Reversible Nucleic Acid Storage in Deconstructable Glassy Polymer Networks.

The rapid decline in DNA sequencing costs has fueled the demand for nucleic acid collection to unravel genomic information, develop treatments for genetic diseases, and track emerging biological threats. Current approaches to maintaining these nucleic acid collections hinge on continuous electricity for maintaining low-temperature and intricate cold-chain logistics. Inspired by the millennia-long preservation of fossilized biological specimens in calcified minerals or glassy amber, we present Thermoset-REinforced Xeropreservation (T-REX): a method for storing DNA in deconstructable glassy polymer networks. Key to T-REX is the development of polyplexes for nucleic acid encapsulation, streamlining the transfer of DNA from aqueous to organic phases, replete with initiators, monomers, cross-linkers, and thionolactone-based cleavable comonomers required to form the polymer networks. This process successfully encapsulates DNA that spans different length scales, from tens of bases to gigabases, in a matter of hours compared to days with traditional silica-based encapsulation. Further, T-REX permits the extraction of DNA using comparatively benign reagents, unlike the hazardous hydrofluoric acid required for recovery from silica. T-REX provides a path toward low-cost, time-efficient, and long-term nucleic acid preservation for synthetic biology, genomics, and digital information storage, potentially overcoming traditional low-temperature storage challenges.

The emergence of valency in colloidal crystals through electron equivalents.

Colloidal crystal engineering of complex, low-symmetry architectures is challenging when isotropic building blocks are assembled. Here we describe an approach to generating such structures based upon programmable atom equivalents (nanoparticles functionalized with many DNA strands) and mobile electron equivalents (small particles functionalized with a low number of DNA strands complementary to the programmable atom equivalents). Under appropriate conditions, the spatial distribution of the electron equivalents breaks the symmetry of isotropic programmable atom equivalents, akin to the anisotropic distribution of valence electrons or coordination sites around a metal atom, leading to a set of well-defined coordination geometries and access to three new low-symmetry crystalline phases. All three phases represent the first examples of colloidal crystals, with two of them having elemental analogues (body-centred tetragonal and high-pressure gallium), while the third (triple double-gyroid structure) has no known natural equivalent. This approach enables the creation of complex, low-symmetry colloidal crystals that might find use in various technologies.

Nanoparticle Superlattices through Template-Encoded DNA Dendrimers.

The chemical interactions that lead to the emergence of hierarchical structures are often highly complex and difficult to program. Herein, the synthesis of a series of superlattices based upon 30 different structurally reconfigurable DNA dendrimers is reported, each of which presents a well-defined number of single-stranded oligonucleotides (i.e., sticky ends) on its surface. Such building blocks assemble with complementary DNA-functionalized gold nanoparticles (AuNPs) to yield five distinct crystal structures, depending upon choice of dendrimer and defined by phase symmetry. These DNA dendrimers can associate to form micelle-dendrimers, whereby the extent of association can be modulated based upon surfactant concentration and dendrimer length to produce a low-symmetry Ti5Ga4-type phase that has yet to be reported in the field of colloidal crystal engineering. Taken together, colloidal crystals that feature three different types of particle bonding interactions-template-dendron, dendrimer-dendrimer, and DNA-modified AuNP-dendrimer-are reported, illustrating how sequence-defined recognition and dynamic association can be combined to yield complex hierarchical materials.

A General DNA-Gated Hydrogel Strategy for Selective Transport of Chemical and Biological Cargos.

The selective transport of molecular cargo is critical in many biological and chemical/materials processes and applications. Although nature has evolved highly efficient in vivo biological transport systems, synthetic transport systems are often limited by the challenges associated with fine-tuning interactions between cargo and synthetic or natural transport barriers. Herein, deliberately designed DNA-DNA interactions are explored as a new modality for selective DNA-modified cargo transport through DNA-grafted hydrogel supports. The chemical and physical characteristics of the cargo and hydrogel barrier, including the number of nucleic acid strands on the cargo (i.e., the cargo valency) and DNA-DNA binding strength, can be used to regulate the efficiency of cargo transport. Regimes exist where a cargo-barrier interaction is attractive enough to yield high selectivity yet high mobility, while there are others where the attractive interactions are too strong to allow mobility. These observations led to the design of a DNA-dendron transport tag, which can be used to universally modify macromolecular cargo so that the barrier can differentiate specific species to be transported. These novel transport systems that leverage DNA-DNA interactions provide new chemical insights into the factors that control selective cargo mobility in hydrogels and open the door to designing a wide variety of drug/probe-delivery systems.

