Radiation damage to biological samples: still a pertinent issue.

An understanding of radiation damage effects suffered by biological samples during structural analysis using both X-rays and electrons is pivotal to obtain reliable molecular models of imaged molecules. This special issue on radiation damage contains six papers reporting analyses of damage from a range of biophysical imaging techniques. For X-ray diffraction, an in-depth study of multi-crystal small-wedge data collection single-wavelength anomalous dispersion phasing protocols is presented, concluding that an absorbed dose of 5 MGy per crystal was optimal to allow reliable phasing. For small-angle X-ray scattering, experiments are reported that evaluate the efficacy of three radical scavengers using a protein designed to give a clear signature of damage in the form of a large conformational change upon the breakage of a disulfide bond. The use of X-rays to induce OH radicals from the radiolysis of water for X-ray footprinting are covered in two papers. In the first, new developments and the data collection pipeline at the NSLS-II high-throughput dedicated synchrotron beamline are described, and, in the second, the X-ray induced changes in three different proteins under aerobic and low-oxygen conditions are investigated and correlated with the absorbed dose. Studies in XFEL science are represented by a report on simulations of ultrafast dynamics in protic ionic liquids, and, lastly, a broad coverage of possible methods for dose efficiency improvement in modalities using electrons is presented. These papers, as well as a brief synopsis of some other relevant literature published since the last Journal of Synchrotron Radiation Special Issue on Radiation Damage in 2019, are summarized below.

Optimization of intracellular macromolecule delivery by nanoparticle-mediated photoporation.

Nanoparticle-mediated photoporation is a novel delivery platform for intracellular molecule delivery. We studied the dependence of macromolecular delivery on molecular weight and sought to enhance delivery efficiency. DU145 prostate cancer cells were exposed to pulsed laser beam in the presence of carbon-black nanoparticles. Intracellular uptake of molecules decreased with increasing molecular weight. Attributing this dependence to molecular diffusivity, we hypothesized that macromolecular delivery efficiency could be enhanced by increasing either laser fluence or laser exposure duration at low fluence. We observed increased percentages of macromolecule uptake by cells in both cases. However, trade-off between cell uptake and viability loss was most favorable at low laser fluence (25-29 mJ/cm2) and longer exposure durations (4-5 min). We conclude that long exposure at low laser fluence optimizes intracellular macromolecule delivery by nanoparticle-mediated photoporation, which may be explained by longer time for macromolecules to diffuse into cells, during and between laser pulses.

Self-Healing Heterometallic Supramolecular Polymers Constructed by Hierarchical Assembly of Triply Orthogonal Interactions with Tunable Photophysical Properties.

Here, we present a method for the building of new bicyclic heterometallic cross-linked supramolecular polymers by hierarchical unification of three types of orthogonal noncovalent interactions, including platinum(II)-pyridine coordination-driven self-assembly, zinc-terpyridine complex, and host-guest interactions. The platinum-pyridine coordination provides the primary driving force to form discrete rhomboidal metallacycles. The assembly does not interfere with the zinc-terpyridine complexes, which link the discrete metallacycles into linear supramolecular polymers, and the conjugation length is extended upon the formation of the zinc-terpyridine complexes, which red-shifts the absorption and emission spectra. Finally, host-guest interactions via bis-ammonium salt binding to the benzo-21-crown-7 (B21C7) groups on the platinum acceptors afford the cross-linked supramolecular polymers. By continuous increase of the concentration of the supramolecular polymer to a relatively high level, supramolecular polymer gel is obtained, which exhibits self-healing properties and reversible gel-sol transitions stimulated by various external stimuli, including temperature, K+, and cyclen. Moreover, the photophysical properties of the supramolecular polymers could be effectively tuned by varying the substituents of the precursor ligands.

State-of-the-Art Preclinical Photoacoustic Imaging in Oncology: Recent Advances in Cancer Theranostics.

