Chemical Communications

The methylation of cytosines at the 5th position (d5mC) is one of the most common epigenetic modifications, and alterations in methylation profile of cells are known to be involved in progression of many diseases including cancer. Increased stability of DNA accompanied by decreased flexibility upon methylation is thought to be a reason behind methylation profiles. The effects of d5mC on DNA stability and structure were investigated via systematic changes in the number and position of d5mCs in DDD. Our results revealed that d5mC substitutions changed DNA conformation and increased the stability only slightly. Next, the effect of DNA methylation on DNA-small molecule interactions was investigated using DDD and fully methylated analogue, DDD8. All the molecules examined (EtBr, Dox, Net and Hoe) had slightly higher affinity to DDD8 compared to DDD. Conversely, their effect, especially Doxs, on DDD structure was more pronounced. Further investigations via MD simulations revealed high selectivity of Dox towards a single intercalation site where the methoxy group of Dox interacts with the methyl group of d5mC and that of two precedent dT to create a highly stable hydrophobic cluster. Hydrophobic cluster formation was not observed upon Dox binding to DDD. Our results rationalize the increased stability of DDD8 over DDD, and open new routes for the design of drugs targeting epigenetic modifications. We suggest, the design of drugs that can engage in hydrophobic interactions with methyl groups in the major groove of a 5-dTdTd5mCdG-3 sequence might lead the way in specific targeting of hypermethylated regions in cancer cells.

Antisense oligonucleotides (ASO) are nucleotide polymers that hybridize to sense strands and have been successful in treating a variety of diseases. A wide range of strategies have been investigated to optimize and develop ASO for clinical studies. A key objective for this study was to provide an overview of the range of detailed data that get be obtained and provide an updated method review on how to design surface plasmon resonance (SPR) kinetic experiments for DNA oligonucleotide hybridization studies that can also be applied to other ASO including peptide nucleic acids (PNA). We describe many lessons learned from published literature and provide a state-of-the-art strategy and methods for generating not only kinetic but also thermodynamic characterizations of oligonucleotide hybridization. In this study we have performed an SPR kinetic and thermodynamic analysis for the hybridization of HIF1 antisense DNA strands to its immobilized Intron2-Exon3 splice site sense DNA strand to provide insight, in general, on the optimal length and insight into optimal design of DNA ASOs. We provide a process on how to design experiments to: 1.) obtain oligonucleotide-length dependent kinetics, 2.) analyze reactions to obtain association and dissociation rate kinetics (ka, kd), assess if hybridization follows a 2-state model and to obtain kinetic dissociation constants (Kd), 3.) perform temperature-dependent hybridization kinetics to obtain thermodynamic values ({Delta}H{degrees}, {Delta}S{degrees} and {Delta}G{degrees}) that can give insight into the molecular interactions driving hybridization, 4.) compare experimental thermodynamic values to values derived from nearest-neighbor prediction models to identify atypical reactions and importantly 5.) enable calculations to predict oligomer hybridization affinity at the physiological 37 {degrees}C temperature to asses if the design of the oligomer will have the required cellular activity for a therapeutic effect. The strategy and results presented throughout the paper are compared to previous SPR reports and suggestions made to optimize kinetic studies.

Transition-metal ion Forster resonance energy transfer (tmFRET) measures short-range distances and conformational changes in proteins, but its distance sensitivity depends on the donor-acceptor pair. Here, we establish the metal-chelating noncanonical amino acid bipyridylalanine (bpyA) as a flexible, genetically encoded acceptor platform whose Forster distance (R0) can be tuned without changing protein labeling. Cu2+, Cu2+ complexed with nitrilotriacetic acid (NTA), and Fe2+ form distinct acceptors at the same bpyA site, allowing distance sensitivity to be adjusted by changing the solution conditions. We incorporated bpyA at two sites in maltose binding protein (MBP) and labeled nearby engineered cysteines with the donor fluorophore BODIPY. The resulting [Cu(bpyA)]2+, [CuNTA(bpyA)]2+, and [Fe(bpyA)]2+ acceptors provided R0 values ranging from 13.9 to 21.2 [A]. Analysis of time-resolved tmFRET data recovered donor-acceptor distance distributions and maltose-dependent changes consistent with structural predictions. At the 237/295 site pair, both copper and iron acceptors resolved the maltose-dependent decrease in distance. At the 309/322 site pair, [Cu(bpyA)]2+ did not appreciably resolve the distance increase, whereas adding NTA shifted R0 toward shorter distances and enabled detection of an increase from 12.1 to 14.4 [A]. These results demonstrate that a single bpyA-labeled protein preparation supports multiple tmFRET acceptors, providing a cysteine-independent acceptor-labeling strategy with adjustable distance sensitivity.

