Chemical Communications

Serotonin is a key neurotransmitter, and aptamer-based tools using the 44 nt Apt44 have been successfully developed for its in vitro and in vivo detection. Nevertheless, the structural basis of recognition by this aptamer remains unclear. Here we report high resolution NMR structures of Apt38, a 6-nt truncated variant in the third loop of Apt44, in free and serotonin-bound states. Both structures reveal a two layered antiparallel chair type G quadruplex core with three edgewise loops and a terminal duplex, forming a G quadruplex duplex hybrid structure. Serotonin binds at the G quadruplex duplex junction, stabilized by stacking, electrostatic attraction, hydrogen bonding, and hydrophobic contacts. Apt38 is preorganized for binding, whereas the longer third loop of Apt44 introduces conformational dynamics into the G quadruplex scaffold, which enables a pronounced binding triggered conformational switch in PBS buffer, explaining its sensing mechanism. Our work reveals the recognition and sensing mechanism of the serotonin aptamer and provides a framework for aptamer design in serotonin biosensing.

The growing number of approved nucleic acid therapeutics illustrates the potential to treat diseases by targeting their genetic blueprints in vivo. The 10-23 DNAzyme is capable of cleaving a wide range of target RNA with high selectivity. However, its poor performance in vivo restricts its therapeutic application as gene silencing agent. Studies on ribozymes have shown that the crowded environment in cells and associated effects can impact ribozyme folding and thermostability, resulting in a change in activity. This opens up the question whether DNAzymes are also affected by molecular crowding. Here, we investigate the functional and structural influence of molecular crowding conditions on the 10-23 DNAzyme. The stability and activity of a PrP-specific 10-23 DNAzyme were examined in presence of PEG, dextran, and osmolytes. Our results indicate that osmolytes decrease DNAzyme activity in a concentration-dependent manner, while certain PEG and dextran concentrations promote activity. To rationalize our observations, we studied the cosolutes effect on physicochemical solution properties and the structure of the DNAzyme:RNA complex using FCS and SAXS. The data reveal that enhanced activity is observed under conditions where a combination of physiochemical properties matches an optimum that seems to be dependent on the metal ion cofactor. Structural influence under such conditions is indicated less. We propose that a certain degree of molecular crowding is required to favor a state, which allows for higher catalytic turnover. In addition, we show that the requirement for magnesium and manganese as a cofactor remains unchanged under the conditions applied. Our work contributes to a better understanding of how the cellular environment affects DNAzyme structure and function.

Fluorescent dyes are critical to visualizing nucleic acids in many applications. SYTOX Orange and SYTOX Green are cyanine dyes, used in dead cell staining and increasingly in single-molecule assays to probe DNA supercoiling and processing. However, their structures and effects on DNA mechanics are not or only partially known. We determine the structure of SYTOX Orange to be (E)-2-((2-(4 ((diethyl(methyl)ammonio)methyl)phenyl)-6-methoxy-1-methylquinolin-4(1H)-ylidene)methyl)-4-methyloxazolo[4,5-b]pyridin-4-ium, identical to SYBR Gold except for an aza-benzoxazol core that is fundamentally different from other dyes in the SYTOX and SYBR families. We report SYTOX Green to be (Z)-2-(bis(3-(trimethylammonio)propyl)amino)-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-1-phenylquinolin-1-ium, similar to PicoGreen. Using magnetic tweezers, we characterize the effect of SYTOX Orange and SYTOX Green on DNA mechanics. They lengthen and unwind DNA consistent with intercalation and the DNA unwinding angles per dye are 21.1(1) degree and 20.5(1) degree for SYTOX Orange and Green, respectively. Both dyes leave the DNA bending persistence length and plectoneme size almost unaltered (<10% change up to 1 uM), which is advantageous in assays probing DNA supercoiling. Their photophysical properties reveal close agreement between single-molecule manipulation and optical absorbance and fluorescence spectroscopy. Our comprehensive set of complementary measurements relates mechanical and optical properties to the molecular structures and provides recommendations for their use in applications.

