Chemical Communications

The intentional targeting of RNA with small molecules is recognized as a viable pathway to new therapeutics with potential to vastly expand the proportion of the human genome considered druggable. The practical considerations to deliberately target RNA are largely under development, including optimal screening methods to identify small molecules for binding to RNA. Native mass spectrometry (nMS) is established as a valuable biophysical method for identifying small molecule hits against diverse biomolecular targets, most frequently proteins and protein-protein interactions. Herein we assess nMS for studying the binding of small molecules with RNA aptamers as a model for nMS screening of RNA as a drug target. We first develop workflows for characterizing the binding of cognate RNA aptamer ligands and then establish a nMS method to screen a small molecule library against the RNA aptamers. nMS analysis permitted identification of binders, quantitation of binding strength and generation of structure-activity relationships with some dependence on the aptamer class. This work demonstrates the utility of nMS as a complementary and efficient target-based biophysical screening method that can characterize RNA-small molecule interactions and the potential of nMS becoming a powerful enabling tool in RNA-targeting drug discovery.

We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for physiologically low pH levels (pH<6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule Forster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the kinetics of i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor establish the time scale for the pH drop in a commonly used oxygen scavenging system.

Studies on fluorophore-tagged peptides help in elucidating the molecular mechanism of amyloidogenesis including their cellular internalization and crosstalk potential. Despite several advantages, unavoidable difficulties including expensive and tedious synthesis-protocols exist in fluorophore-based tools. Importantly, covalently-tagged fluorophores could introduce structural constraints which may influence the conformation of the monomeric and aggregated forms of protein. To resolve this problem, we describe a robust yet simple method to make fluorescent amyloid fibrils through non-covalent incorporation of fluorophores into amyloid fibrils. We used aggregation protocol in which a small amount of fluorophore is incorporated into the amyloids, and this protocol does not alter the aggregation kinetics and the characteristic {beta}-sheet-conformers of the generated amyloid fibrils. We have successfully prepared fluorescent amyloid fibrils of Insulin, Lysozyme and A{beta}1-42, and the noncovalently incorporated fluorophores remained intact in the amyloid fibrils without leaching, even after serial-dilutions and prolonged-storage. Further, this method enables successful monitoring of cellular-internalization of the fluorescent amyloids into SH-SY5Y and A549 cells, and it also detects FRET-signals during interfibrillar interactions. The findings establish a simple and affordable protocol to prepare fluorescent amyloid structures, which may significantly help amyloid researchers working on both in vitro and animal model systems.

The ribosome is responsible for assembling proteins using 20 naturally occurring L-handed amino acids. However, incorporating non-natural amino acids into a protein is a challenging process needs improvement. In this study, we report a new possible approach to creating nonnatural peptides using ribozymes inspired by the peptidyl transfer center. These RNA scaffolds, which are approximately 100 nucleotides in length, bind to RNase T1 truncated tRNA-like chimeras and bring them into close proximity to facilitate peptide ligation. We used single-molecule fluorescence resonance energy transfer (smFRET) to show close distances between RNA-RNA, tRNALys-tRNALys, and RNA-tRNALys pairs, which strongly suggests that the mechanism of peptide ligation is due to the proximity of the substrate through dimerization of the enzymes. Mass spectrometry analysis confirmed the detection of oligopeptides from four amino acids, including L-Lysine, D-Lysine, L-Phenylalanine, and D-Phenylalanine. These results indicate that ribozymes have greater flexibility in accommodating nonnatural amino acids. Our findings pave the way for potentially new avenues in the synthesis of nonnatural peptides, beyond the limitations of ribosomal peptide synthesis and other existing methods. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/538729v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@121044corg.highwire.dtl.DTLVardef@a11b2eorg.highwire.dtl.DTLVardef@ffa0b9org.highwire.dtl.DTLVardef@90f512_HPS_FORMAT_FIGEXP M_FIG C_FIG

