had profound effects on hybridization kinetics. ONs containing 12- and 13-membered rings exhibited faster on-rates (up to 5-fold) and off-rates (up to 161-fold). In contrast, ONs using the larger ring size macrocycles generally exhibited smaller changes in binding kinetics relative to unmodified DNA. Interestingly, several of the analogues retained significant binding affinity for RNA based on their dissociation constants, despite being modestly destabilizing in the thermal denaturation experiments, highlighting the potential utility of measuring dissociation constants versus duplex thermal stability when evaluating novel nucleic acid analogues. Overall, our results provide additional insights into the ability of backbone-constrained macrocyclic nucleic acid analogues to modulate hybridization kinetics of modified ONs with RNA.

Drop-off-reinitiation at the amino termini of nascent peptides and its regulation by IF3, EF-G, and RRF.

In translation initiation in prokaryotes, IF3 recognizes the interaction between the initiator codon of mRNA and the anticodon of fMet-tRNAini and then relocates the fMet-tRNAini to an active position. Here, we have surveyed 328 codon-anticodon combinations for the preference of IF3. At the first and second base of the codon, only Watson-Crick base pairs are tolerated. At the third base, stronger base pairs, for example, Watson-Crick, are more preferred, but other types of base pairs, for example, G/U wobble, are also tolerated; weaker base pairs are excluded by IF3. When the codon-anticodon combinations are unfavorable for IF3 or the concentration of IF3 is too low to recognize any codon-anticodon combinations, IF3 fails to set the P-site fMet-tRNAini at the active position and causes its drop-off from the ribosome. Thereby, translation reinitiation occurs from the second aminoacyl-tRNA at the A site to yield a truncated peptide lacking the amino-terminal fMet. We refer to this event as the amino-terminal drop-off-reinitiation. We also showed that EF-G and RRF are involved in disassembling such an aberrant ribosome complex bearing inactive fMet-tRNAini Thereby EF-G and RRF are able to exclude unfavorable codon-anticodon combinations with weaker base pairs and alleviate the amino-terminal drop-off-reinitiation.

Dynamics and conformational heterogeneity of the PIM1 G-quadruplex from polarizable molecular dynamics simulations.

Reproductive cancers, such as triple-negative breast cancer (TNBC), are often challenging to treat due to a lack of specific chemotherapeutic targets. Overproduction of PIM1, an anti-apoptotic protein, is common in TNBC. Thus, counteracting this overproduction could serve as a viable therapeutic strategy against TNBC. The promoter region of the PIM1 gene can adopt two G-quadruplexes (GQs) with distinct topologies. GQs are higher-order, noncanonical nucleic acid structures that are rich in guanine and have become intensely explored as potential novel drug targets due to their proposed involvement in transcriptional regulation and other critical cellular processes. Two GQ-duplex hybrid structures have been found to exist in equilibrium in the promoter region of PIM1. The dominant GQ structure, form 1, contains conventional guanine tetrads and a duplex loop region of three Watson-Crick base pairs, while form 2 adopts a fold containing a mixed guanine-cytosine (GCGC) tetrad via slippage of two canonical Watson-Crick base pairs, and also contains a duplex loop consisting of two additional Watson-Crick pairs. We performed unbiased simulations and Gaussian-accelerated molecular dynamics simulations of both GQs with the Drude-2017 polarizable force field to better understand the properties of these GQs and how they might interconvert. Given the role of ion binding and noncanonical base pairing in stabilizing GQ structures, we monitored ion coordination and electronic properties (base dipole moments and electric fields). We observed an increased dipole moment for cytosine bases in the GCGC mixed tetrad of form 2, suggesting electronic plasticity as a factor in structural rearrangement between forms 1 and 2. We also report on conformational sampling in each structure to provide atomistic details that will be essential in the future design of more specific chemotherapeutic agents that target PIM1 on the nucleic acid level.

Non-canonical interactions tune condensation and kinetically arrested material states of short RNA homopolymers.