Cell penetrating thyclotides (CPTs) are synthetic molecules that promote highly efficient cellular uptake and endosomal escape of bioactive peptides. While peptides are valuable as medicinal agents, their translation to therapies is often limited by their inability to cross cell membranes. CPTs have a unique combination of chiral tetrahydrofurans and polar sidechains within a molecular scaffold that can be optimized to efficiently deliver peptide cargo into cells. The cellular uptake and endosomal escape of two peptides with anticancer biological activities but low bioavailabilities were remarkably improved after conjugation to a CPT. Using CPTs to overcome barriers to cellular uptake represents a new direction for the intracellular delivery of bioactive molecules, and will accelerate drug development for new medical therapies.

mRNA technology has emerged as a powerful new class of medicines. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, including treatments for cancer, metabolic disorders, cardiovascular conditions and autoimmune diseases. The 3'-polyadenylated (poly(A)) tail of mRNA is required for ribosome initiation, translation, and mRNA stability and is considered a critical quality attribute. In this study, novel direct mass spectrometry approaches were used for the analysis of both the DNA template and corresponding mRNA generated via in vitro transcription. Nucleotide resolution of the poly(A/T) sequence of the DNA template and mRNA poly(A) tail was achieved. The results show that the mRNA poly(A) tail length and heterogeneity is impacted by the heterogeneity of the DNA template, the DNA template design and RNA manufacturing conditions, including relative NTP concentrations. These results provide further important mechanistic insight into the poly(A) tail length and heterogeneity of mRNAs synthesised in vitro, including the identification of 3'-end additions of cytidine to mRNA poly(A) tails. The ability to rapidly assess DNA template quality, combined with monitoring mRNA poly(A) tail length and heterogeneity, is important as part of the characterisation of mRNA precision medicines and ensuring consistent quality of mRNA from manufacturing processes.

The inhibition of A{beta}42 ({beta}-amyloid) fibril formation is a key therapeutic strategy in Alzheimer's disease research. Paddlewheel diruthenium complexes have shown promising activity against A{beta}42 aggregation and preformed fibril disaggregation, yet their molecular mode of action remains poorly understood. In this work, we perform atomistic simulations to explore how charge modulation influences the interactions of three analogous paddlewheel diruthenium complexes, the parent neutral complex [Ru2Cl(D-p-FPhF)(O2CCH3)3], and its anionic [Ru2Cl2(D-p-FPhF)(O2CCH3)3]- and cationic [Ru2(D-p-FPhF)(O2CCH3)3]+ counterparts (D-p-FPhF- is the N,N' -bis(4-fluorophenyl)formamidinato ligand) with A{beta}42. Our results indicate that electrostatic tuning governs binding affinity and the extent of interaction across the A{beta}42 fibril surface. As the complexes' charge changes from -1 to +1, the interaction pattern shifts from localized contacts to widespread, multi-site engagement encompassing key charged, aromatic, and hydrophobic regions of A{beta}42. This enhanced binding correlates with longer-lived, thermodynamically stable interactions at the fibril interface, which effectively lower the free energy penalty for fibril disassembly. Overall, our findings propose a mechanism in which charge-dependent activation through ligand exchange enhances fibril recognition and promotes disruptive binding modes, demonstrating the potential of charge-tunable diruthenium complexes as therapeutic modulators of A{beta}42 fibril stability.