The optical imaging plays an increasing role in preclinical studies, particularly in cancer biology. The combined ultrasound and optical imaging, named photoacoustic imaging (PAI), is an emerging hybrid technique for real-time molecular imaging in preclinical research and recently expanding into clinical setting. PAI can be performed using endogenous contrast, particularly from oxygenated and deoxygenated hemoglobin and melanin, or exogenous contrast agents, sometimes targeted for specific biomarkers, providing comprehensive morphofunctional and molecular information on tumor microenvironment. Overall, PAI has revealed notable opportunities to improve knowledge on tumor pathophysiology and on the biological mechanisms underlying therapy. The aim of this review is to introduce the principles of PAI and to provide a brief overview of current PAI applications in preclinical research, highlighting also on recent advances in clinical translation for cancer diagnosis, staging, and therapy.

Room-Temperature and Selective Triggering of Supramolecular DNA Assembly/Disassembly by Nonionizing Radiation.

Recent observations have suggested that nonionizing radiation in the microwave and terahertz (THz; far-infrared) regimes could have an effect on double-stranded DNA (dsDNA). These observations are of significance owing to the omnipresence of microwave emitters in our daily lives (e.g., food preparation, telecommunication, and wireless Internet) and the increasing prevalence of THz emitters for imaging (e.g., concealed weapon detection in airports, skin cancer screenings) and communication technologies. By examining multiple DNA nanostructures as well as two plasmid DNAs, microwaves were shown to promote the repair and assembly of DNA nanostructures and single-stranded regions of plasmid DNA, while intense THz pulses had the opposite effect (in particular, for short dsDNA). Both effects occurred at room temperature within minutes, showed a DNA length dependence, and did not affect the chemical integrity of the DNA. Intriguingly, the function of six proteins (enzymes and antibodies) was not affected by exposure to either form of radiation under the conditions examined. This particular detail was exploited to assemble a fully functional hybrid DNA-protein nanostructure in a bottom-up manner. This study therefore provides entirely new perspectives for the effects, on the molecular level, of nonionizing radiation on biomolecules. Moreover, the proposed structure-activity relationships could be exploited in the field of DNA nanotechnology, which paves the way for designing a new range of functional DNA nanomaterials that are currently inaccessible to state-of-the-art assembly protocols.

Near-infrared excited cooperative upconversion in luminescent Ytterbium(ΙΙΙ) bioprobes as light-responsive theranostic agents.

Near-infrared (NIR) Ytterbium(ΙΙΙ) complexes namely [Yb(dpq)(DMF)2Cl3] (1), [Yb(dppz)(DMF)2Cl3] (2), [Yb(dpq)(ttfa)3] (3) and [Yb(dppz)(ttfa)3] (4) based on photosensitizing antenna: dipyrido-[3,2-d:2',3'-f]-quinoxaline (dpq), dipyrido-[3,2-a:2',3'-c]-phenazine (dppz) and 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (Httfa), were designed as NIR bioimaging agents utilizing cooperative upconversion luminescence (CUCL) of Yb(III). Their structures, detailed photophysical properties, biological interactions, photo-induced DNA cleavage, NIR photocytotoxicity and cellular internalization and bioimaging properties were examined. Discrete mononuclear complexes adopt a seven-coordinated {LnN2O2Cl3} mono-capped octahedron (1, 2) and eight-coordinated {LnN2O6} distorted square antiprism geometry (3, 4) with bidentate N, N-donor dpq, dppz and O,O-donor ttfa ligands. The designed Yb(III) probes (3, 4) having advantages of dual sensitizing antennae (dpq/dppz and Httfa) to modulate the desirable optical properties in NIR region for bioimaging in biologically transparent window and light-responsive intracellular damage with spatiotemporal control. The lack of inner-sphere water (q = 0), remarkable photostability, large Stokes' shifts, presence of energetically rightly poised ligand 3T states allows efficient energy transfer (ET) to the emissive 2F5/2 state of Yb(ΙΙΙ). The unique cooperative upconversion luminescence (CUCL) of Yb(III) was observed in 1-4 in the visible blue region (λem = 490 nm) upon NIR excitation at 980 nm, makes them special candidates for NIR-to-visible or NIR-to-NIR cellular imaging probes. The CUCL property of Yb(III) were observed in the discrete mononuclear complexes both in solid state and solution. We elegantly utilized this remarkable property of Yb(III) for cellular imaging application for the first time to the our knowledge including potential uses in CUC/multiphoton excitation microscopy. The complexes exhibit significant binding propensity to DNA, HSA and BSA (K ∼ 105 M-1). They effectively cleave supercoiled (SC) DNA to its nicked circular (NC) form at 365 nm via photoredox pathways. The cellular uptake studies evidently displayed cytosolic and nuclear localization of the complexes. Finally, the capability of Yb(III) complexes usage for PDT were demonstrated through significant near-IR photocytotoxicity at 980 nm CW laser. The results depicted here offers an intelligent strategy towards developing light-responsive highly photostable Yb(III) probes for NIR theranostic application in the biologically transparent phototherapeutic window.

