Nucleic Acid-Binding Dyes as Versatile Photocatalysts for Atom-Transfer Radical Polymerization.

Nucleic acid-binding dyes (NuABDs) are fluorogenic probes that light up after binding to nucleic acids. Taking advantage of their fluorogenicity, NuABDs have been widely utilized in the fields of nanotechnology and biotechnology for diagnostic and analytical applications. We demonstrate the potential of NuABDs together with an appropriate nucleic acid scaffold as an intriguing photocatalyst for precisely controlled atom-transfer radical polymerization (ATRP). Additionally, we systematically investigated the thermodynamic and electrochemical properties of the dyes, providing insights into the mechanism that drives the photopolymerization. The versatility of the NuABD-based platform was also demonstrated through successful polymerizations using several NuABDs in conjunction with diverse nucleic acid scaffolds, such as G-quadruplex DNA or DNA nanoflowers. This study not only extends the horizons of controlled photopolymerization but also broadens opportunities for nucleic acid-based materials and technologies, including nucleic acid-polymer biohybrids and stimuli-responsive ATRP platforms.

Biomass RNA for the Controlled Synthesis of Degradable Networks by Radical Polymerization.

Nucleic acids extracted from biomass have emerged as sustainable and environmentally friendly building blocks for the fabrication of multifunctional materials. Until recently, the fabrication of biomass nucleic acid-based structures has been facilitated through simple crosslinking of biomass nucleic acids, which limits the possibility of material properties engineering. This study presents an approach to convert biomass RNA into an acrylic crosslinker through acyl imidazole chemistry. The number of acrylic moieties on RNA was engineered by varying the acylation conditions. The resulting RNA crosslinker can undergo radical copolymerization with various acrylic monomers, thereby offering a versatile route for creating materials with tunable properties (e.g., stiffness and hydrophobic characteristics). Further, reversible-deactivation radical polymerization methods, such as atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT), were also explored as additional approaches to engineer the hydrogel properties. The study also demonstrated the metallization of the biomass RNA-based material, thereby offering potential applications in enhancing electrical conductivity. Overall, this research expands the opportunities in biomass-based biomaterial fabrication, which allows tailored properties for diverse applications.

Red-Light-Driven Atom Transfer Radical Polymerization for High-Throughput Polymer Synthesis in Open Air.

Photoinduced reversible-deactivation radical polymerization (photo-RDRP) techniques offer exceptional control over polymerization, providing access to well-defined polymers and hybrid materials with complex architectures. However, most photo-RDRP methods rely on UV/visible light or photoredox catalysts (PCs), which require complex multistep synthesis. Herein, we present the first example of fully oxygen-tolerant red/NIR-light-mediated photoinduced atom transfer radical polymerization (photo-ATRP) in a high-throughput manner under biologically relevant conditions. The method uses commercially available methylene blue (MB+) as the PC and [X-CuII/TPMA]+ (TPMA = tris(2-pyridylmethyl)amine) complex as the deactivator. The mechanistic study revealed that MB+ undergoes a reductive quenching cycle in the presence of the TPMA ligand used in excess. The formed semireduced MB (MB•) sustains polymerization by regenerating the [CuI/TPMA]+ activator and together with [X-CuII/TPMA]+ provides control over the polymerization. This dual catalytic system exhibited excellent oxygen tolerance, enabling polymerizations with high monomer conversions (>90%) in less than 60 min at low volumes (50-250 µL) and high-throughput synthesis of a library of well-defined polymers and DNA-polymer bioconjugates with narrow molecular weight distributions (D < 1.30) in an open-air 96-well plate. In addition, the broad absorption spectrum of MB+ allowed ATRP to be triggered under UV to NIR irradiation (395-730 nm). This opens avenues for the integration of orthogonal photoinduced reactions. Finally, the MB+/Cu catalysis showed good biocompatibility during polymerization in the presence of cells, which expands the potential applications of this method.

Synthesis of RNA-Amphiphiles via Atom Transfer Radical Polymerization in the Organic Phase.