Phase separation related processes have been frequently linked to biological compartmentalization and organization, and associated with cellular responses to stress, development, and disease. As a result, understanding biomolecular condensates and the parameters that modulate material and chemical properties has been an area of interest. Although much initial work focused on the role of proteins in phase separation, increasingly RNA-RNA interactions are being recognized for their potential to tune the condensate properties, influencing material properties, morphology, and dynamic organization. While many of these studies have focused on Watson-Crick base pairing as the primary networking interaction, previous work has also suggested a potential for non-canonical RNA-RNA interactions in long homopolymers to promote phase separation and modulate overall material properties. Here, we examine the phase separation of short RNA homopolymers in the presence of divalent cations and disordered peptides. Our studies reveal that particular short homopolymers form internally kinetically arrested droplets with limited monomer mixing despite continued fusion. We also probe the combined cooperative effects of various ions in modulating phase behavior and investigate the potential for homopolymer tracts in slightly more complex sequences to tune the material and phase separation propensities of different oligomers. Collectively, our results suggest a potential for tracts predominantly interacting through non-canonical interactions to tune the phase behavior and mesoscale properties of simplified condensates, as well as offer insight into the fundamental biophysics of RNA-based condensate systems and short homopolymer tracts in more complex biological systems.

Determining the confromational ensemble of the HIV-1 LTR G-quadruplexes through gaussian-accelerated molecular dynamics using the drude polarizable force field.

Human immunodeficiency virus (HIV) impacts nearly 37.6 million people globally, but there is no effective cure for the infections it causes. HIV-1 is a retrovirus containing a single-stranded RNA genome. After reverse transcription, the 5’ long terminal repeat (LTR) acts as a promoter for viral replication. Two DNA G-quadruplexes (GQs), known as LTR-III and LTR-IV, form within this promoter and inversely regulate viral gene expression. The LTR-III GQ downregulates viral expression and is a quadruplex: duplex hybrid whereas LTR-IV upregulates viral expression and is a parallel GQ containing a single-nucleotide bulge. Their distinct topologies and functional roles suggest their potential to act as selective drug targets for HIV-1.To gain a greater understanding of structure and dynamics of the LTR-III and LTR-IV GQs, we performed conventional molecular dynamics simulations using the Drude polarizable force field. The loop nucleotides in both structures were highly flexible, sampling a variety of conformational states. The LTR-IV loop also formed base pair interactions that limited ion access to its loop cavity and further highlighted two distinct ion binding sites near the backbone tetrad guanines. Within the LTR-III duplex loop, an additional Watson-Crick base pair formed between Ade4:Thy14 and persisted for 40% of the snapshots collected in the total 4-μs simulation time. Analysis of the electric field exerted on Thy14 indicates a preference for base pairing with Ade4 compared to being unpaired as in the NMR ensemble. Gaussian-accelerated molecular dynamics simulations were also performed to identify high and low energy conformational states for subsequent drug targeting. Our results emphasize the importance of electronic polarization in GQ dynamics and can guide future development of antiviral therapeutics.

Direct and high-throughput detection of DNA methylation and demethylation intermediates using biological nanopore.

This paper describes a nanopore sensing approach for 5-methylcytosine (5mC) positions with numbers and demethylation intermediates directly in a high-throughput way. Methylation-demethylation processes are crucial for mammalian development and aberrant methylation patterns could lead to human diseases. The general method of 5mC detection, the bisulfite conversion, requires multiple procedures, and the incomplete conversion suggests the inappropriateness of reliable epigenetic analysis. For detecting demethylation intermediates, restriction enzymes and antibodies are used. However, these methods are hard to recognize the intermediaries at a single molecular level. Here, we report a nanopore-based electrical sensor that utilized epigenetic modulation of cytosine dynamics in dsDNA, which destabilizes the Watson-Crick base pair to allow modified cytosine to interact with the acidic anion acids in nanopore for directly detecting 5mC, accurately to its position, number and variants without chemical conversion and modification, or enzyme amplification.A probe DNA hybridizes with the target DNA, then the dsDNA is grasped in nanopore, which reconstitutes the artificial bilayers, and passes through the pore under an applied voltage with unzipping strands electrophoretically. Furthermore, to realize high throughput determination, we mounted this system on MinION, a commercial device measuring more than 2,000 nanopores simultaneously.To determine 5mC information, we analyzed the unzipping behaviors, including unzipping time and blocking ratio, then shown in scatter plot graph. Moreover, to improve the detection accuracy of 5mC variants, we changed K+ to the lower mobility ion Li+ to decrease the speed of DNA translocation. As a result, the scatter plot graph showed the possibility of detecting 5mC and its variants using our method. Using MinION showed that our system could work as a high-throughput nanopore epigenetic detection.Our method offers a direct and high-throughput tool for detecting 5mC position, number, and demethylation intermediates.