Mannoside and 1,2-mannobioside ß-cyclodextrin-scaffolded NO-photodonors for targeting antibiotic resistant bacteria.

Two ß-cyclodextrin derivatives randomly appended on the primary face with both the nitric oxide (NO) photodonor 4-nitro-3-(trifluoromethyl)aniline and a mannose or α(1→2)mannobioside residue are reported to construct targeted NO photoreleasing nanocarriers. 2D ROESY and PGSE NMR suggested supramolecular homodimerization in water by inclusion of the nitroaniline group into the facing macrocycle cavities. Isothermal titration calorimetry on their concanavalin A lectin binding showed an exothermic binding event to the lectin and an endothermic process during the dilution of the conjugates. Both α(1→2)mannobioside and the nitroaniline moieties significantly enhanced the binding to the lectin. These effects might arise from a better fit within the carbohydrate-recognition site in the former case and a multivalent effect caused by homodimerization in the latter. Direct detection of NO by amperometric technique shows that both ß-cyclodextrin derivatives release this radical upon excitation with visible light with higher efficiency than the unfunctionalized NO photodonor.

Ultra-Bright Rhodamines with Sulfobutylether-ß-Cyclodextrin: A Viable Supramolecular Dye Laser in Aqueous Medium.

Although aqueous dye lasers are much sought after, they have been of no practical use, as laser dyes show a strong tendency for aggregation in water, thus diminishing their optical output. Contributing towards this shortcoming, we studied the noncovalent interactions of two prominent laser dyes, namely, rhodamine 6G and rhodamine B, with a water soluble macrocyclic host, sulfobutylether-ß-cyclodextrin (SBE7 ßCD). Spectral changes in the absorption and fluorescence behavior of dyes in presence of the SBE7 ßCD host indicated adequate complex formation between dye and host (K∼104  M-1 ). A combination of various photophysical parameters evaluated from measurements such as Job plot, changes in the fluorescence lifetime/anisotropy values, and favorable thermodynamic parameters from isothermal titration calorimetric measurements adjudicated a 1 : 1 stoichiometric complex formation between dye and SBE7 ßCD host. Consequently, SBE7 ßCD prevents dye aggregation/adsorption and present rhodamine dyes in their monomeric forms with enhanced fluorescence yield and brightness. These vital parameters were utilized to optimize and demonstrate cost-effective supramolecular broad-band and narrow-band aqueous dye laser systems with improved lasing efficiencies (∼25 % higher for the SBE7 ßCD : RhB system and ∼10 % higher for SBE7 ßCD : Rh6G system), better beam profile, and enhanced durability compared to the respective dyes in optically matched ethanol solutions.

Supramolecular nanofiber of pyrene-lactose conjugates and its two-photon fluorescence imaging.