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

Site-specifically labeling proteins with multiple dyes or molecular moieties is an important yet non-trivial task for many research, such as when using Foster resonance energy transfer (FRET) to study dynamics of protein conformational change. Many strategies have been devised, but usually done on a case-by-case basis. Expanded genetic code provided a general platform to incorporate non-canonical amino acids (ncAA), which can also enable multiple site-specific labeling, but its technically complicated and not suitable for some applications. Here we present a streamlined method that could enable dual site-specific protein labeling by using a tryptophan auxotroph of Escherichia coli to incorporate a naturally found tryptophan analog, 5-hydroxytryptophan into a recombinant protein. As a demonstration, we incorporated 5-hydroxytryptophan into E. coli release factor 1 (RF1), a protein known to possess two different conformations, and site-specifically attached two different fluorophores, one on 5-hydroxytryptophan and another on a cysteine residue. This method is simple, generally applicable, efficient, and can serve as an alternative way for researchers who want to install an additional labeling site in their proteins.

Dopamine (DA) is an essential neurotransmitter modulating motor and cognitive functions. Several neurological disorders, including Parkinsons disease (PD) and drug addiction, are the result of DA system dysfunction; however, it remains incomplete understood of why DA neuron is selectively more vulnerable than other neurons. Here we utilize the spectral feature of human MAO B (monoamine oxidase B) to design a genetic-amenable, GFP-based fluorescent probe CyDAP. Upon genetic and pharmacological manipulations to elevate the cytosolic DA levels in cells and Drosophila models, CyDAP shows enhanced GFP emission, suggesting this probe is feasible for DA detection. Furthermore, we observe that expressing human -Synuclein in Drosophila elicited GFP emission from CyDAP, suggesting a link between cytosolic DA imbalance and regional vulnerability in PD context. Importantly, CyDAP can detect the change of cytosolic DA in live Drosophila brains, as demonstrated by time-lapse and the 4D light-sheet confocal recording. CyDAP may serve as a tool for evaluating metabolic deregulation of DA in brain models of PD and other DA system-related psychiatric disorders.

While SARS-CoV-2 Mpro and PLpro proteases are known to cleave polyproteins pp1a and pp1ab at multiple sites, these have not been comprehensively characterized in living cells. Here we engineered a two-color Bioluminescence Resonance Energy Transfer (BRET)-based, dual protease (DuProSense) biosensor platform relying on a proximity-dependent energy transfer from a luciferase donor to two spectrally separated fluorescent protein acceptors enabling simultaneous monitoring of processing of two cleavage sites in a single assay with high specificity. DuProSense revealed a similar Mpro and PLpro cleavage kinetics for their N-terminal autocleavage sites. Importantly, systematic characterization of various Mpro and PLpro cleavage sites using DuProSense revealed significant differences in cleavage rates and nirmatrelvir potency of Mpro cleavage sites but no correlation between the cleavage rates and nirmatrelvir IC50 values. Overall, our results provide deeper insights into the proteolytic processing of SARS-CoV-2 polyproteins and the dual color BRET platform will find wider applications in the future. HighlightsO_LIEngineered a two-color BRET-based, dual protease biosensor (DuProSense) C_LIO_LIDuProSense biosensor enabled simultaneous and specific monitoring of Mpro and PLpro activities C_LIO_LIDuProSense platform revealed differential cleavage kinetics of Mpro cleavage sites in live cells C_LIO_LIDuProSense platform revealed Mpro cleavage site-dependent nirmatrelvir potency in live cells C_LI

Intermediates along the fibrillation pathway are generally considered to be the toxic species responsible for the pathologies of amyloid diseases. However, structural studies of these species have been hampered by heterogeneity and poor stability in standard aqueous conditions. Here, we report a novel methodology for producing stable, on-pathway oligomers of the human Type-2 Diabetes-associated islet amyloid polypeptide (hIAPP, or amylin) using the mechanical forces associated with magic angle spinning (MAS). The species were a heterogeneous mixture of globular and short rod-like species with significant {beta}-sheet content and the capability of seeding hIAPP fibrillation. We used MAS NMR to demonstrate that the nature of the species was sensitive to sample conditions including peptide concentration, ionic strength, and buffer. The methodology should be suitable for studies of other aggregating systems.