The combination of hydrophobic polymers with nucleic acids is a fascinating way to engineer the self-assembly behavior of nucleic acids into diverse nanostructures such as micelles, vesicles, nanosheets, and worms. Here we developed a robust route to synthesize a RNA macroinitiator with protecting groups on the 2'-hydroxyl groups in the solid phase using an oligonucleotide synthesizer. The protecting groups successfully solubilized the RNA macroinitiator, enabling atom transfer radical polymerization (ATRP) of hydrophobic monomers. As a result, the RNA-polymer hybrids obtained by ATRP exhibited enhanced chemical stability by suppressing cleavage. In addition, we demonstrated evidence of controlled polymerization behavior as well as control over the molecular weight of the hydrophobic polymers grown from RNA. We envision that this methodology will expand the field of RNA-polymer conjugates while vastly enhancing the possibility to alter and engineer the properties of RNA-based polymeric materials.

RNA-Polymer Hybrids via Direct and Site-Selective Acylation with the ATRP Initiator and Photoinduced Polymerization.

Combining synthetic polymers with RNA paves the way for creating RNA-based materials with non-canonical functions. We have developed an acylation reagent that allows for direct incorporation of the atom transfer radical polymerization (ATRP) initiator into both short synthetic oligoribonucleotides and natural biomass RNA extracted from torula yeast. The acylation was performed in a quantitative yield. The resulting initiator-functionalized RNAs were used for grafting polymer chains from the RNA by photoinduced ATRP, resulting in RNA-polymer hybrids with narrow molecular weight distributions. The RNA initiator was used for the polymerization of oligo(ethylene oxide) methyl ether methacrylate, poly(ethylene glycol) dimethacrylate, and N-isopropylacrylamide monomers, resulting in RNA bottlebrushes, hydrogels, and stimuli-responsive materials. This approach, readily applicable to both post-synthetic and nature-derived RNA, can be used to engineer the properties of a variety of RNA-based macromolecular hybrids and assemblies providing access to a wide variety of RNA-polymer hybrids.

Visible-Light-Mediated Controlled Radical Branching Polymerization in Water.

Hyperbranched polymethacrylates were synthesized by green-light-induced atom transfer radical polymerization (ATRP) under biologically relevant conditions in the open air. Sodium 2-bromoacrylate (SBA) was prepared in situ from commercially available 2-bromoacrylic acid and used as a water-soluble inibramer to induce branching during the copolymerization of methacrylate monomers. As a result, well-defined branched polymethacrylates were obtained in less than 30 min with predetermined molecular weights (36 000

Open-air green-light-driven ATRP enabled by dual photoredox/copper catalysis.

Photoinduced atom transfer radical polymerization (photo-ATRP) has risen to the forefront of modern polymer chemistry as a powerful tool giving access to well-defined materials with complex architecture. However, most photo-ATRP systems can only generate radicals under biocidal UV light and are oxygen-sensitive, hindering their practical use in the synthesis of polymer biohybrids. Herein, inspired by the photoinduced electron transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization, we demonstrate a dual photoredox/copper catalysis that allows open-air ATRP under green light irradiation. Eosin Y was used as an organic photoredox catalyst (PC) in combination with a copper complex (X-CuII/L). The role of PC was to trigger and drive the polymerization, while X-CuII/L acted as a deactivator, providing a well-controlled polymerization. The excited PC was oxidatively quenched by X-CuII/L, generating CuI/L activator and PC˙+. The ATRP ligand (L) used in excess then reduced the PC˙+, closing the photocatalytic cycle. The continuous reduction of X-CuII/L back to CuI/L by excited PC provided high oxygen tolerance. As a result, a well-controlled and rapid ATRP could proceed even in an open vessel despite continuous oxygen diffusion. This method allowed the synthesis of polymers with narrow molecular weight distributions and controlled molecular weights using Cu catalyst and PC at ppm levels in both aqueous and organic media. A detailed comparison of photo-ATRP with PET-RAFT polymerization revealed the superiority of dual photoredox/copper catalysis under biologically relevant conditions. The kinetic studies and fluorescence measurements indicated that in the absence of the X-CuII/L complex, green light irradiation caused faster photobleaching of eosin Y, leading to inhibition of PET-RAFT polymerization. Importantly, PET-RAFT polymerizations showed significantly higher dispersity values (1.14 ≤ D ≤ 4.01) in contrast to photo-ATRP (1.15 ≤ D ≤ 1.22) under identical conditions.