A lactose modified pyrene derivative (Py-Lac) was synthesized, with which novel twisted supramolecular nanofibers in diameter about 20 nm were constructed by self-assembly. The nanofibers showed solid-state fluorescence between 400 nm and 650 nm with the maximum emission at 495 nm. Furthermore, its recognition reaction with PNA lectin was investigated by fluorescence spectra and turbidity assays. It is interesting found that the supramolecular assembly as multivalent glycocluster exhibited unique and selectively binding interactions with PNA lectin with the binding constant of 5.74 × 106 M-1. Moreover, compound Py-Lac showed two-photon fluorescence imaging with Hep G2 cells.

X-ray and UV radiation-damage-induced phasing using synchrotron serial crystallography.

Specific radiation damage can be used to determine phases de novo from macromolecular crystals. This method is known as radiation-damage-induced phasing (RIP). One limitation of the method is that the dose of individual data sets must be minimized, which in turn leads to data sets with low multiplicity. A solution to this problem is to use data from multiple crystals. However, the resulting signal can be degraded by a lack of isomorphism between crystals. Here, it is shown that serial synchrotron crystallography in combination with selective merging of data sets can be used to determine high-quality phases for insulin and thaumatin, and that the increased multiplicity can greatly enhance the success rate of the experiment.

Ultrafast Electron Transfer across a Nanocapsular Wall: Coumarins as Donors, Viologen as Acceptor, and Octa Acid Capsule as the Mediator.

Results of our study on ultrafast electron transfer (eT) dynamics from coumarins (coumarin-1, coumarin-480, and coumarin-153) incarcerated within octa acid (OA) capsules as electron donors to methyl viologen dissolved in water as acceptor are presented. Upon photoexcitation, coumarin inside the OA capsule transfers an electron to the acceptor electrostatically attached to the capsule leading to a long-lived radical-ion pair separated by the OA capsular wall. This charge-separated state returns to the neutral ground state via back electron transfer on the nanosecond time scale. This system allows for ultrafast electron transfer processes through a molecular wall from the apolar capsular interior to the highly polar (aqueous) environment on the femtosecond time scale. Employing femtosecond transient absorption spectroscopy, distinct rates of both forward (1-25 ps) and backward eT (700-1200 ps) processes were measured. Further understanding of the energetics is provided using Rehm-Weller analysis for the investigated photoinduced eT reactions. The results provide the rates of the eT across a molecular wall, akin to an isotropic solution, depending on the standard free energy of the reaction. The insights from this work could be utilized in the future design of efficient electron transfer processes across interfaces separating apolar and polar environments.

Non-ionising UV light increases the optical density of hygroscopic self assembled DNA crystal films.

We report on ultraviolet (UV) light induced increases in the UV optical density of thin and optically transparent crystalline DNA films formed through self assembly. The films are comprised of closely packed, multi-faceted and sub micron sized crystals. UV-Vis spectrophotometry reveals that DNA films with surface densities up to 0.031 mg/mm2 can reduce the transmittance of incident UVC and UVB light by up to 90%, and UVA transmittance by up to 20%. Subsequent and independent film irradiation with either UVA or UVB dosages upwards of 80 J/cm2 both reduce UV transmittance, with reductions scaling monotonically with UV dosage. To date the induction of a hyperchromic effect has been demonstrated using heat, pH, high salt mediums, and high energy ionising radiation. Both hyperchromicity and increased light scattering could account for the increased film optical density after UV irradiation. Additional characterisation of the films reveal they are highly absorbent and hygroscopic. When coated on human skin, they are capable of slowing water evaporation and keeping the tissue hydrated for extended periods of time.

Water-Soluble Pd8L4 Self-assembled Molecular Barrel as an Aqueous Carrier for Hydrophobic Curcumin.