Programming Fluorogenic DNA Probes for Rapid Detection of Steroids.

The ability of aptamers to recognize a variety of different molecules has fueled their emergence as recognition agents to probe complex media and cells. Many detection strategies require aptamer binding to its target to result in a dramatic change in structure, typically from an unfolded to a folded state. Here, we report a strategy based on forced intercalation (FIT) that increases the scope of aptamer recognition by transducing subtle changes in aptamer structures into fluorescent readouts. By screening a library of green-fluorescent FIT-aptamers whose design is guided by computational modeling, we could identify hits that sense steroids like dehydroepiandrosterone sulfate (DHEAS) down to 1.3 µM with no loss in binding affinity compared to the unmodified aptamer. This enabled us to study DHEAS in clinical serum samples with several advantages over gold standard methods, including rapid readout (<30 min), simple instrumentation (plate-reader), and low sample volumes (10 µL).

Multi-State Dynamic Coordination Complexes Interconverted through Counterion-Controlled Phase Transfer.

We studied a series of dynamic weak-link approach (WLA) complexes that can be shuttled between two immiscible solvents and switched between two structural states via ion exchange. Here, we established that hydrophobic anions transfer cationic, amphiphilic complexes from the aqueous phase to the organic phase, while a chloride source reverses the process. As a result of the dynamic metal coordination properties of WLA complexes, the denticity of these complexes (mono- to bi-) can be modulated as they partition into different phases. In addition, we discovered that heteroligated complexes bearing ligands of different donor strengths preferentially rearrange into two homoligated complexes that are phase-partitioned to maximize the number of stronger coordination bonds. This behavior is not observed in systems with one solvent, highlighting the dynamic and stimuli-responsive nature of hemilabile ligands in a multiphasic solvent environment. Taken together, this work shows that the highly reconfigurable WLA modality can enable the design of biphasic reaction networks or chemical separations driven by straightforward salt metathesis reactions.

Electron-Equivalent Valency through Molecularly Well-Defined Multivalent DNA.

Oligonucleotide-functionalized nanoparticles (NPs), also known as "programmable atom equivalents" (PAEs), have emerged as a class of versatile building blocks for generating colloidal crystals with tailorable structures and properties. Recent studies have shown that, at small size and low DNA grafting density, PAEs can also behave as "electron equivalents" (EEs), roaming through and stabilizing a complementary PAE sublattice. However, it has been challenging to obtain a detailed understanding of EE-PAE interactions and the underlying colloidal metallicity because there is inherent polydispersity in the number of DNA strands on the surfaces of these NPs; thus, the structural uniformity and tailorability of NP-based EEs are somewhat limited. Herein, we report a strategy for synthesizing colloidal crystals where the EEs are templated by small molecules, instead of NPs, and functionalized with a precise number of DNA strands. When these molecularly precise EEs are assembled with complementary NP-based PAEs, X-ray scattering and electron microscopy reveal the formation of three distinct "metallic" phases. Importantly, we show that the thermal stability of these crystals is dependent on the number of sticky ends per EE, while lattice symmetry is controlled by the number and orientation of EE sticky ends on the PAEs. Taken together, this work introduces the notion that, unlike conventional electrons, EEs that are molecular in origin can have a defined valency that can be used to influence and guide specific phase formation.

Protein Spherical Nucleic Acids for Live-Cell Chemical Analysis.

We report the development of a new strategy for the chemical analysis of live cells based on protein spherical nucleic acids (ProSNAs). The ProSNA architecture enables analyte detection via the highly programmable nucleic acid shell or a functional protein core. As a proof-of-concept, we use an i-motif as the nucleic acid recognition element to probe pH in living cells. By interfacing the i-motif with a forced-intercalation readout, we introduce a quencher-free approach that is resistant to false-positive signals, overcoming limitations associated with conventional fluorophore/quencher-based gold NanoFlares. Using glucose oxidase as a functional protein core, we show activity-based, amplified sensing of glucose. This enzymatic system affords greater than 100-fold fluorescence turn on in buffer, is selective for glucose in the presence of close analogs (i.e., glucose-6-phosphate), and can detect glucose above a threshold concentration of ∼5 µM, which enables the study of relative changes in intracellular glucose concentrations.

