Electrostatically Engineered Tetraphenylethylene-Based Fluorescence Sensor for Protamine and Trypsin: Leveraging Aggregation-Induced Emission for Enhanced Sensitivity and Selectivity.

The accurate detection of Protamine and Trypsin, two biomolecules with significant clinical and biological relevance, presents a substantial challenge because of their structural peculiarities, low abundance in physiological fluids, and potential interference from other substances. Protamine, a cationic protein, is crucial for counteracting heparin overdoses, whereas Trypsin, a serine protease, is integral to protein digestion and enzyme activation. This study introduces a novel fluorescence sensor based on a (4-(1,2,2-tris(4-phosphonophenyl)vinyl)phenyl)phosphonic acid octasodium salt (TPPE), leveraging aggregation-induced emission (AIE) characteristics and electrostatic interactions to achieve selective and sensitive detection of these biomolecules. Through comprehensive optical characterization, including ground-state absorption, steady-state, and time-resolved emission spectroscopy, the interaction mechanisms and aggregation dynamics of TPPE with Protamine and Trypsin were elucidated. The sensor exhibits very high sensitivity (LOD: 1.45 nM for Protamine and 32 pM for Trypsin), selectivity, and stability, successfully operating in complex biological matrices, such as human serum and urine. Importantly, this sensor design underscores the synergy between the AIE phenomena and biomolecular interactions, offering a promising alternative for analytical applications in biomedical research and clinical diagnostics. The principles outlined herein open new avenues for the development of other AIE-based sensors, expanding the toolkit available for detecting a wide range of biomolecules using similar design strategies.

Enzymatic response of heparin-protamine complex: Spectroscopic investigation and application for lung adenocarcinoma cells detection.

Though the heparin-protamine complex (HP complex) is a crucial system utilized in clinical settings, the metabolic pathways of this complex remain inadequately understood. Herein, the enzymatic degradation of the heparin-protamine complex by trypsin and its broader implications were investigated. By utilizing fluorescent gold nanoclusters liganded with the HP complex (AuNCs-HP complex), we observed significant morphological and spectral changes during enzymatic degradation. Experiments showed that AuNCs-HP complex could be degraded and cleaved into small fragments by trypsin. Moreover, the AuNCs-HP complex demonstrated its potential as a highly sensitive spectral sensing platform, enabling precise measurement of trypsin activity with an outstanding detection limit (0.34 ng mL-1). Additionally, we explored its utility for specific tumor cell detection, focusing on lung adenocarcinoma cells, and successfully identified their presence through distinctive fluorescence changes. These remarkable findings not only contribute valuable insights into targeted degradation systems but also offer promising opportunities for cancer biomarker detection. The AuNCs-HP complex serves as an innovative tool for real-time trypsin activity monitoring, paving the way for advanced biomedical applications.

Design of "Off-On-Off" fluorescence sensors for Heparin detection by precise modulation of molecular structure.

In this strategy, the fluorescence sensor Nap-Co-T1 employing the fluorescence resonance energy transfer (FRET) mechanism was designed and synthesized to have an efficient response to Heparin, and the FRET mechanism was explored for different excitation-emission wavelengths with different distances between the energy acceptor and the energy donor (comparing with fluorescence sensor Nap-TPA-T2). Upon the addition of Heparin, the fluorescence emission of Nap-Co-T1 was turned on at 565 nm, and the fluorescence color changed of the solution from colorless to bright yellow. The limit of detection (LOD) was as low as 0.04 µg/mL. With the addition of antagonistic protamine (PRTM) to the sensor complex with Heparin, the fluorescence emission was turned off to a certain extent, and the reversibility of the "off-on-off" system was maintained for five cycles or more. In addition, Nap-Co-T1 provides rapid and sensitive detection of Heparin in human serum albumin solution and artificial urine and is highly sensitive to environmental viscosity.

Dual-mode upconversion sensors for detecting differently charged biotargets based on the oxidase-mimicking activity of Ce4+ and electrostatic control.