Modeling and simulating human mitochondrial RNA polymerase nucleotide addition cycle using computational microscope.

Human mitochondrial RNA polymerase (POLRMT) is essential to the transcription of mitochondrial DNA and cell metabolism. This work contributes to an all-atom structural dynamics model for individual steps of the nucleotide addition cycle of POLRMT. We aim to establish a computational model using all-atom molecular dynamics to reveal Watson-Crick base pair regulation of natural nucleotide and nucleotide analog binding and insertion in POLRMT, and a model for the post-catalytic mechanochemical coupling between product release and POLRMT translocation. The models are currently constructed combining the available high-resolution structures of POLRMT in the open and closed conformations with the bound NTP and Mg2+ ions detected in structurally similar T7 bacteriophage RNA polymerase, and by additionally using curve interpolation to place a missing non-template DNA segment. The models have been evaluated for protein subdomain stability and the presence of the appropriate protein conformation, open or closed, via subdomain motion regulation on the active site. Ongoing work will establish a model for the post-translocation mechanochemical regulations that reset for a new nucleotide addition cycle. Since POLRMT is structurally similar to viral RNA polymerases, the studies are particularly important for antiviral drug design, where nucleotide analog drug candidates are expected to be well screened for POLRMT toxicity.

Unusual Paradigm for DNA-DNA Recognition and Binding: "Socket-Plug" Complementarity.

DNA is the key informational polymer in biology by virtue of its precisely defined self-assembling properties. Watson-Crick complementarity, which underlies DNA's self-assembly, is required not only in biology but has also proved powerful in the field of nanoscience, where it has been utilized to assemble complex 2D and 3D architectures and nanodevices built from the DNA double-helix. Aside from Watson-Crick base-pairing, however, DNA also participates in alternative base pairing schemes, giving rise to DNA triplexes and G-quadruplexes. Herein, we describe "sticky-ended" DNA triplex-quadruplex composites that specifically recognize and bind to each other using a wholly different logic, "socket-plug" complementarity, a shape-sensing fitting of guanine "prongs" into guanine-lacking "cavities." A remarkable property of this kind of complementarity is the key role played in it by specific counter-cations: thus, exclusive "self" socket-plug recognition occurs over "other" in sodium salt solutions while precisely the reverse occurs in potassium salt solutions. We have used gel electrophoresis, Förster resonance energy transfer, alkylation protection, and structural modeling to study this remarkable fundamental property of DNA, that we anticipate will find wide practical application.

Transferability of the Electrostatic Parameters of the Polarizable Gaussian Multipole Model.