Discovering small molecules targeting proteins represents a major effort in drug development. RNA, however, as a class of macromolecule that carrying out important regulatory roles in the cell as drug target, only received attention recently. Although several methods have been proposed, an easy to operate, fast and robust method is still lacking. We designed a generic florescence screening method by fusing the target RNA with a florescent aptamer (fusion RNA) and then carried out screening using high-throughput format (Fluorescent Aptamer Screening, FAS). In this work, we chose SL5 on SARS-Cov-2 5’UTR as the test target. SL5 is a conserved motif across several corona virus family members whose core is not prone to mutation. We screened 9528 compounds, successfully identified four molecules (Sertraline (hydrochloride), Samuraciclib (hydrochloride), Minocycline (hydrochloride), JG-98 bind direct to the full-length SL5 at micromolar or higher affinity. The design of FAS could be easily adapted to structured RNA motifs without prior knowledge of its 3D structural information. In addition, this work showed the possibility of developing generic drugs for RNA virus by targeting the conserved viral RNA genome and paved a new way for the discovery of small molecule drugs in combating human diseases.

Flavins are organic redox cofactors central to metabolism and uniquely capable of acting as extracellular electron shuttles. For life to have emerged, it must have disengaged itself from its stationary geochemical environment, a step requiring mobile redox-active components. The role of flavins at life's origin has been debated for decades, centered on their capacity for both one- and two-electron chemistry, distinguishing them from nicotinamides and iron-sulfur clusters. Here we chart the abiotic reduction of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and riboflavin under hydrothermal conditions (40 {degrees}C, 1 bar N2 or 5 bar H2, pH 6, 8, and 10) by nickel (Ni) and iron (Fe). Flavins show greater environmental versatility than hydride carriers such as NAD and can harvest electrons from metals that would otherwise reduce water's protons to H2. Reduction is favoured under acidic conditions, while increasing molecular charge at higher pH impedes electron transfer. Ni acts as a hydrogenation catalyst, reducing deprotonated flavins via hydride transfer, suggesting mineral composition could have influenced geochemical selection of early electron carriers. Reduced FMNH2 and FADH2 were tested as electron shuttles toward Fe3+-containing minerals, revealing that FMNH2 enables faster mineral dissolution than FADH2. We further demonstrate complete redox cycling of FMN through Ni-assisted H2 reduction and subsequent oxidation by magnetite (Fe3O4) under inert atmosphere, releasing Fe2+. This study highlights the versatility, stability and redox chemical capabilities of flavins in prebiotic context.

Protein cages provide useful scaffolds for nanoscale engineering due to their highly ordered structures and in vivo self-assembly. These scaffolds are amendable to late-stage conjugation, enabling expansion in functionality. However, many conjugation techniques either lack site-selectivity, require unnatural amino acid incorporation, or have bulky recognition motifs to facilitate ligation reactions. Here, an asparaginyl endopeptidase (AEP) enzyme with ligase activity is employed for the highly efficient functionalization of virus-like particles (VLPs) from Salmonella Typhimurium bacteriophage P22. The capacity of this enzyme to conjugate peptides and proteins onto assembled P22 VLPs under mild reaction conditions, via a minimal extension to the P22 coat protein C-terminus, is demonstrated. We extend the reaction efficiency to facilitate a one-pot dual-functionalization reaction whereby two therapeutically relevant receptor targeting domains are conjugated to P22 VLPs in a single step. Finally, we demonstrate the potential for AEP-mediated bioconjugation to bestow P22 VLPs with receptor-binding functionality in vitro. This work demonstrates the efficacy of AEP ligases as bioconjugation tools for site-selective functionalization of large molecular assemblies like VLPs.