The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and appropriate localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, FusionRed-MQV, which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (~140,000 M-1cm-1), increased radiative rate constant and reduced non-radiative rate constant with respect to its precursor. The properties of FusionRed-MQV derive from three mutations - M42Q, C159V and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the phenol and the acylimine ends of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the phenol end of the chromophore were mutated. The M42Q mutation is located near the acylimine end of the chromophore and was discovered using site-directed mutagenesis guided by x-ray crystal structures. FusionRed-MQV exhibits 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells (based on FACS) compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells.

Recent outbreak of novel coronavirus (COVID-19) caused around 7 million deaths people worldwide and still afflicting on the global health, economy and social setup. Timely detection and diagnosis are crucial steps to reduce the spread and prevention of any pandemic. Different types of diagnosis methos has been used. In last decade nanomaterials and metal organic frameworks (MOFs) based biosensors has been developed to detect the other viruses. We have designed the Zeolitic imidazolate framework-8 (ZIF-8) based biosensor to detect the COVID-19. ZIF-8 work as fluorescence quenching and re-emergence platform to detect the COVID-19 RNA sequences. ZIF-8 platform is highly sensitive which can distinguish the highly conserved single strand RNA and with 200 pM concentrations. It can distinguish down to the single mismatch nucleotide in RNA sequences.

Rapid and reproducible optical transitions of a fluorescent protein (FP) can be achieved with a Genetically Encoded Voltage Indicator (GEVI) via manipulation of the membrane potential. These transitions revealed novel effects of internal mutations near the chromophore that would not be detected under steady state conditions. Mutating an internal threonine (T203) affected the speed of the voltage-dependent fluorescence transition suggesting a conformational change inside the protein. These optical transitions also demonstrated interplay between internal and externally oriented sidechains of the {beta}-can structure. Replacing the steric hindrance of a phenylalanine near the chromophore with threonine (F165T) did not alter the resting fluorescence but resulted in a more complex fluorescent transition providing evidence for a flexible chromophore undergoing conformational changes. F165T orientation was influenced by the flanking external amino acids at positions 164 and 166 with 164F/165T/166T exacerbating the complexity of the voltage-dependent transition while 164T/165T/166F reduced the flexibility of the chromophore resembling the transition pattern of the original F165 version. Alphafold predictions reveal a threonine switch with different orientations of the F165T internal side chain depending on the direction of the offset in polarity at external positions 164 and 166. The crystal structures of the pH-sensitive FP, Super Ecliptic pHluorin and two derivatives solved in varying pH conditions also indicate interactions between the external protein surface and the internal environment providing another example of a threonine switch near the chromophore at T203. This ability to orient internal sidechains has led to the development of a novel GEVI that gets brighter upon depolarization of the plasma membrane, works at low light levels, is less susceptible to physiological pH, and provides in vivo signals. These observations affecting fluorescent transitions should also prove valuable to the development of any FP-based biosensor.

Fibrillar aggregates of the natively disordered protein -synuclein (S) are hallmarks of Parkinsons disease and related neurodegenerative disorders termed synucleinopathies. Here, we used micromap ({micro}Map) photo-proximity labeling to determine the interactomes of S monomers and fibrils in mouse brain lysate to better understand both the loss of healthy function and gain of toxic function aspects of synucleinopathies. Several S variants were synthesized and characterized, showing that the small size (1 kDa) of the Ir catalyst attached through a Cys-maleimide linkage makes it minimally-perturbing to S, with a narrow labeling radius that allows one to identify interactome differences between different regions of S. Monomer and fibril interactomes were compared to each other and to previous proximity labeling data sets for validation and several examples of further investigations are demonstrated, including Western blotting, super-resolution microscopy, and {micro}Map in primary neurons.