Accuracy and transferability are the two highly desirable properties of molecular mechanical force fields. Compared with the extensively used point-charge additive force fields that apply fixed atom-centered point partial charges to model electrostatic interactions, polarizable force fields are thought to have the advantage of modeling the atomic polarization effects. Previous works have demonstrated the accuracy of the recently developed polarizable Gaussian multipole (pGM) models. In this work, we assessed the transferability of the electrostatic parameters of the pGM models with (pGM-perm) and without (pGM-ind) atomic permanent dipoles in terms of reproducing the electrostatic potentials surrounding molecules/oligomers absent from electrostatic parameterizations. Encouragingly, both the pGM-perm and pGM-ind models show significantly improved transferability than the additive model in the tests (1) from water monomer to water oligomer clusters; (2) across different conformations of amino acid dipeptides and tetrapeptides; (3) from amino acid tetrapeptides to longer polypeptides; and (4) from nucleobase monomers to Watson-Crick base pair dimers and tetramers. Furthermore, we demonstrated that the double-conformation fittings using amino acid tetrapeptides in the αR and β conformations can result in good transferability not only across different tetrapeptide conformations but also from tetrapeptides to polypeptides with lengths ranging from 1 to 20 repetitive residues for both the pGM-ind and pGM-perm models. In addition, the observation that the pGM-ind model has significantly better accuracy and transferability than the point-charge additive model, even though they have an identical number of parameters, strongly suggest the importance of intramolecular polarization effects. In summary, this and previous works together show that the pGM models possess both accuracy and transferability, which are expected to serve as foundations for the development of next-generation polarizable force fields for modeling various polarization-sensitive biological systems and processes.

Detection of Oxidatively Modified Base Lesion(s) in Defined DNA Sequences by FLARE Quantitative PCR.

Assessment of DNA base and strand damage can be determined using a quantitative PCR assay that is based on the concept that damage blocks the progression of a thermostable polymerase thus resulting in decreased amplification. However, some of the mutagenic DNA base lesions cause little or no distortion in Watson-Crick base pairing. One of the most abundant such lesion is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo(d)Gua), although it affects the thermodynamic stability of the DNA, duplex 8-oxo(d)Gua does not inhibit DNA synthesis or arrest most of DNA or RNA polymerases during replication and transcription. When unrepaired, it is a pre-mutagenic base as it pairs with adenine in anti-syn conformation. Recent studies considered 8-oxo(d)Gua as an epigenetic-like mark and along with 8-oxoguanine DNA glycosylase1 (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1) has roles in gene expression via nucleating transcription factor's promoter occupancy. Here, we introduce its identification through fragment length analysis with repair enzyme (FLARE)-coupled quantitative (q)-PCR. One of the strengths of the assay is that 8-oxo(d)Gua can be identified within short stretches of nuclear and mitochondrial DNA in ng quantities. Bellow we describe the benefits and limits of using FLARE qPCR to assess DNA damage in mammalian cells and provide a detailed protocol of the assay.

Non-canonical base pairing

Non-canonical base pairs are planar hydrogen-bonded pairs of nucleobases, having hydrogen bonding patterns which differ from the patterns observed in Watson-Crick base pairs, as in the classic double-helical DNA. The structures of polynucleotide strands of both DNA and RNA molecules can be understood in terms of sugar-phosphate backbones consisting of phosphodiester-linked D 2’ deoxyribofuranose (D ribofuranose in RNA) sugar moieties, with purine or pyrimidine nucleobases covalently linked to them. Here, the N9 atoms of the purines, guanine and adenine, and the N1 atoms of the pyrimidines, cytosine and thymine (uracil in RNA), respectively, form glycosidic linkages with the C1’ atom of the sugars. These nucleobases can be schematically represented as triangles with one of their vertices linked to the sugar, and the three sides accounting for three edges through which they can form hydrogen bonds with other moieties, including with other nucleobases. As also explained in greater detail later in this article, the side opposite to the sugar-linked vertex is traditionally called the Watson-Crick edge, since they are involved in forming the Watson-Crick base pairs which constitute building blocks of double-helical DNA. The two sides adjacent to the sugar-linked vertex are referred to, respectively, as the Sugar and Hoogsteen (C-H for pyrimidines) edges. Each of the four different nucleobases is characterized by distinct edge-specific distribution patterns of their respective hydrogen bond donor and acceptor atoms, complementarity with which, in turn, define the hydrogen bonding patterns involved in base pairing. The double helical structures of DNA or RNA are generally known to have base pairs between complementary bases, Adenine: Thymine (Adenine: Uracil in RNA) or Guanine: Cytosine. They involve specific hydrogen bonding patterns corresponding to their respective Watson-Crick edges and are considered Canonical Base Pairs. At the same time, the helically twisted backbones in the double helical duplex DNA form two grooves, major and minor, through which the hydrogen bond donor and acceptor atoms corresponding respectively to the Hoogsteen and sugar edges are accessible for additional potential molecular recognition events. Experimental evidence reveals that the nucleotide bases are also capable of forming a wide variety of pairing between bases in various geometries, having hydrogen bonding patterns different from those observed in Canonical Base Pairs [Figure 1]. These base pairs, which are generally referred to as Non-Canonical Base Pairs, are held together by multiple hydrogen bonds and are mostly planar and stable. Most of these play very important roles in shaping the structure and function of different functional RNA molecules. In addition to their occurrences in several double-stranded stem regions, most of the loops and bulges that appear in single-stranded RNA secondary structures form recurrent 3D motifs, where non-canonical base pairs play a central role. Non-canonical base pairs also play crucial roles in mediating the tertiary contacts in RNA 3D structures.