Heparin is a highly negatively charged sulfated linear polymer glycosaminoglycan that has been widely used as an anticoagulant in medicine. Protamine is a cationic protein rich in arginine that is used to treat the blood-brain barrier during excess heparin surgery. Trypsin is the most important digestive enzyme-encoding generated by the pancreas and can specifically cleave the carboxyl ends of arginine and lysine residues. Heparin, protamine, and trypsin interact and constrain each other, and their fluctuations reflect the body's dysfunction. Therefore, it is necessary to develop a fast, sensitive, and highly selective assay for regularly monitoring the levels of heparin, protamine, and trypsin in serum. Herein, a fluorescent and colorimetric dual-mode upconversion nanoparticle (UCNP) biosensor was used for the determination of heparin, protamine, and trypsin based on the oxidase-mimicking activity of Ce4+ and electrostatic control. The biosensor exhibited sensitive detection of heparin, protamine, and trypsin with low limits of detection (LODs) of 16 ng/mL, 87 ng/mL and 31 ng/mL, respectively. Furthermore, the designed biosensor could eliminate autofluorescence, which not only effectively increased the accuracy of the sensor but also provided a new sensing pathway for the detection of differently charged biotargets.

Fine-tuning of liposome integrity for differentiated transcytosis and enhanced antitumor efficacy.

Transcytosis-inducing nanomedicines have been developed to improve tumor extravasation. However, the fate during transcytosis across multicell layers and the structural integrity of the nanomedicines before reaching tumor cells could impact antitumor therapy. Here, a BAY 87-2243 (a hypoxia-inducible factor-1 inhibitor)-loaded liposomal system (HA-P-LBAY) modified by low molecular weight protamine (LMWP) and crosslinked by hyaluronic acid (HA) was constructed. This system could accomplish differentiate cellular transport in endothelial and tumor cells by fine-tuning its structural integrity, i.e. transcytosis across the endothelial cells while preserving structural integrity, facilitating subsequent retention and drug release within tumor cells via degradation-induced aggregation. In vitro cellular uptake and transwell studies demonstrated that HA-P-LBAY were internalized by endothelial cells (bEnd.3) via an active, caveolin and heparin sulfate proteoglycan (HSPG)-mediated endocytosis, and subsequently achieved transcytosis mainly through the ER/Golgi pathway. Moreover, the fluorescence resonance energy transfer (FRET) study showed that HA-crosslinking maintained higher integrity of HA-P-LBAY after transcytosis, more efficiently than electrostatic coating of HA (HA/P-LBAY). In addition, more HA-P-LBAY was retained in tumor cells (4T1) compared to HA/P-LBAY corresponding to its enhanced in vitro cytotoxicity. This may be attributed to better integrity of HA-P-LBAY post endothelial transcytosis and more degradation of HA in tumor cells, leading to more liposome aggregation and inhibition of their transcytosis, which was inferred by both TEM images and the HAase responsiveness assay proved by FRET. In vivo, HA-P-LBAY exhibited more potency in tumor suppression than the other formulations in both low and high permeability tumor models. This highlighted that fine-tuning of structural integrity of nanocarriers played a key role no matter whether the transcytosis of nanocarriers contributed to cellular transport. Collectively, this study provides a promising strategy for antitumor therapies by fine-tuning liposome integrity to achieve active trans-endothelial transport with structural integrity and selective aggregation for prolonged tumor retention.

Brain Delivery of Protein Therapeutics by Cell Matrix-Inspired Biomimetic Nanocarrier.