Fluorescent base analogues have proven to be valuable probes of RNA structure. Pyrrolocytosine (pC), an analogue of cytosine, exhibits highly environment-sensitive fluorescence within RNA and is strongly quenched in hydrogen-bonded contexts. In this work, we incorporated pC into the aptamer domain of the B. subtilis guanine riboswitch to determine if this quenching mechanism could allow for sensing of RNA-small molecule interactions. We utilized complementary fluorescence spectroscopy methods to under-stand the functional impacts and ligand-sensitivity of pC substitutions distal to and within the binding pocket. We found that pC substitution within a static structural region exhibits the expected ligand-independent fluorescence properties. When placed in the ligand binding pocket, pC exhibits a moderately decreased affinity for the ligands guanine (G) and hypoxanthine (Hx) while maintaining high specificity for G. We found that pC behaves as a turn-off sensor, with distinct changes in fluorescence upon G or Hx binding. These differences are dependent on the number of hydrogen bonds formed with pC and are resolvable in both time-dependent and steady-state fluorescence measurements. Our results show that pC can be used to study RNA-small molecule interactions and can discriminate between similar ligands even when they have identical binding modes.

Fluorescent molecular rotors for sensing local microviscosity have become indispensable tools for better understanding the mechanisms of living systems. Current probes relay on intensity or lifetime measurements, while ratiometric molecular rotors are lacking. Here, we introduce a new design concept of molecular rotor using xanthene scaffold with freely rotating 9-aryl group. We studied three rhodamine derivatives, where 2-carboxyphenyl group was replaced with 4-methoxyphenyl, 2-thienyl and 2-benzofuryl. We found that five membered 9-aryl ring provides two major effects. First, free rotation of thienyl derivative, confirmed by theoretical calculations, makes it classical molecular rotor with intensity and lifetime-based response to viscosity. Second, benzofuryl derivative exhibits second emission band in near-infrared region and fluorescence ratiometric response to viscosity. Theoretical calculations suggest that the benzofuryl derivative can attain planar conformation in the excited state, which explains appearance of the NIR emitting band. Both new dyes efficiently target mitochondria, while the 9-benzofuryl derivative enable quantitative ratiometric measurement of viscosity of inner mitochondrial membrane. Ratiometric imaging revealed mitochondria heterogeneity with local viscosity values ranging from 250 to 460 cP. Oxidative stress induced significant rise in the local viscosity in mitochondria in addition to their morphological changes. Overall, we propose a concept that converts bright rhodamine dyes into molecules rotors with a valuable ratiometric response to viscosity, which opens a bunch of potential biological applications.

Tryptophan is a biologically important redox-active amino acid whose functions in proteins, including long-range electron transfer, protection against oxidative damage, and environmental sensing, are governed not only by its chemical identity but also by its precise structural organization. Inspired by this biological principle, we investigated whether controlling the organization of tryptophan within crystalline materials could provide a strategy for modulating its physicochemical properties and molecular accessibility. Using identical molecular components but distinct assembly pathways, tryptophan was organized either as a confined guest within a preformed Zn-imidazolate framework, yielding a star-shaped crystalline architecture, or as an integral coordination component during framework growth, producing a distinct layered Zn- tryptophan crystalline framework. Although assembled from the same building blocks, these two organization modes generated fundamentally different crystal structures, morphologies, and mechanisms of biomolecule incorporation. In both architectures, incorporation of tryptophan into the crystalline environment preserved its intrinsic fluorescence while producing robust fluorescence under multiple excitation wavelengths, highlighting the strong influence of molecular organization on its optical response. The structural modes also exhibited distinct encapsulation efficiencies and pH-dependent molecular accessibility, while secondary calcium-alginate fixation provided an additional level of control over guest retention without disrupting the underlying crystalline architecture. These results demonstrate that engineering the structural organization of tryptophan provides a versatile strategy for tuning the optical behavior, molecular accessibility, and functional integration of a biologically important redox-active amino acid in crystalline materials, establishing a foundation for future biomimetic redox architectures, responsive sensing platforms, and controlled molecular delivery.