Non-canonical amino acids (ncAAs) are valuable tools in chemical biology and biochemistry for labeling, probing, and tracking biomolecules. ncAAs that can be recombinantly incorporated using native E. coli machinery are particularly useful because they allow for global protein incorporation and avoid complex genetic code expansion. Here, we demonstrate successful incorporation of a methionine analog, L-cyanohomoalanine (Cha), by the methionyl-tRNA synthetase of E. coli into mutant superfolder GFP (sfGFP) expressed in methionine auxotroph bacterial cultures. We compare to methionine auxotroph bacterial cultures supplemented with L-methionine (Met) or L-azidohomoalanine (Aha). In control prototrophic E. coli, bacterial growth rates are inhibited with high concentrations of Aha but not Cha. However, less sfGFP is produced in auxotrophic cells supplemented with Cha compared to Aha and Met. Thus, while Cha is non-toxic to E. coli it is incorporated less efficiently into proteins than Aha or Met. Mass spectrometry confirmed that N-terminal Cha, Aha, and Met are cleaved, as expected for the sfGFP mutants. Other sites of Cha and Aha incorporation were confirmed by mass spectrometry, with labeling efficiency varying by position. Thermal melts of purified sfGFPs demonstrate that Cha and Aha labeling does not significantly perturb the protein stability. In the future, Cha may be useful for proteome labeling by wild-type methionyl-tRNA synthetase and could be implemented in metabolic pulse-labeling of newly synthesized proteins with other methionine analogs. Additionally, the nitrile moiety of Cha may be used to perform reactions orthogonal to azide/alkyne click chemistry or could serve as a vibrational reporter of the environment.

Cryptic binding sites generated by local conformational dynamics have become an important concept in protein-targeted ligand discovery, yet their energetic accessibility and relevance to RNA recognition remain less well understood. Here, we use the env8 cobalamin (Cbl) riboswitch as a model system to investigate the energetic consequences of cryptic-site formation through base displacement. Structural analysis revealed that binding of {beta}-axial substituted Cbl derivatives displaces a conserved adenosine (A20) from the RNA core, exposing a previously hidden binding site that is subsequently occupied by the {beta}-axial substituent. Using selective abasic substitution at this position, we quantified the energetic contributions associated with A20 in the native RNA core and with base displacement. Isothermal titration calorimetry and fluorescence measurements revealed that cryptic-site formation incurs a modest energetic penalty of ~1.4 kcal mol-1. Guided by this experimentally derived framework, computational conformational sampling recapitulated cryptic-site formation in the Cbl riboswitch and identified analogous cryptic sites in structurally unrelated RNAs from HIV-1 and HCV. These cryptic-site conformers were identified within low-energy conformational windows and exposed ligand-accessible surfaces through local base displacement. Finally, a ligand previously identified to target the env8 cryptic site bound both RNAs and yielded docking poses consistent with engagement of the newly exposed binding surfaces. Together, these results indicate that cryptic RNA binding sites can be both energetically accessible and chemically addressable, expanding the range of conformational states that may contribute to RNA ligandability.

Single-particle tracking (SPT) over time enables direct observation of molecular transport and interactions in living cells. Fluorescence-based SPT has provided insights into intracellular processes such as endocytosis, receptor signaling, and drug delivery. Extending the observation window to several hours and beyond is critical for capturing slow intracellular dynamics, including the full course of endosomal trafficking, the long-term accumulation of particles within subcellular compartments, and transitions between transport modes that occur on hour-scale timescales. However, long-term intracellular SPT under visible-wavelength excitation remains challenging because fluorescence probes generally suffer from photobleaching and phototoxicity. While near-infrared (NIR) excitation can simultaneously mitigate these issues, generally weak emission of NIR-emitting dyes has hindered its wide application in long-term SPT. Here, we demonstrate long-term NIR SPT using atomically precise gold quantum needles, Au42(PET)32 (PET = 2-phenylethanethiolate). Continuous tracking of intracellular particles in living HEK293 cells was achieved for up to 12 h. Trajectory analysis revealed temporal transitions between directional and diffusive transport, as well as the accumulation of multiple particles within localized intracellular domains over several-hour timescales. The high photostability of Au42, combined with low phototoxicity of NIR excitation, enables visualization of intracellular transport dynamics over timescales difficult to access using conventional visible fluorescent probes. These results establish Au42-based NIR imaging as a platform for long-term, low-phototoxicity intracellular SPT and provide a framework for investigating slow intracellular dynamics in living systems.