Sulfoxide-Containing Polyacrylamides Prepared by PICAR ATRP for Biohybrid Materials.

Water-soluble and biocompatible polymers are of interest in biomedicine as the search for alternatives to PEG-based materials becomes more important. In this work, the synthesis of a new sulfoxide-containing monomer, 2-(methylsulfinyl)ethyl acrylamide (MSEAM), is reported. Well-defined polymers were prepared by photoinduced initiators for continuous activator regeneration atom transfer radical polymerization (PICAR ATRP). The polymerizations were performed in water under biologically relevant conditions in a small volume without degassing the reaction mixture. DNA-PMSEAM and protein-PMSEAM hybrids were also synthesized. The lower critical solution temperature (LCST) of PMSEAM was estimated to be approximately 170 °C by extrapolating the LCST for a series of copolymers with variable content of N-isopropylacrylamide. The cytotoxicity studies showed excellent biocompatibility of PMSEAM, even at concentrations up to 2.5 mg/mL. Furthermore, the MSEAM monomer exhibited relatively lower toxicity than similar (meth)acrylate-based monomers at comparable concentrations.

Controlled Release of Exosomes Using Atom Transfer Radical Polymerization-Based Hydrogels.

Exosomes are 30-200 nm sized extracellular vesicles that are increasingly recognized as potential drug delivery vehicles. However, exogenous exosomes are rapidly cleared from the blood upon intravenous delivery, which limits their therapeutic potential. Here, we report bioactive exosome-tethered poly(ethylene oxide)-based hydrogels for the localized delivery of therapeutic exosomes. Using cholesterol-modified DNA tethers, the lipid membrane of exosomes was functionalized with initiators to graft polymers in the presence of additional initiators and crosslinker using photoinduced atom transfer radical polymerization (ATRP). This strategy of tethering exosomes within the hydrogel network allowed their controlled release over a period of 1 month, which was much longer than physically entrapped exosomes. Exosome release profile was tuned by varying the crosslinking density of the polymer network and the use of photocleavable tethers allowed stimuli-responsive release of exosomes. The therapeutic potential of the hydrogels was assessed by evaluating the osteogenic potential of bone morphogenetic protein 2-loaded exosomes on C2C12 and MC3T3-E1 cells. Thus, ATRP-based exosome-tethered hydrogels represent a tunable platform with improved efficacy and an extended-release profile.

Biocompatible photoinduced CuAAC using sodium pyruvate.

Sodium pyruvate, a natural intermediate produced during cellular metabolism, is commonly used in buffer solutions and media for biochemical applications. Here we show the use of sodium pyruvate (SP) as a reducing agent in a biocompatible aqueous photoinduced azide-alkyne cycloaddition (CuAAC) reaction. This copper(I)-catalyzed 1,3-dipolar cycloaddition is triggered by SP under UV light irradiation, exhibits oxygen tolerance and temporal control, and provides a convenient alternative to current CuAAC systems, particularly for biomolecular conjugations.

Engineering exosome polymer hybrids by atom transfer radical polymerization.

Exosomes are emerging as ideal drug delivery vehicles due to their biological origin and ability to transfer cargo between cells. However, rapid clearance of exogenous exosomes from the circulation as well as aggregation of exosomes and shedding of surface proteins during storage limit their clinical translation. Here, we demonstrate highly controlled and reversible functionalization of exosome surfaces with well-defined polymers that modulate the exosome's physiochemical and pharmacokinetic properties. Using cholesterol-modified DNA tethers and complementary DNA block copolymers, exosome surfaces were engineered with different biocompatible polymers. Additionally, polymers were directly grafted from the exosome surface using biocompatible photo-mediated atom transfer radical polymerization (ATRP). These exosome polymer hybrids (EPHs) exhibited enhanced stability under various storage conditions and in the presence of proteolytic enzymes. Tuning of the polymer length and surface loading allowed precise control over exosome surface interactions, cellular uptake, and preserved bioactivity. EPHs show fourfold higher blood circulation time without altering tissue distribution profiles. Our results highlight the potential of precise nanoengineering of exosomes toward developing advanced drug and therapeutic delivery systems using modern ATRP methods.