Protein therapeutics are anticipated to offer significant treatment options for central nervous system (CNS) diseases. However, the majority of proteins are unable to traverse the blood-brain barrier (BBB) and reach their CNS target sites. Inspired by the natural environment of active proteins, the cell matrix components hyaluronic acid (HA) and protamine (PRTM) are used to self-assemble with proteins to form a protein-loaded biomimetic core and then incorporated into ApoE3-reconstituted high-density lipoprotein (rHDL) to form a protein-loaded biomimetic nanocarrier (Protein-HA-PRTM-rHDL). This cell matrix-inspired biomimetic nanocarrier facilitates the penetration of protein therapeutics across the BBB and enables their access to intracellular target sites. Specifically, CAT-HA-PRTM-rHDL facilitates rapid intracellular delivery and release of catalase (CAT) via macropinocytosis-activated membrane fusion, resulting in improved spatial learning and memory in traumatic brain injury (TBI) model mice (significantly reduces the latency of TBI mice and doubles the number of crossing platforms), and enhances motor function and prolongs survival in amyotrophic lateral sclerosis (ALS) model mice (extended the median survival of ALS mice by more than 10 days). Collectively, this cell matrix-inspired nanoplatform enables the efficient CNS delivery of protein therapeutics and provides a novel approach for the treatment of CNS diseases.

Intranasal delivery of low-dose anti-CD124 antibody enhances treatment of chronic rhinosinusitis with nasal polyps.

Frequent injections of anti-CD124 monoclonal antibody (αCD124) over long periods of time are used to treat chronic rhinosinusitis with nasal polyps (CRSwNP). Needle-free, intranasal administration (i.n.) of αCD124 is expected to provide advantages of localized delivery, improved efficacy, and enhanced medication adherence. However, delivery barriers such as the mucus and epithelium in the nasal tissue impede penetration of αCD124. Herein, two novel protamine nanoconstructs: allyl glycidyl ether conjugated protamine (Nano-P) and polyamidoamine-linked protamine (Dendri-P) were synthesized and showed enhanced αCD124 penetration through multiple epithelial layers compared to protamine in mice. αCD124 was mixed with Nano-P or Dendri-P and then intranasally delivered for the treatment of severe CRSwNP in mice. Micro-CT and pathological changes in nasal turbinates showed that these two nano-formulations achieved ∼50 % and ∼40 % reductions in nasal polypoid lesions and eosinophil count, respectively. Both nano-formulations provided enhanced efficacy in suppressing nasal and systemic Immunoglobulin E (IgE) and nasal type 2 inflammatory biomarkers, such as interleukin 13 (IL-13) and IL-25. These effects were superior to those in the protamine formulation group and subcutaneous (s.c.) αCD124 given at a 12.5-fold higher dose. Intranasal delivery of protamine, Nano-P, or Dendri-P did not induce any measurable toxicities in mice.

"Rich arginine and strong positive charge" antimicrobial protein protamine: From its action on cell membranes to inhibition of bacterial vital functions.

Protamine, an antimicrobial protein derived from salmon sperm with a molecular weight of approximately 5 kDa, is composed of 60-70 % arginine and is a highly charged protein. Here, we investigated the mechanism of antimicrobial action of protamine against Cutibacterium acnes (C. acnes) focusing on its rich arginine content and strong positive charge. Especially, we focused on the attribution of dual mechanisms of antimicrobial protein, including membrane disruption or interaction with intracellular components. We first determined the dose-dependent antibacterial activity of protamine against C. acnes. In order to explore the interaction between bacterial membrane and protamine, we analyzed cell morphology, zeta potential, membrane permeability, and the composition of membrane fatty acid. In addition, the localization of protamine in bacteria was observed using fluorescent-labeled protamine. For investigation of the intracellular targets of protamine, bacterial translation was examined using a cell-free translation system. Based on our results, the mechanism of the antimicrobial action of protamine against C. acnes is as follows: 1) electrostatic interactions with the bacterial cell membrane; 2) self-internalization into the bacterial cell by changing the composition of the bacterial membrane; and 3) inhibition of bacterial growth by blocking translation inside the bacteria. However, owing to its strong electric charge, protamine can also interact with DNA, RNA, and other proteins inside the bacteria, and may inhibit various bacterial life processes beyond the translation process.