5-Formylcytosine weakens the G-C pair and imparts local conformational fluctuations to DNA duplexes.

DNA epigenetic modifications such as 5-methyl (5mC), 5-hydroxymethyl (5hmC), 5-formyl (5fC) and 5-carboxyl (5caC) cytosine have unique and specific biological roles. Crystallographic studies of 5mC containing duplexes were conducted in the A-, B- or the intermediate E-DNA polymorphic forms. 5fC-modified duplexes initially observed in the disputed F-DNA architecture were subsequently crystallized in the A-form, suggesting that epigenetic modifications enable DNA sequences to adopt diverse conformational states that plausibly contribute to their function. Solution-state studies of these modifications were found in the B-DNA form, with marked differences in the conformational flexibility of 5fC containing duplexes in comparison to C/5mC containing duplexes, compromising the DNA duplex's stability. Herein, we systematically evaluate sensitive and commonly inaccessible NMR parameters to map the subtle differences between C, 5mC, and their oxidized (5hmC/5fC) counterparts. We observe that 15N/1H chemical shifts effectively report on the weakening of 5fC-G Watson-Crick base-pair H-bonding, extending the instability beyond any achievable within the sequence-specific changes in DNA. Triple 5fC containing sequences propagate the destabilization farther from the site of modifications, explaining reduced duplex stability upon multiple modifications. Additionally, scalar and residual dipolar coupling measurements unravel local sugar pucker fluctuations. One-bond 13C-1H scalar coupling measurements point towards a significant deviation away from the anticipated C2'-endo pucker for the 5fC modified nucleotide. Structural models obtained employing 13C-1H residual dipolar couplings and inter-proton distances corroborate the sugar pucker's deviation for 5fC modified DNA duplexes. The changes in the sugar pucker equilibria remain local to the 5fC modified nucleotide sans additive/long-range effects arising from multiple contiguous modifications. These observations highlight the impact of a major groove modification that alters the physical properties of DNA duplex without disturbing the Watson-Crick face. The changes observed in our studies for the 5fC containing DNA contrast with the perturbations induced by damage/lesion highlight the varied conformational preferences that modified nucleobases impart to the DNA duplex. As sequence-specific DNA transactions are rooted in the base-pair stability and pucker deviations, the observed structural perturbations for 5fC-modified DNA potentially play critical functional roles, such as protein-DNA recognition and interactions.

Aptamers as Functional Modules for DNA Nanostructures.

Watson-Crick base-pairing of DNA allows the nanoscale fabrication of biocompatible synthetic nanostructures for diagnostic and therapeutic biomedical purposes. DNA nanostructure design elicits exquisite control of shape and conformation compared to other nanoparticles. Furthermore, nucleic acid aptamers can be coupled to DNA nanostructures to allow interaction and response to a plethora of biomolecules beyond nucleic acids. When compared to the better-known approach of using protein antibodies for molecular recognition, nucleic acid aptamers are bespoke with the underlying DNA nanostructure backbone and have various other stability, synthesis, and cost advantages. Here, we provide detailed methodologies to synthesize and characterize aptamer-enabled DNA nanostructures. The methods described can be generally applied to various designs of aptamer-enabled DNA nanostructures with a wide range of applications both within and beyond biomedical nanotechnology.