DNA nanostructures can be tailored to perform a wide variety of functions, with continued interest in biological applications. Some aspects of DNA nanostructure assembly can hinder the ability of nanostructures to be useful in physiological environments. Typical assembly methods employ magnesium ions to stabilize the structure, which leave the structure susceptible to damage by nucleases in body fluids. Further, DNA nanostructure assembly typically involves a thermal annealing protocol in which DNA strands are heated in a specific buffer to a high temperature and cooled slowly at specific rates, preventing convenient encapsulation of temperature-sensitive guest molecules. In this work, we demonstrate the assembly of a wide variety of DNA nanostructures and 3D crystals in a hydrated ionic liquid (choline dihydrogen phosphate, CDHP) instead of magnesium at constant moderate temperatures, thus avoiding thermal annealing. CDHP-assembled structures show enhanced biostability against a variety of nucleases. Molecular dynamics simulations show that choline ions stabilize DNA nanostructures by a direct and close-range interaction in contrast to the predominantly water-mediated interactions of Mg2+, leading to enhanced nuclease resistance in CDHP-containing environments. CDHP-assembled structures do not affect the viability of HepG2 cells and show higher cell internalization. Overall, this work develops a potential method to construct more biostable DNA nanostructures and 3D crystals in a simple one pot process. Assembly of DNA nanostructures under isothermal conditions is desirable for scaffolding biomolecules and to reduce the need for thermal annealing instruments, allowing nanostructure preparation in low-resource settings.

The development of NIR fluorescent dyes with favorable optical properties is of great importance especially for applications within the field of optical imaging. In this study, we report the design, synthesis and photophysical characterization of novel BODIPY-Xanthene hybrid dyes obtained via Knoevenagel condensation between an asymmetrical BODIPY core and the functionalized xanthene aldehydes intermediates. The synthesized dyes ex-hibit absorption (667 nm) and emission bands (720-722 nm) within the NIR region. Detailed UV-Vis and fluo-rescence studies were performed and molar extinction coefficients and stokes shifts also calculated. Moreover, findings highlighting the influence of structural modifications on the optical behavior of the hybrid dyes and their potential utility in optical imaging applications are reported.

A platform method to associate native proteins and peptides onto a liposome membrane using site-specific N-terminal alkylation based on 1H-1,2,3-triazole-4-carbaldehyde (TA4C) is developed. The TA4C reagent reacts with the N-terminal -amino group of native proteins under mild aqueous conditions in a single step, without genetic engineering or protecting group strategies. Equipping TA4C with hexyl and nonyl chains provides a direct handle for tuning the association between the protein and membrane. N-terminal alkylation of green fluorescent protein (GFP) as a model proceeds in high yield (93% for the hexyl group and 70% for the nonyl group), and tethering the N-terminal alkyl group on GFP efficiently associates the protein with the liposomal membrane, as confirmed by confocal laser scanning microscopy and dynamic light scattering. We extended this strategy to an investigation of the GE11 peptide, a ligand for the epidermal growth factor receptor (EGFR). The liposome immobilized with GE11 peptide possessing an N-terminal alkyl group enables active targeting with EGFR-overexpressing A431 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/742023v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1f4095org.highwire.dtl.DTLVardef@1c91509org.highwire.dtl.DTLVardef@841647org.highwire.dtl.DTLVardef@1d2909b_HPS_FORMAT_FIGEXP M_FIG C_FIG

Ultrashort (US) cell-free DNA (cfDNA) is a population of approximately 50-nucleotide single-stranded DNA molecules in human plasma.1-3 Although it holds biological and diagnostic potential, US cfDNA escapes detection by conventional double-stranded library preparation methods.1-3 Independent studies have linked US cfDNA to regulatory genomic regions and to sequences predicted to form noncanonical structures, prompting the hypothesis that higher-order DNA structure contributes to its molecular properties. This interpretation, however, has so far rested on computational prediction rather than experimental evidence. Here we test this hypothesis using computational, biophysical and biochemical approaches. In silico size-selected US cfDNA from 20 healthy donors was selectively enriched at putative quadruplex sequences (PQS) that overlap both experimentally observed quadruplex sequences and accessible chromatin of blood cells. Synthetic oligonucleotides corresponding to the most enriched of these loci adopted predominantly parallel G-quadruplex (G4) structures, as revealed by circular dichroism. Endogenous nucleic acids captured directly from pooled plasma by poly(A)-tailing and immobilization, without extraction, denaturation or annealing at any step, displayed folded G4 structures. Two orthogonal probes detected these structures: the BG4 antibody and the fluorogenic ligand N-methyl mesoporphyrin IX. Reciprocal competition with a third, chemically unrelated G4 ligand, pyridostatin, confirmed the signal. Together, these experiments provide direct experimental evidence for the presence of folded G4 structures in human blood plasma.