Chemically modified nucleic acids have become a powerful platform for basic research and applied technologies. Universal nucleobases are used in PCR,sequencing, and the design of nanodevices and aptamers. Fluorescent universal nucleobases have an even wider range of applications, including the development of nucleic acid-based sensors, switches, and relay logic gates. However, few such nucleobases have been proposed to date, and most of them have suboptimal optical properties. Here, we propose an adenine-based molecular rotor, 7,8-dihydro-8-oxo-6-(3-methylbenzo[d]thiazol-2(3H)-ylidene)adenine (oxo-Ade BZT), as a new, remarkably bright and potent fluorescent universal nucleobase. Its brightness in both oligodeoxyribonucleotides (ODNs) and DNA duplexes (4200 - 10000 M-1 x cm-1) originates from a high molar extinction coefficient (averaged{varepsilon} 368 37000 M-1 x cm-1), provided by the appended 3-methylbenzo[d]thiazolyl moiety, and a relatively high quantum yield (0.11 - 0.27). Melting temperature variations observed upon the incorporation of oxo-Ade BZT opposite native nucleobases in a duplex context did not exceed 10%. The basis of these universal hybridizing properties was unveiled using computational methods. According to molecular dynamics simulations, oxo-Ade BZT pushes the opposite nucleobase out of the DNA double helix and forms multiple hydrophobic contacts with the flanking base pairs. At the same time, the rotational mobility of the bonds between the oxo-Ade BZT-constituting heterobicycles decreases, and oxo-Ade BZT adopts a planar conformation in both ODNs and their duplexes, resulting in the light-up effect. These properties make oxo-Ade BZT a promising molecular tool for analytical, biophysical and biochemical studies.

In this study, we develop and apply a directed evolution approach to engineer the optical sensing properties of DNA-wrapped single-walled carbon nanotubes (DNA-SWCNTs) towards mycotoxins, a class of molecules critical to detect in the food industry. We successfully demonstrate the creation of sensors for the detection of both the aflatoxin B1 (AFB1) and fumonisin B1 (FB1) mycotoxins based on the specific response of the (9,4) and (7,5) SWCNT chirality fluorescence peaks, respectively. The resulting chirality-specific responsivity was used to demonstrate the multimodal detection of both mycotoxins at different wavelengths of light in the presence of complex food medium. Moreover, we show that directed evolution can be used not only to improve the chiral-dependent selectivity of our sensors to the mycotoxins, but also the sensor sensitivity and fluorescence intensity through multiple rounds of evolution. The approach demonstrated in this study is versatile and could be generalized to other SWCNT sensors as well as other nanosensors comprising a biological element.

Riboswitch is a non-coding messenger RNA (m-RNA) whose aptamer domain binds cognate ligands and subsequently undergoes conformational changes in the expression platform leading to the regulation of gene expression. Fluoride riboswitch has immense pharmacological potential due to its presence in some human bacterial pathogens. Several experimental studies shed light on the bacterial defense mechanism of Fluoride riboswitch upon binding of F- cognate ligand in the presence of Mg2+. However, the structural and thermodynamic basis of ligand binding with Fluoride riboswitch aptamer is not well known. This fascinates us for investigating the conformational stability of (i) the holo form of T. Petrophila fluoride riboswitch aptamer (RNA+F-+Mg2++K+) with respect to (ii) the apo form of fluoride riboswitch (RNA in the absence of F- +Mg2++K+). Conformational thermodynamics results derived from molecular dynamics simulation reveal that the holo form of the Fluoride riboswitch aptamer is stabilized by ion recognition site, pseudoknot, and stem1. However, Stem2, Loop1, Loop2, and most of the unpaired bases show significant disorder and destabilization. Molecular docking study validates the thermodynamically destabilized and disordered residues from Loop1 and Stem2 of the Fluoride riboswitch aptamer to serve as putative binding sites for non-cognate ligands. The global health system in the current century faces a serious crisis to counteract bacterial infection due to the severe emergence of bacterial resistance to antibiotics. Consequently, a need for a new generation of antibiotics against resistant bacteria is critically acclaimed. Our work hopefully improves the design of new ligands and aptamers which may be helpful in nucleic acid-targeted therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/600262v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@145b5beorg.highwire.dtl.DTLVardef@19eeb58org.highwire.dtl.DTLVardef@6d0e6corg.highwire.dtl.DTLVardef@1ce0c10_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIConformational stability of (i) the holo form of T. Petrophila fluoride riboswitch aptamer (RNA+F-+Mg2++K+) with respect to (ii) the apo form of fluoride riboswitch (RNA in the absence of F- +Mg2++K+) is studied. C_LIO_LIHolo Fluoride riboswitch aptamer gets energetically and entropically stable at Pseudoknot, Stem1, and Ion recognition sites whereas Stem2, Loop1, Loop2, and most of the unpaired bases show significant disorder and destabilization. C_LIO_LIThe hydrogen bond network for Pseudoknot, Stem1, and Stem2 in apo Fluoride riboswitch is significantly weak. C_LIO_LIMolecular Docking study confirms that the thermodynamically destabilized and disordered residues from Stem2 and Loop1 of the holo Fluoride riboswitch aptamer serve as putative binding sites for non-cognate ligands. C_LI