Atom Transfer Radical Polymerization for Biorelated Hybrid Materials.

Proteins, nucleic acids, lipid vesicles, and carbohydrates are the major classes of biomacromolecules that function to sustain life. Biology also uses post-translation modification to increase the diversity and functionality of these materials, which has inspired attaching various other types of polymers to biomacromolecules. These polymers can be naturally (carbohydrates and biomimetic polymers) or synthetically derived and have unique properties with tunable architectures. Polymers are either grafted-to or grown-from the biomacromolecule's surface, and characteristics including polymer molar mass, grafting density, and degree of branching can be controlled by changing reaction stoichiometries. The resultant conjugated products display a chimerism of properties such as polymer-induced enhancement in stability with maintained bioactivity, and while polymers are most often conjugated to proteins, they are starting to be attached to nucleic acids and lipid membranes (cells) as well. The fundamental studies with protein-polymer conjugates have improved our synthetic approaches, characterization techniques, and understanding of structure-function relationships that will lay the groundwork for creating new conjugated biomacromolecular products which could lead to breakthroughs in genetic and tissue engineering.

Capturing the Mechanism Underlying TOP mRNA Binding to LARP1.

The RNA-binding protein La-related protein 1 (LARP1) plays a central role in ribosome biosynthesis. Its C-terminal DM15 region binds the 7-methylguanosine (m7G) cap and 5' terminal oligopyrimidine (TOP) motif characteristic of transcripts encoding ribosomal proteins and translation factors. Under the control of mammalian target of rapamycin complex 1 (mTORC1), LARP1 regulates translation of these transcripts. Characterizing the dynamics of DM15-TOP recognition is essential to understanding this fundamental biological process. We use molecular dynamics simulations, biophysical assays, and X-ray crystallography to reveal the mechanism of DM15 binding to TOP transcripts. Residues C-terminal to the m7G-binding site play important roles in cap recognition. Furthermore, we show that the unusually static pocket that recognizes the +1 cytosine characteristic of TOP transcripts drives binding specificity. Finally, we demonstrate that the DM15 pockets involved in TOP-specific m7GpppC-motif recognition are likely druggable. Collectively, these studies suggest unique opportunities for further pharmacological development.

The Rad51 paralogs facilitate a novel DNA strand specific damage tolerance pathway.

Accurate DNA replication is essential for genomic stability and cancer prevention. Homologous recombination is important for high-fidelity DNA damage tolerance during replication. How the homologous recombination machinery is recruited to replication intermediates is unknown. Here, we provide evidence that a Rad51 paralog-containing complex, the budding yeast Shu complex, directly recognizes and enables tolerance of predominantly lagging strand abasic sites. We show that the Shu complex becomes chromatin associated when cells accumulate abasic sites during S phase. We also demonstrate that purified recombinant Shu complex recognizes an abasic analog on a double-flap substrate, which prevents AP endonuclease activity and endonuclease-induced double-strand break formation. Shu complex DNA binding mutants are sensitive to methyl methanesulfonate, are not chromatin enriched, and exhibit increased mutation rates. We propose a role for the Shu complex in recognizing abasic sites at replication intermediates, where it recruits the homologous recombination machinery to mediate strand specific damage tolerance.

Rapid On-Demand Extracellular Vesicle Augmentation with Versatile Oligonucleotide Tethers.

Exosomes show potential as ideal vehicles for drug delivery because of their natural role in transferring biological cargo between cells. However, current methods to engineer exosomes without negatively impacting their function remain challenging. Manipulating exosome-secreting cells is complex and time-consuming, while direct functionalization of exosome surface proteins suffers from low specificity and low efficiency. We demonstrate a rapid, versatile, and scalable method with oligonucleotide tethers to enable diverse surface functionalization on both human and murine exosomes. These exosome surface modifiers, which range from reactive functional groups and small molecules to aptamers and large proteins, can readily and efficiently enhance native exosome properties. We show that cellular uptake of exosomes can be specifically altered with a tethered AS1411 aptamer, and targeting specificity can be altered with a tethered protein. We functionalize exosomes with an immunomodulatory protein, FasL, and demonstrate their biological activity both in vitro and in vivo. FasL-functionalized exosomes, when bioprinted on a collagen matrix, allows spatial induction of apoptosis in tumor cells and, when injected in mice, suppresses proliferation of alloreactive T cells. This oligonucleotide tethering strategy is independent of the exosome source and further circumvents the need to genetically modify exosome-secreting cells.