The formation of magnesium pyrophosphate (Mg2P2O7) in nucleic acid amplification and cell-free transcription systems has attracted considerable attention, since Mg2P2O7 serves as a reliable indicator of reaction efficiency. However, real-time monitoring of Mg2P2O7 remains challenging, relying largely on time-consuming analytical techniques or end-point detection methods. Here, we report a Mg2P2O7-driven co-aggregation mechanism involving glutathione-capped gold nanoclusters (GSH-AuNCs) that induces fluorescence enhancement, enabling real-time crystal formation monitoring. The mechanism was first investigated in simplified mixtures containing pyrophosphate (P2O74-) and magnesium (Mg2+) ions. Real-time fluorescence profiles revealed that the GSH-AuNCs/Mg2P2O7 co-aggregation can be correlated with crystal formation/growth/solubilization and solution turbidity, while distinct kinetic patterns can be indicative of the crystal size at the end of the reaction. As a next level of complexity, we examined the effects of common components in an enzymatic amplification reaction, i.e., dithiothreitol (DTT), ammonium sulfate ((NH4)2SO4), deoxynucleotides (dNTPs) and Bst polymerase, on Mg2P2O7 formation through real-time GSH-AuNCs fluorescence variations. Guided by the above results, we studied and selected the experimental parameters for the design of an optimized qualitative (end-point) or quantitative (real-time) genetic test. Finally, the loop-mediated isothermal amplification (LAMP) was used as a platform to demonstrate the quantification of Influenza A RNA within the range of 102-108 copies/reaction. The resulting one-tube, contamination-free assay was shown to have a response time of <25 min even in a crude saliva sample. Beyond diagnostics, this crystallization-activated fluorescence strategy may also support real-time investigation of Mg2P2O7 formation in other biotechnological processes, including in vitro transcription and Mg2P2O7-bioorganic composites synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/744482v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@96dd88org.highwire.dtl.DTLVardef@aa122dorg.highwire.dtl.DTLVardef@18f4abforg.highwire.dtl.DTLVardef@745f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Reactive metal-oxygen intermediates are central to metalloenzyme catalysis, yet their low populations and short lifetimes make vibrational characterization under aqueous reaction conditions challenging. Here, we establish shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) as a powerful vibrational approach for visualizing reaction-associated metal-ligand signatures of reactive metalloenzyme states. Across the heme and non-heme iron enzymes TauD, VioC, CYP119, and TqaLNc, isotope-sensitive features provide vibrational information on reactive coordination states. In TauD and VioC, isotope-sensitive features provide vibrational characterization of Fe(IV)=O intermediates. In CYP119, an isotope-sensitive Fe(IV)=O vibration is assigned to P450 Compound I generated by m-CPBA, including under lauric-acid turnover conditions. Most notably, in TqaLNc, combined 18O- and 15N-sensitive vibrational responses support formation of the proposed (R)HN-Fe(IV)=O coordination state and the dynamic-coordination mechanism underlying selective C-N bond formation. These results demonstrate that SHINERS can serve as a powerful technique for the in situ vibrational visualization of reactive intermediates and their coordination states in metalloenzyme catalysis.

Plasma membranes (PM) have been traditionally studied by fluorescent probes by either labelling lipid or protein parts. Based on our MemGraft concept, we report a fluorescent probe based on solvatochromic dye Nile Red (MemGraft-NR) for sensing lipid organization in the immediate surrounding of proteins, i.e. at the protein-lipid interface. Two sequentially attached MemGraft scaffolds are required to ensure robust grafting of Nile Red to PM. In contrast to classical lipid-bound membrane probes, MemGraft-NR exhibits high resistance to the presence of serum and fixation with permeabilization. Two-color confocal imaging of MemGraft-NR in live cells reveals higher polarity at the protein-lipid interface compared to lipid-bound probe NR12A. Surprisingly, MemGraft-NR displays higher sensitivity to variation of cholesterol content in PM compared to NR12A, suggesting that protein-lipid interface is particularly impacted by changes in the lipid composition. MemGraft-NR also enabled in situ monitoring of lipid exchange and permeabilization of PM, something impossible for classical solvatochromic PM probes. Unexpectedly, MemGraft-NR reveals a dramatic decrease in the local polarity in early endosomes, suggesting that endosomal negative curvature provides strong impact on organization of the protein-lipid interface. The developed probe opens a new way to study biological membranes by sensing lipid surrounding of membrane proteins.