Past few decades witnessed a boom in pharmaceutical and bioproduct industry with the help of bioprocess technology. Industrially important bioproducts can be produced in large scale for commercialization with the help of fermenters. Here in, pharmaceutically valuable bioproduct melanin, synthesized from Pseudomonas stutzeri strain BTC109 by using two different sized bioreactors. Under controlled conditions the bacteria were allowed to synthesis melanin nanoparticles. The important parameters to be monitored here are pH, dissolved oxygen, agitation, aeration, melanin production and cell biomass concentration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/737634v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@d92fe5org.highwire.dtl.DTLVardef@d76c07org.highwire.dtl.DTLVardef@f5516forg.highwire.dtl.DTLVardef@1b5864b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPharmaceutical and bioproduct development industries witnessed a shoot up due to bioprocess technology. C_LIO_LIIndustrially important bioproduct like melanin can be produced in large scale with the help of industrial fermentation technology. C_LIO_LIThe product thus obtained was found to be nano sized and it can be commercialized. C_LI

Fluorescence correlation spectroscopy (FCS) provides valuable information about molecular dynamics, however, experimental setup typically requires labour-intensive passivation to prevent non-specific binding of molecules to sample containers. Furthermore, precious samples can be wasted by having to use relatively high sample volumes in existing sample containers. We overcome these major issues using a simple method of sample encapsulation into water-in-oil droplets, using purified proteins and cell lysates as proof-of-concept. FCS of fluorescently labelled protein samples in the nanomolar (nM) range confirmed that water-in-oil droplets yield more accurate measurements than conventional open-chamber methods. We first optimized the droplet composition to prevent protein coating at the water-oil interface using pegylated-lipids. We then utilized FCS to accurately measure protein concentrations and diffusion speeds in nanolitre volumes. Additionally, we used fluorescence cross-correlation spectroscopy (FCCS) to measure enzymatic cleavage of substrate inside our droplet system, demonstrating the capacity of this platform to measure biological processes at the nanoscale. Overall, conducting FCS in droplets offers a cost-effective, robust, and accessible alternative for measuring molecular dynamics, with promising potential for high-throughput and resource-limited applications. TOC Image + Text O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/734730v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@e0023dorg.highwire.dtl.DTLVardef@b32cb0org.highwire.dtl.DTLVardef@13ad832org.highwire.dtl.DTLVardef@47dc12_HPS_FORMAT_FIGEXP M_FIG C_FIG Conventional single-molecule fluorescence requires slow, expensive glass passivation procedures to prevent proteins adsorbing to surfaces. By encapsulating proteins in lipid-coated nanolitre water droplets, the passivation requirement is removed, enabling accurate measurement of protein dynamics in low nanolitre volumes. Water-in-oil droplets thus provide a passivation-free platform for fluorescence correlation spectroscopy.

The incorporation of non-canonical amino acids (ncAAs) into proteins, developed in the past 20 years, has opened new avenues with respect to protein structure, protein modification, protein-protein interaction or enzyme catalysis beyond what is possible with the 20 proteinogenic AAs. Although >300 unusual building blocks including several ncAAs have been described in nonribosomal peptides (NRPs) naturally, we aimed to further expand the scope of the underlying nonribosomal peptide synthetases (NRPS) to incorporate ncAAs beyond the naturally available ones. We have therefore systematically screened for ncAA accepting NRPS systems, applied NRPS engineering to transfer the respective ncAA-accepting parts into other NRPSs and thereby created novel peptides that were further derivatized in post-enzymatic chemical synthesis reactions directly in bacterial culture extracts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/738027v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@90552forg.highwire.dtl.DTLVardef@1c8a5e0org.highwire.dtl.DTLVardef@2549dorg.highwire.dtl.DTLVardef@1012911_HPS_FORMAT_FIGEXP M_FIG C_FIG