DNA Packaging and Polycation Length Determine DNA Susceptibility to Free Radical Damage in Condensed DNA.

In nature, DNA exists primarily in a highly compacted form. The compaction of DNA in vivo is mediated by cationic proteins: histones in somatic nuclei and protamines in sperm chromatin. The extreme, nearly crystalline packaging of DNA by protamines in spermatozoa is thought to be essential for both efficient genetic delivery as well as DNA protection against damage by mutagens and oxidative species. The protective role of protamines is required in sperm, as they are sensitive to ROS damage due to the progressive loss of DNA repair mechanisms during maturation. The degree to which DNA packaging directly relates to DNA protection in the condensed state, however, is poorly understood. Here, we utilized different polycation condensing agents to achieve varying DNA packaging densities and quantify DNA damage by free radical oxidation within the condensates. Although we see that tighter DNA packaging generally leads to better protection, the length of the polycation also plays a significant role. Molecular dynamics simulations suggest that longer polyarginine chains offer increased protection by occupying more space on the DNA surface and forming more stable interactions. Taken together, our results suggest a complex interplay among polycation properties, DNA packaging density, and DNA protection against free radical damage within condensed states.

Protamine-grafted carboxymethyl chitosan based hydrogel with adhesive and long-term antibacterial properties for hemostasis and skin wound healing.

In this study, we developed a tissue-adhesive and long-term antibacterial hydrogel consisting of protamine (PRTM) grafted carboxymethyl chitosan (CMC) (PCMC), catechol groups modified CMC (DCMC), and oxidized hyaluronic acid (OHA), named DCMC-OHA-PCMC. According to the antibacterial experiments, the PCMC-treated groups showed obvious and long-lasting inhibition zones against E. coli (and S. aureus), and the corresponding diameters varied from 10.1 mm (and 15.3 mm) on day 1 to 9.8 mm (and 15.3 mm) on day 7. The DCMC-OHA-PCMC hydrogel treated groups also exhibited durable antibacterial ability against E. coli (and S. aureus), and the antibacterial rates changed from 99.3 ± 0.21 % (and 99.6 ± 0.36 %) on day 1 to 76.2 ± 1.74 % (and 84.2 ± 1.11 %) on day 5. Apart from good mechanical and tissue adhesion properties, the hydrogel had excellent hemostatic ability mainly because of the grafted positive-charged PRTM. As the animal assay results showed, the hydrogel was conducive to promoting the deposition of new collagen (0.84 ± 0.03), the regeneration of epidermis (98.91 ± 6.99 µm) and wound closure in the process of wound repairing. In conclusion, the presented outcomes underline the prospective potential of the multifunctional CMC-based hydrogel for applications in wound dressings.

M-MDSCs mediated trans-BBB drug delivery for suppression of glioblastoma recurrence post-standard treatment.

We found that immunosuppressive monocytic-myeloid-derived suppressor cells (M-MDSCs) were more likely to be recruited by glioblastoma (GBM) through adhesion molecules on GBM-associated endothelial cells upregulated post-chemoradiotherapy. These cells are continuously generated during tumor progression, entering tumors and expressing PD-L1 at a high level, allowing GBM to exhaust T cells and evade attack from the immune system, thereby facilitating GBM relapse. αLy-6C-LAMP is composed of (i) drug cores with slightly negative charges condensed by cationic protamine and plasmids encoding PD-L1 trap protein, (ii) pre-formulated cationic liposomes targeted to Ly-6C for encapsulating the drug cores, and (iii) a layer of red blood cell membrane on the surface for effectuating long-circulation. αLy-6C-LAMP persistently targets peripheral, especially splenic, M-MDSCs and delivers secretory PD-L1 trap plasmids, leveraging M-MDSCs to transport the plasmids crossing the blood-brain barrier (BBB), thus expressing PD-L1 trap protein in tumors to inhibit PD-1/PD-L1 pathway. Our proposed drug delivery strategy involving intermediaries presents an efficient cross-BBB drug delivery concept that incorporates live-cell targeting and long-circulating nanotechnology to address GBM recurrence.