Thioamides are natural post-translational modifications of the peptide backbone and can be introduced synthetically to probe protein folding or functionalize peptides for translational applications. In this work, we demonstrate that thioamide-containing peptides with C-terminal thioesters can be efficiently generated using Knorr pyrazole activation and used in subsequent native chemical ligation reactions to generate thioamide containing proteins. We compare this method to acyl azide activation and find that both routes provide similar yields. We also investigate ultrasound-mediated desulfurization of the ligation site cysteine for potential advantages over chemical radical initiators. Scaling up our syntheses allows us to study thioamide perturbations to the {beta}-sheet region of the B1 domain of protein G (GB1) as well as {beta}-strand interactions in amyloid fibrils of the Parkinsons disease protein -synuclein. In both contexts, we observe dramatic destabilization of the {beta}-sheet networks, manifested in decreased GB1 thermal stability and altered folding and slowed aggregation of -synuclein. These findings illustrate the impact that a single atom substitution can have on cooperative hydrogen bonding networks and prompt future study of both systems.

RNA interactions are a key contributor to the formation and disassembly of intracellular protein condensates. Although some proteins utilize specific RNA-binding domains, these processes can also be mediated by charge interactions with intrinsically disordered regions. Due to the dynamic nature of these systems, investigating the underlying specificity and stoichiometry remains challenging. Here, we demonstrate that single-molecule mass measurements with mass photometry can capture RNA-protein interactions in phase-separated protein systems. Using the approach to investigate RNA-mediated phase shifts of tau condensates, we find that increasing the RNA concentration, which promotes phase re-entry, results in RNA-mediated tau multimerization, where each tau monomer binds a linear RNA sequence of approximately 30 nucleotides. Solution NMR and native mass spectrometry confirm the formation of stable complexes between RNA and the basic proline-rich and repeat domains of tau, which have a net charge of -29. Our findings demonstrate that mass photometry can distinguish between charge neutralization, which drives coacervation, and complex formation, which mediates phase re-entry, making it a highly complementary tool for the study of RNA-mediated phase separation.

Neuropeptides regulate a wide range of physiological processes throughout the nervous and endocrine systems, yet their spatiotemporal dynamics in vivo remain poorly understood. While recently developed intensiometric sensors can detect neuropeptides, their use in vivo is often confounded by artifacts induced by hemodynamic changes, pH fluctuations, and motion. Here, we present a general strategy for engineering dual-excitation ratiometric (Ex-ratiometric) neuropeptide sensors by tuning the excited-state proton transfer properties of the fluorescent reporter. Dual excitation at 405 nm and 488 nm produces a ratiometric signal that intrinsically corrects fluorescence fluctuations unrelated to ligand binding. Using an AlphaFold3-guided in silico design approach combined with experimental validation, we developed a suite of Ex-ratiometric sensors. As a representative example, Ex-NTS2.0 enables robust detection of neurotensin (NTS) in vivo while remaining largely resistant to hemodynamics, pH, and motion-induced artifacts. Overall, these findings establish a scalable and mechanistically grounded platform for developing new Ex-ratiometric tools and provide a broadly applicable strategy for sensitive detection of neuropeptide dynamics in living systems.

Achieving predictable, tunable, and temporal control over mRNA function would grant direct regulation of gene expression, facilitating the development of new therapeutics and biotechnologies. Although several approaches for stimuli-responsive control of nucleic acids have been explored, most are limited to short oligonucleotides, lack a timed-release mechanism, or both. We envisioned a complementary method using glyoxal as a caging reagent. Glyoxal readily reacts with amidine groups found on the faces of nucleobases to give stable bis-hemiaminal adducts, directly disrupting hydrogen bonding. Fortuitously, this reaction is readily reversible, enabling spontaneous time-release decaging that varies with temperature. However, when applied previously to full-length mRNAs, the sequence length and excessive adduct formation resulted in no reactivation under relevant physiological conditions. To address this challenge, we developed chemical lithography in which portions of longer RNAs are "masked" through hybridization to complementary DNAs, permitting selective caging on only non-masked regions and preventing excessive adduct formation. We present an optimized glyoxalation protocol applied to EGFP as a model mRNA sequence and evaluate masking effectiveness through qualitative and quantitative studies. Using EGFP fluorescence, we monitored and assessed the ability of selective glyoxalation to control gene expression over time in vitro. We demonstrate the direct dependence of both the initial inhibited expression and the respective activity recovery based on the amount and location of glyoxalation. We also highlight distinct caging patterns exhibiting total inhibition upon initial treatment and complete reactivation following decaging. We anticipate that this approach will improve the mechanistic study of mRNA and gene expression and also facilitate new investigations and methods within chemical biology and biomedicine.