Biocatalytic "Oxygen-Fueled" Atom Transfer Radical Polymerization.

Atom transfer radical polymerization (ATRP) can be carried out in a flask completely open to air using a biocatalytic system composed of glucose oxidase (GOx) and horseradish peroxidase (HRP) with an active copper catalyst complex. Nanomolar concentrations of the enzymes and ppm amounts of Cu provided excellent control over the polymerization of oligo(ethylene oxide) methyl ether methacrylate (OEOMA500 ), generating polymers with high molecular weight (Mn >70 000) and low dispersities (1.13≤D≤1.27) in less than an hour. The continuous oxygen supply was necessary for the generation of radicals and polymer chain growth as demonstrated by temporal control and by inducing hypoxic conditions. In addition, the enzymatic cascade polymerization triggered by oxygen was used for a protein and DNA functionalized with initiators to form protein-b-POEOMA and DNA-b-POEOMA bioconjugates, respectively.

Accessibility of Densely Localized DNA on Soft Polymer Nanoparticles.

The dense localization of DNA on soluble nanoparticles can lead to effects distinct from equivalent amounts of the DNA in solution. However, the specific effect may depend on the nature of the assembly and the nanoparticle core. Here we examine the accessibility of densely packed DNA duplexes that extend from a bottle-brush polymer core. We find that unlike spherical nucleic acids, the DNA duplex bristles on the bottle-brush polymer remain accessible to sequence-specific cleavage by endonucleases. In addition, the hybridized strand of the duplex can be displaced through a toehold-mediated strand exchange even at the polymer interface. These results demonstrate that the DNA on bottle-brush polymer remains sufficiently flexible to allow enzymatic degradation or DNA hybridization.

Eliminating Spurious Zero-Efficiency FRET States in Diffusion-Based Single-Molecule Confocal Microscopy.

Single-molecule Förster resonance energy transfer (smFRET) of freely diffusing biomolecules using confocal microscopy is a simple and powerful technique for measuring conformation and dynamics. However, a spurious zero-FRET population can significantly distort the measured histograms and lead to incorrect results, particularly in measurements of intrinsically low-FRET systems. Using a model system consisting of duplex DNAs, we show that there are two important contributions to the zero-FRET state: (1) formation of a dark triplet state of the acceptor dye and (2) the presence of donor-only strands due to incomplete hybridization between donor- and acceptor-labeled strands. The combined strategy of using Trolox as a triplet-state quencher and labeling the same DNA strand with donor and acceptor dyes effectively eliminates the zero-FRET population, even for constructs with intrinsically low FRET efficiencies. This strategy allows us to perform smFRET experiments using a simple confocal microscope with improved accuracy.

Synthesis of Polymer Bioconjugates via Photoinduced Atom Transfer Radical Polymerization under Blue Light Irradiation.

A rapid blue-light-induced atom transfer radical polymerization (ATRP) was conducted in a biologically friendly environment. Well-controlled polymerization of oligo(ethylene oxide) methyl ether methacrylate (OEOMA) was successfully performed in aqueous media (1X PBS) under irradiation by blue LED strips. With 10.0 mW/cm2 intensity output at 450 nm, >90% conversion was achieved in 2 h in the presence of a system comprising glucose, glucose oxidase, and sodium pyruvate. Poly-(OEOMA) was synthesized with predetermined M n and low dispersities using low ppm of Cu catalysts. Importantly, secondary structures of proteins, as analyzed by circular dichroism (CD), were preserved under blue-light irradiation due to its lower energy output. The aqueous blue-light ATRP technique was applied to biological systems by synthesizing well-defined protein-polymer and DNA-polymer hybrids by the "grafting-from" method.

Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn2+ /Mn2+ heterobinucleation.

The RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2'-5' phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni2+ , Mn2+ , Mg2+ , Fe2+ , and Zn2+ . While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn2+ ion), recent structures determined a Zn2+ /Fe2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn2+ /Mn2+ occupancy. ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.