Facile Expansion of the Variety of Orthogonal Ligand/Aptamer Pairs for Artificial Riboswitches.

An RNA aptamer that induces suitable conformational changes upon binding to a user-defined ligand allows us to artificially construct a riboswitch, a ligand-dependent and cis-acting gene regulatory RNA. Although such an aptamer can be obtained through in vitro selection, it is still challenging to rationally expand the variety of orthogonal ligand/aptamer (ligand/riboswitch) pairs. To achieve this in a facile, selection-free way, we herein focused on a specific type of ligand, 6-nt nanosized DNA (nDNA) and its aptamer that was previously selected to construct a eukaryotic artificial riboswitch. Specifically, we merely mutated one or more possible Watson-Crick base pairs in the nDNA/aptamer (nDNA/riboswitch) interactions into another base pair or pairs. Using two sets that each had 16 comprehensive mutations, we obtained three groups of several orthogonal nDNA/riboswitch pairs. These pairs could be used to create complex gene circuits, including multiple simultaneous and/or multistep cascading regulations in synthetic biology.

Watson-Crick Base-Pairing Requirements for ssDNA Recognition and Processing in Replication-Initiating HUH Endonucleases.

Replication-initiating HUH endonucleases (Reps) are sequence-specific nucleases that cleave and rejoin single-stranded DNA (ssDNA) during rolling-circle replication. These functions are mediated by covalent linkage of the Rep to its substrate post cleavage. Here, we describe the structures of the endonuclease domain from the Muscovy duck circovirus Rep in complex with its cognate ssDNA 10-mer with and without manganese in the active site. Structural and functional analyses demonstrate that divalent cations play both catalytic and structural roles in Reps by polarizing and positioning their substrate. Further structural comparisons highlight the importance of an intramolecular substrate Watson-Crick (WC) base pairing between the -4 and +1 positions. Subsequent kinetic and functional analyses demonstrate a functional dependency on WC base pairing between these positions regardless of the pair's identity (i.e., A·T, T·A, G·C, or C·G), highlighting a structural specificity for substrate interaction. Finally, considering how well WC swaps were tolerated in vitro, we sought to determine to what extent the canonical -4T·+1A pairing is conserved in circular Rep-encoding single-stranded DNA viruses and found evidence of noncanonical pairings in a minority of these genomes. Altogether, our data suggest that substrate intramolecular WC base pairing is a universal requirement for separation and reunion of ssDNA in Reps. IMPORTANCE Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses are a ubiquitous group of viruses that infect organisms across all domains of life. These viruses negatively impact both agriculture and human health. All members of this viral family employ a multifunctional nuclease (Rep) to initiate replication. Reps are structurally similar throughout this family, making them targets of interest for viral inhibition strategies. Here, we investigate the functional dependencies of the Rep protein from Muscovy duck circovirus for ssDNA interaction. We demonstrate that this Rep requires an intramolecular Watson-Crick base pairing for origin of replication (Ori) recognition and interaction. We show that noncognate base pair swaps are well tolerated, highlighting a local structural specificity over sequence specificity. Bioinformatic analysis found that the vast majority of CRESS-DNA Oris form base pairs in conserved positions, suggesting this pairing is a universal requirement for replication initiation in the CRESS-DNA virus family.

Fluorous-Directed Assembly of DNA Origami Nanostructures.

An orthogonal, noncovalent approach to direct the assembly of higher-order DNA origami nanostructures is described. By incorporating perfluorinated tags into the edges of DNA origami tiles we control their hierarchical assembly via fluorous-directed recognition. When we combine this approach with Watson-Crick base-pairing we form discrete dimeric constructs in significantly higher yield (8x) than when either molecular recognition method is used in isolation. This integrated "catch-and-latch" approach, which combines the strength and mobility of the fluorous effect with the specificity of base-pairing, provides an additional toolset for DNA nanotechnology, one that enables increased assembly efficiency while requiring significantly fewer DNA sequences. As a result, our integration of fluorous-directed assembly into origami systems represents a cheap, atom-efficient means to produce discrete superstructures.