Doxorubicin (DOX) is a clinically important anthracycline whose anticancer activity is closely associated with DNA binding. However, the structural basis by which metal coordination modulates DOX-DNA association remains poorly understood. Here, an integrated computational and spectroscopic approach was employed to elucidate the role of Cu(II)-coordination in modulating the structure of DOX and its DNA binding. DFT, molecular docking, and molecular dynamics simulations established a stable intercalative association of DOX with DNA. At the same time, DFT-based characterization of Cu(II)-DOX revealed coordination induced structural and electronic changes consistent with reduced conformational freedom and increased rigidity. This led to the hypothesis that Cu(II) coordination may favor an intercalation-compatible configuration of DOX. UV-Vis and competitive fluorescence studies experimentally supported Cu(II)-dependent modulation of DOX-DNA association, with UV-Vis analysis indicating a substantial tenfold increase in apparent DNA-binding affinity and fluorescence measurements confirming pronounced perturbation of DNA-associated ethidium bromide. Collectively, the findings establish a coherent relationship between Cu(II) coordination, DOX conformational modulation, and DNA recognition, highlighting metal coordination as a potential strategy for tuning the biomolecular interactions of anthracycline therapeutics.

Sequence-specific cleavage of viral RNA by deoxyribozymes offers a direct approach for suppressing viral gene expression, although limited cellular accessibility remains a major barrier to their functional application. In this study, a panel of eight deoxyribozymes representing different catalytic motifs and target regions was designed against SARS-CoV-2 Spike RNA and comparatively evaluated through time-dependent in vitro cleavage assays. Substantial target-site-dependent differences were observed even among candidates sharing the same catalytic core, demonstrating that catalytic performance was strongly influenced by the selected RNA target region. Among the tested sequences, 8-17-769 exhibited the fastest and most reproducible cleavage profile and the highest observed rate constants, and was therefore selected as the catalytic module for subsequent chimera development. To evaluate its activity in a cellular context, 8-17-769 was combined with the nucleolin-binding aptamer AS1411 in two opposite linear orientations. In an A549-based Spike expression model, both AS1411-containing chimeras were associated with reduced S gene expression, with 8-17-769-AS1411 producing the strongest response, corresponding to an approximately tenfold reduction relative to the reference group and reaching statistical significance (p < 0.05). The functional difference between the two orientations was further examined structurally. Circular dichroism spectroscopy showed that 8-17-769-AS1411 more closely preserved the spectral characteristics of the individual components, while computational analyses indicated a dynamic organization more similar to the free deoxyribozyme-RNA complex and a nucleolin-compatible docking configuration. Collectively, these findings identify 8-17-769 as a lead Spike RNA-cleaving deoxyribozyme and demonstrate that the linear organization of aptamer and catalytic modules can substantially influence the structural and functional properties of aptamer-deoxyribozyme chimeras.

Magnetic readout-based assays are compatible with unprocessed biological samples as unbound background molecules do not interfere with magnetic signal. Yet, a true challenge is their poor specificity and susceptibility of magnetic nanoparticles (MNPs) to clusters in complex biological media, hampering their true advancement. Here, we demonstrate that the spatial organization of functional groups at the external periphery of custom magnetic nanoparticles by harnessing ultra-dense double-stranded DNA results in an efficient antibody conjugation with good accessibility toward antigen. By labeling our MNPs with anti-S protein neutralizing IgG antibody, we showcase the detection of S1 subunit of SARS-CoV-2 Spike protein in a wash-free fashion in less than five minutes in nM regime using magnetic particle spectrometer. By mixing our IgG-labelled MNPs with DMEM cell culture (10-20% FBS serum), we sense the S1 proteins in a one-pot fashion with high specificity. Our results show that by having the ultra-dense dsDNA shell on MNPs, the entropic cost of an irreversible protein binding to particle surface is high, thus allowing the formation of dynamic protein corona on the DNA shell that can be replaced with S1 protein with high affinity. When the azide moieties are placed at the close proximity of MNPs by using non-functional dsDNA, antibody conjugation becomes inefficient, to a level not sufficient for S1 protein detection. Our study highlights the importance of spatial organization of functional moieties on the nanoscale on magnetic nanoparticles for highly specific assays in biologically complex media.