Thermodynamic characterization of biomolecular interactions is essential for understanding the enthalpic and entropic driving forces of molecular recognition, but established label-free techniques are limited either by bulk refractive-index sensitivity or by the lengthy thermal equilibration required to suppress it. Here, we used focal molography to investigate the temperature-dependent binding of the protein kinase A regulatory subunit (PKA-R) to cyclic AMP (cAMP) derivatives and to derive apparent thermodynamic signatures from kinetic measurements. We first validated the diffractometric readout under conditions that challenge refractometric sensors: the coherent mass density channel strongly suppressed temperature-induced bulk refractive-index effects and resolved binding in 50% human serum despite measurable non-specific adsorption, reducing the need for lengthy equilibration and buffer matching. We then combined focal molography with DNA-directed immobilization (DDI), allowing five cAMP derivatives to be presented in parallel on the same multiplexed chip and followed across five temperatures. This format yielded distinct, internally consistent apparent thermodynamic fingerprints for each derivative, separating ligands with similar affinities by their enthalpic and entropic contributions. Together, these results establish focal molography with DDI as a multiplexed workflow for comparative thermodynamic fingerprinting of biomolecular interactions at higher throughput.

Actin turnover is a fundamental cellular process essential for cell dynamics, whose control is critical for both medicine and biotechnology. However, conventional small molecules modifying actin turnover scramble the structure of actin filaments and display high cellular toxicity. MICAL enzymes oxidizing methionine (Met) residues in actin can potentially address this challenge, but their large size and multiple required cofactors make MICALs manufacturing and utilization difficult. Here we show that redox-active chiral decavanadate nanoclusters with tartaric acid are capable of site-selective actin modulation, mimicking MICALs, while requiring no cofactors, displaying high biocompatibility and being membrane permeable. Decavanadate nanoclusters serve as atomically precise "nano-enzymes" oxidizing three Met residues in globular actin, including Met-176; the latter inhibits the opening of the backdoor segment and prevents depolymerization of actin filaments. The structure of actin filaments formed after nanocluster treatment revealed no structural disturbances as confirmed by cryo-electron microscopy. The biocompatibility and bioactivity of chiral decavanadate nanoclusters was demonstrated by modulation of actin in living NG108-15 cells. Taking advantage of atomically precise structure of the nanoclusters, we show that their docking into actin can be predicted computationally, indicating the possibility of programmable actin modulation using the tools of nanochemistry.

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II' (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

The function of a protein depends not only on its sequence but on post-translational modifications and folding that produce functionally distinct proteoforms. Single-molecule methods for protein identification, such as nanopore sequencing, typically require denaturation or proteolysis, sacrificing conformational information that contributes to proteoform diversity. Here, we identify intact, folded proteins by recording an optical fingerprint of their local surface chemistry using a single covalent label. The signal is produced by a spontaneously blinking fluorophore attached to the protein through established bioconjugation reactions. The thermodynamics and kinetics of its switching between a fluorescent and a dark state are influenced by the immediate protein environment in a chemically interpretable manner. Further discriminative information can be extracted using deep learning to achieve excellent identification accuracy. Using this approach, we distinguish different proteins, different pockets of the same protein, and the presence of a single post-translational modification, in each case tracing the classification back to a distinct physicochemical mechanism. These results establish single-molecule fluorescence blinking as both a protein fingerprinting method and a probe of local chemistry on the surface of folded proteins.

Recent advances in the discovery, characterisation, and engineering of urethanases provide new opportunities for the sustainable biocatalytic degradation of polyurethane waste. A mechanistic understanding of enzyme-plastic interactions is essential for structure-based engineering to enhance urethanase activity. However, the extremely complex and hydrophobic nature of polyurethane makes it challenging to elucidate the structural basis of enzyme-plastic interactions. Here, we used a fragment-based approach to characterise the active sites of two novel urethanases with different catalytic scaffolds, employing both a crystallographic fragment-screening (FASE) campaign and soluble fragments of plastic-like analogues that mimic the substrate, transition state, or product. FASE identified new substrate-binding subpockets while interactions of plastic mimetics in the active site provided a mechanistic understanding of the recognition and binding of polyurethane fragments by these subpockets. These results highlight a diversity of binding modes among urethanases toward different polyurethane fragments.