A tetrafacial water-soluble molecular barrel (1) was synthesized by coordination driven self-assembly of a symmetrical tetrapyridyl donor (L) with a cis-blocked 90° acceptor [cis-(en)Pd(NO3)2] (en = ethane-1,2-diamine). The open barrel structure of (1) was confirmed by single crystal X-ray diffraction. The presence of a hydrophobic cavity with large windows makes it an ideal candidate for encapsulation and carrying hydrophobic drug like curcumin in an aqueous medium. The barrel (1) encapsulates curcumin inside its molecular cavity and protects highly photosensitive curcumin from photodegradation. The photostability of encapsulated curcumin is due to the absorption of a high proportion of the incident photons by the aromatic walls of 1 with a high absorption cross-sectional area, which helps the walls to shield the guest even against sunlight/UV radiations. As compared to free curcumin in water, we noticed a significant increase in solubility as well as cellular uptake of curcumin upon encapsulation inside the water-soluble molecular barrel (1) in aqueous medium. Fluorescence imaging confirmed that curcumin was delivered into HeLa cancer cells by the aqueous barrel (1) with the retention of its potential anticancer activity. While free curcumin is inactive toward cancer cells in aqueous medium at room temperature due to negligible solubility, the determined IC50 value of ∼14 µM for curcumin in aqueous medium in the presence of the barrel (1) reflects the efficiency of the barrel as a potential curcumin carrier in aqueous medium without any other additives. Thus, two major challenges of increasing the bioavailability and stability of curcumin in aqueous medium even in the presence of UV light have been addressed by using a new supramolecular water-soluble barrel (1) as a drug carrier.

UV-Visible Absorption Spectroscopy Enhanced X-ray Crystallography at Synchrotron and X-ray Free Electron Laser Sources.

This review describes the use of single crystal UV-Visible Absorption micro-Spectrophotometry (UV-Vis AS) to enhance the design and execution of X-ray crystallography experiments for structural investigations of reaction intermediates of redox active and photosensitive proteins. Considerations for UV-Vis AS measurements at the synchrotron and associated instrumentation are described. UV-Vis AS is useful to verify the intermediate state of an enzyme and to monitor the progression of reactions within crystals. Radiation induced redox changes within protein crystals may be monitored to devise effective diffraction data collection strategies. An overview of the specific effects of radiation damage on macromolecular crystals is presented along with data collection strategies that minimize these effects by combining data from multiple crystals used at the synchrotron and with the X-ray free electron laser.

Near infrared light-responsive and injectable supramolecular hydrogels for on-demand drug delivery.

A near infrared (NIR) light-responsive supramolecular hydrogel consisting of α-cyclodextrin and poly(ethylene glycol)-modified dendrimer-encapsulated platinum nanoparticles was developed. Upon NIR irradiation, this hydrogel underwent a photothermo-sensitive degradation to release the entrapped therapeutic agents in an on-demand and dose-tunable fashion.

Interaction prolonged DNA translocation through solid-state nanopores.

An interesting smooth blocked nanopore and corresponding "current ladder" phenomenon was observed in DNA translocation experiments through solid-state nanopores. The ionic current shows several drop steps (current levels in the current ladder) with an identical drop interval, which corresponds to an individual unfolded DNA translocation event. This indicates that multiple anchored DNA molecules have one end inside the nanopore to cause such a current ladder. On each current level, normal DNA translocation events were detected. The event duration time increases as the level number increases, which means DNA translocates more slowly when more DNA molecules are inside the nanopore due to DNA-DNA interactions. The Langevin dynamic model was used to explain the experimental observations. This finding strongly suggests that DNA-DNA interactions greatly impact the translocation dynamics when DNA passes through the nanopores.

Radiation damage to macromolecules: kill or cure?

