The Development And Application Of Bioinspired Peptidyl Rainbow Nanoparticle Based On Green Fluorescent Proteins

As an invaluable tool for biomedical research,green fluorescent proteins(GFP)are derived from the bioluminescent jellyfish Aequorea victoria.The core motif of the GFP is a p-hydroxybenzylidene-2,3-dimethylimidazolinone(p-HOBDI)chromophore that is encapsulated by an extensive hydrogen-bonding network and is responsible for the green fluorescence.To mimic the fluorescence of GFP,various compounds,such as GFP chromophore analogues,hydrogen bond-rich proteins,and aromatic peptidyl nanostructures that preclude free rotation of the aryl-alkene bond,have been developed to adapt them for a fantastic range of applications.Over the past decades,there is a growing enthusiasm to mimic the luminous properties of GFP with intrinsic fluorescent peptidyl nanostructures owing to their structural simplicity,biocompatibility,simple synthesis,and capacity to self-assemble into a variety of functional nanostructures.However,monochromatic fluorescent peptidyl nanomaterials and a narrow range of applications show limitations as luminescent agents in biomedical fields.To address these issues,a bioinspired polychromatic colorful toolbox was developed,combined with an in-depth theoretical analysis of the fluorescence mechanism,expanding the application scenario to biomolecule monitoring,biological structures imaging,targeting delivery,and viral detection.(1)Bioinspired fluorescent peptidyl nanoparticles in rainbow colors were developed.To address these issues,inspired by GFP and its variants,a series of nonfluorescent oligopeptides have been developed to expand the color palette to encompass blue,green,yellow,orange,and red regions.The non-fluorescent oligopeptides can aggregate into fluorescent nanoparticles with rainbow colors,termed the peptidyl rainbow kit(PRK).Due to extensive intermolecular bonds and red edge excitation shift(REES)effect,the PRK oligopeptides emit intrinsic photoluminescent.Similar to that of GFP,the PRK oligopeptides are biocompatible and photostable under physiological conditions.Notably,PRK oligopeptides are more suitable for non-technical users and less cost,making the PRK a promising fluorescence candidate for biomedical applications.(2)The rainbow colors of the PRK originate from the aggregation-induced emissions.Combined with careful characterization,the coarse-grained molecular dynamics(CG-MD)simulations for PRK oligopeptides were performed to explore the underlying mechanism of intrinsic fluorescence.The CG-MD simulation results support the characterization of intramolecular bonding and provide structural illustrations for the extensive intermolecular hydrogen bonds and hydrophobic interactions within the selfassembled nanoparticles.Based on these results,the fluorescent mechanism was proposed that intermolecular bonds restricted the intramolecular motions of the oligopeptide residues,which provided a barrier to non-radiative conformational relaxation pathways,leading to aggregation-induced emissive(AIE)fluorescence.(3)The first application of PRK is the construction of fluorescent peptidyl nanoparticles for the monitoring of nucleic acid delivery.To evaluate the potential application of the PRK oligopeptides,we designed a nonpathogenic peptidyl virus-like nanoparticles(pVLPs)based on PRK nanoassemblies by mimicking the simian virus 40 and human immunodeficiency virus.The pVLPs were co-assembled spontaneously using two ferrocene-diphenylalaninebased(Fc-FF)peptides: Fc-FF based nuclear localization signal(Fc-FF-NLS)and FcFF based third variable loop(Fc-FF-V3).The bioactive pVLPs show stable luminescence in the green spectral range and has been endowed with novel function.The bio-safe,biocompatible,photostable,and cell-penetrating properties allow the fluorescent pVLPs system for monitoring the delivery of nucleic acid.(4)Rationally designed peptidyl virus-like particles enable targeted delivery of genetic cargo.Based on the above research,two Fc-FF based viral peptides with both visible fluorescence and cell/nucleus targeting capabilities were further explored.These peptides could co-assemble in their active conformations into well-defined nanoparticles and could enclose the DNA of clustered regularly interspaced short palindromic repeat-associated proteins 9(CRISPR/ Cas9),forming biodegradable pVLPs with excellent cell targeting ability and biocompatibility.The pVLPs can penetrate the cellular membrane and deliver genetic cargos into the nucleus through the viral entry route.(5)Bioinspired fluorescent peptidyl nanoparticles for long-term cell imaging.Furthermore,the tyrosine-based peptide that shows intrinsic luminescence in the aggregation state was constructed based on PRK oligopeptides.Similar to the optical property of GFP,the tyrosine-based peptidyl nanoparticles are stabilized by intermolecular hydrogen bonding and emit fluorescence when the Tyr residues bear phenolic anions.Besides,the tyrosine-based peptide is cell-permeable and endosomeescaped when conjugated with the GPGR motif of human immunodeficiency virus and can be used for long-term cell imaging due to its excellent photostability,pH-sensitivity,and biocompatibility in physiological conditions.(6)Bioinspired octopus-like DNA for enzyme-free HIV Detection.To further broaden the application of PRK oligopeptides,an octopus-like DNA(OLD)for rapid enzyme-free detection of the HIV-1 genome and visualize virus nucleic acid during HIV infection was constructed,which can act as the genetic cargo of pVLPs.The OLD is compatible with both HIV genetic fragments and single-strand genome detection,which should be a valuable technique for the rapid detection of HIV infection and further be used for biomedical application.Results above come to show us the PRK rainbow oligopeptides expand the color palette to encompass visible range due to AIE and show excellent bioactivity,biocompatibility,photostability,and pH-sensitivity in physiological conditions,which can be applied for tracking and monitoring the genetic biological process,targeting delivery genetic drugs,long-term cell imaging,and potential viral detection.