Various oligomeric species of amyloid-beta have been proposed to play different immunogenic roles in the cellular pathology of Alzheimers Disease. However, investigating the role of a homogenous single oligomeric species has been difficult due to highly dynamic oligomerization and fibril formation kinetics that convert between many species. Here we report the design and construction of a quantum dot mimetic for larger spherical oligomeric amyloid species as an "endogenously" fluorescent proxy for this cytotoxic species to investigate its role in inducing inflammatory and stress response states in neuronal and glial cell types.

Vascular Endothelial Growth Factor-A (VEGF-A), a pluripotent cytokine and angiogenic growth factor mediates the switch to an angiogenic phenotype in cancer cells. The interaction of VEGF-A protein with the VEGF receptors (VEGFR-1and VEGFR-2) starts downstream effect that promotes angiogenesis by mediating migration and increasing the permeability of endothelial cells. A cis-regulatory elements consisting of a polypurine/polypyrimidine (pPu/pPy) tract in the proximal 36-bp region (-85 to -50), can participate in the formation of a stable higher order G-quadruplex structure (G4) which is essential for VEGF promoter activity. During cancer progression the VEGF-A G4 succumbs to cellular pressure and fails to maintain the stable structure. This shifts the balance to form duplex structure thereby increasing the rate of transcription. Earlier research has tried to develop small-molecule ligands to target and stabilize G4, however they either lack specificity or non-toxicity. Peptide on the other hand are very less studied. Here we used bioinformatics in-silico tool to develop peptides which can successfully bind and stabilize the VEGF-A G4 while reducing its gene expression. This further alters the expression fate of the VEGF-A signalling cascade and prevents angiogenesis in cancer cells. We used high resolution Nuclear magnetic resonance and molecular dynamics simulation to map the chemistry of the interaction while the qPCR and western blot allowed us to check the expression pattern of the molecules of VEGF-A signalling cascade. In this investigation, we navigate the complex interplay between peptides and quadruplex structures, unravelling valuable insights that can enhance the crafting of pharmacophores directed at the dynamic quadruplex structure. The outcomes of our study are promising, paving the way for progress in the realms of research, characterization, and optimization of peptides binding to G-quadruplexes, with potential implications for therapeutic applications.

Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtainedin vivo and in vitro, with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments.

Photodynamic Therapy (PDT), which involves the combined action of a drug and its activation by suitable light, is a particularly attractive novel cancer therapy method due to less systemic side-effects. However, the delivery and accumulation of the PDT drug into cancer cells is still problematic. Here, by using -scale molecular dynamic simulations combined with quantum mechanics/molecular mechanics approaches, we examine the behavior of a PDT drug functionalized with a folic acid unit targeting the folate receptor (FR-), which is overexpressed in ovarian cancer cells. We show that the PDT drug forms a stable complex with the folate receptor, albeit slightly disrupting the main interaction patterns as compared to the parent folate ligand. Furthermore, we also show that the optical properties of the PDT drug are not altered by its interaction with the protein. Our results confirm that coupling with folate is an attractive strategy for selective active delivery of PDT agents.