Radiation damage induced by X-ray beams during macromolecular diffraction experiments remains an issue of concern in structural biology. While advances in our understanding of this phenomenon, driven in part by a series of workshops in this area, undoubtedly have been and are still being made, there are still questions to be answered. Eight papers in this volume give a flavour of ongoing investigations, addressing various issues. These range over: a proposed new metric derived from atomic B-factors for identifying potentially damaged amino acid residues, a study of the relative damage susceptibility of protein and DNA in a DNA/protein complex, a report of an indication of specific radiation damage to a protein determined from data collected using an X-ray free-electron laser (FEL), an account of the challenges in FEL raw diffraction data analysis, an exploration of the possibilities of using radiation damage induced phasing to solve structures using FELs, simulations of radiation damage as a function of FEL temporal pulse profiles, results on the influence of radiation damage during scanning X-ray diffraction measurements and, lastly, consideration of strategies for minimizing radiation damage during SAXS experiments. In this short introduction, these contributions are briefly placed in the context of other current work on radiation damage in the field.

Identifying and quantifying radiation damage at the atomic level.

Radiation damage impedes macromolecular diffraction experiments. Alongside the well known effects of global radiation damage, site-specific radiation damage affects data quality and the veracity of biological conclusions on protein mechanism and function. Site-specific radiation damage follows a relatively predetermined pattern, in that different structural motifs are affected at different dose regimes: in metal-free proteins, disulfide bonds tend to break first followed by the decarboxylation of aspartic and glutamic acids. Even within these damage motifs the decay does not progress uniformly at equal rates. Within the same protein, radiation-induced electron density decay of a particular chemical group is faster than for the same group elsewhere in the protein: an effect known as preferential specific damage. Here, BDamage, a new atomic metric, is defined and validated to recognize protein regions susceptible to specific damage and to quantify the damage at these sites. By applying BDamage to a large set of known protein structures in a statistical survey, correlations between the rates of damage and various physicochemical parameters were identified. Results indicate that specific radiation damage is independent of secondary protein structure. Different disulfide bond groups (spiral, hook, and staple) show dissimilar radiation damage susceptibility. There is a consistent positive correlation between specific damage and solvent accessibility.

Limiting radiation damage for high-brilliance biological solution scattering: practical experience at the EMBL P12 beamline PETRAIII.

Radiation damage is the general curse of structural biologists who use synchrotron small-angle X-ray scattering (SAXS) to investigate biological macromolecules in solution. The EMBL-P12 biological SAXS beamline located at the PETRAIII storage ring (DESY, Hamburg, Germany) caters to an extensive user community who integrate SAXS into their diverse structural biology programs. The high brilliance of the beamline [5.1 × 10(12) photons s(-1), 10 keV, 500 (H) µm × 250 (V) µm beam size at the sample position], combined with automated sample handling and data acquisition protocols, enable the high-throughput structural characterization of macromolecules in solution. However, considering the often-significant resources users invest to prepare samples, it is crucial that simple and effective protocols are in place to limit the effects of radiation damage once it has been detected. Here various practical approaches are evaluated that users can implement to limit radiation damage at the P12 beamline to maximize the chances of collecting quality data from radiation sensitive samples.

New methods in time-resolved Laue pump-probe crystallography at synchrotron sources.

Newly developed methods for time-resolved studies using the polychromatic and in particular the pink-Laue technique, suitable for medium and small-size unit cells typical in chemical crystallography, are reviewed. The order of the sections follows that of a typical study, starting with a description of the pink-Laue technique, followed by the strategy of data collection for analysis with the RATIO method. Novel procedures are described for spot integration, orientation matrix determination for relatively sparse diffraction patterns, scaling of multi-crystal data sets, use of Fourier maps for initial assessment and analysis of results, and least-squares refinement of photo-induced structural and thermal changes. In the calculation of Fourier maps a ground-state structure model, typically based on monochromatic results, is employed as reference, and the laser-ON structure factors for the Fourier summations are obtained by multiplying the reference ground-state structure factors by the square root of the experimental ON/OFF ratios. A schematic of the procedure followed is included in the conclusion section.