Forced Intercalation (FIT)-Aptamers.

Aptamers are oligonucleotide sequences that can be evolved to bind to various analytes of interest. Here, we present a general design strategy that transduces an aptamer-target binding event into a fluorescence readout via the use of a viscosity-sensitive dye. Target binding to the aptamer leads to forced intercalation (FIT) of the dye between oligonucleotide base pairs, increasing its fluorescence by up to 20-fold. Specifically, we demonstrate that FIT-aptamers can report target presence through intramolecular conformational changes, sandwich assays, and target-templated reassociation of split-aptamers, showing that the most common aptamer-target binding modes can be coupled to a FIT-based readout. This strategy also can be used to detect the formation of a metallo-base pair within a duplexed strand and is therefore attractive for screening for metal-mediated base pairing events. Importantly, FIT-aptamers reduce false-positive signals typically associated with fluorophore-quencher based systems, quantitatively outperform FRET-based probes by providing up to 15-fold higher signal to background ratios, and allow rapid and highly sensitive target detection (nanomolar range) in complex media such as human serum. Taken together, FIT-aptamers are a new class of signaling aptamers which contain a single modification, yet can be used to detect a broad range of targets.

A Redox-Switchable, Allosteric Coordination Complex.

A redox-regulated molecular tweezer complex was synthesized via the weak-link approach. The PtII complex features a redox-switchable hemilabile ligand (RHL) functionalized with a ferrocenyl moiety, whose oxidation state modulates the opening of a specific coordination site. Allosteric regulation by redox agents gives reversible access to two distinct structural states-a fully closed state and a semi-open state-whose interconversion was studied via multinuclear NMR spectroscopy, cyclic voltammetry, and UV-vis-NIR spectroscopy. Two structures in this four-state system were further characterized via SCXRD, while the others were modeled through DFT calculations. This fully reversible, RHL-based system defines an unusual level of electrochemical control over the occupancy of a specific coordination site, thereby providing access to four distinct coordination states within a single system, each defined and differentiated by structure and oxidation state.

An Allosterically Regulated, Four-State Macrocycle.

Macrocycles capable of host-guest chemistry are an important class of structures that have attracted considerable attention because of their utility in chemical separations, analyte sensing, signal amplification, and drug delivery. The deliberate design and synthesis of such structures are rate-limiting steps in utilizing them for such applications, and coordination-driven supramolecular chemistry has emerged as a promising tool for rapidly making large classes of such systems with attractive molecular recognition capabilities and, in certain cases, catalytic properties. A particularly promising subset of such systems are stimuli-responsive constructs made from hemilabile ligands via the weak-link approach (WLA) to supramolecular coordination chemistry. Such structures can be reversibly toggled between different shapes, sizes, and charges based upon small-molecule and elemental-anion chemical effectors. In doing so, one can deliberately change their recognition properties and both stoichiometric and catalytic chemistries, thereby providing mimics of allosteric enzymes. The vast majority of structures made to date involve two-state systems, with a select few being able to access three different states. Herein, we describe the synthesis of a new allosterically regulated four-state macrocycle assembled via the WLA. The target structure was made via the stepwise assembly of ditopic bidentate hemilabile N-heterocyclic carbene thioether (NHC,S) and phosphino thioether (P,S) ligands at PtII metal nodes. The relatively simple macrocycle displays complex dynamic behavior when addressed with small-molecule effectors, and structural switching can be achieved with several distinct molecular cues. Importantly, each state was fully characterized by multinuclear NMR spectroscopy and, in some cases, single-crystal X-ray diffraction studies and density functional theory computational models. This new structure opens the door to complex multicue switching reminiscent of multistate chemoswitches that could be important in controlling stoichiometric and catalytic transformations as well as generating molecular logic systems.