External Trigger Free Charge Switchable Cationic Ligands in the Design of Safe and Effective Universal Heparin Antidote.

Thrombosis, the formation of blood clots within a blood vessel, can lead to severe complications including pulmonary embolism, cardiac arrest, and stroke. The most widely administered class of anticoagulants is heparin-based anticoagulants such as unfractionated heparin, low-molecular weight heparins (LMWHs), and fondaparinux. Protamine is the only FDA-approved heparin antidote. Protamine has limited efficacy neutralizing LMWHs and no reversal activity against fondaparinux. The use of protamine can lead to complications, including excessive bleeding, hypotension, and hypersensitivity, and has narrow therapeutic window. In this work, a new concept in the design of a universal heparin antidote: switchable protonation of cationic ligands, is presented. A library of macromolecular polyanion inhibitors (MPIs) is synthesized and screened to identify molecules that can neutralize all heparins with high selectivity and reduced toxicity. MPIs are developed by assembling cationic binding groups possessing switchable protonation states onto a polymer scaffold. By strategically selecting the identity and modulating the density of cationic binding groups on the polymer scaffold, a superior universal heparin reversal agent is developed with improved heparin-binding activity and increased hemocompatibility profiles leading to minimal effect on hemostasis. The activity of this heparin antidote is demonstrated using in vitro and in vivo studies.

Protamines and the sperm nuclear basic proteins Pandora's Box of insects.

Insects are the largest group of animals when it comes to the number and diversity of species. Yet, with the exception of Drosophila, no information is currently available on the primary structure of their sperm nuclear basic proteins (SNBPs). This paper represents the first attempt in this regard and provides information about six species of Neoptera: Poecillimon thessalicus, Graptosaltria nigrofuscata, Apis mellifera, Nasonia vitripennis, Parachauliodes continentalis, and Tribolium castaneum. The SNBPs of these species were characterized by acetic acid urea gel electrophoresis (AU-PAGE) and high-performance liquid chromatography fractionated. Protein sequencing was obtained using a combination of mass spectrometry sequencing, Edman N-terminal degradation sequencing and genome mining. While the SNBPs of several of these species exhibit a canonical arginine-rich protamine nature, a few of them exhibit a protamine-like composition. They appear to be the products of extensive cleavage processing from a precursor protein which are sometimes further processed by other post-translational modifications that are likely involved in the chromatin transitions observed during spermiogenesis in these organisms.

DNA groove preference shift upon phosphorylation of a protamine-like cationic peptide.

Protamines, arginine-rich DNA-binding proteins, are responsible for chromatin compaction in sperm cells, but their DNA groove preference, major or minor, is not clearly identified. We herein study the DNA groove preference of a short protamine-like cationic peptide before and after phosphorylation, using all-atom molecular dynamics and umbrella sampling simulations. According to various thermodynamic and structural analyses, a peptide in its non-phosphorylated native state prefers the minor groove over the major groove, but phosphorylation of the peptide bound to the minor groove not only reduces its binding affinity but also brings a serious deformation of the minor groove, eliminating the minor-groove preference. As protamines are heavily phosphorylated before binding to DNA, we expect that the structurally disordered phosphorylated protamines would prefer major grooves to enter into DNA during spermatogenesis.

Protamine folds DNA into flowers and loop stacks.

DNA in sperm undergoes an extreme compaction to almost crystalline packing levels. To produce this dense packing, DNA is dramatically reorganized in minutes by protamine proteins. Protamines are positively charged proteins that coat negatively charged DNA and fold it into a series of toroids. The exact mechanism for forming these ∼50-kbp toroids is unknown. Our goal is to study toroid formation by starting at the "bottom" with folding of short lengths of DNA that form loops and working "up" to more folded structures that occur on longer length scales. We previously measured folding of 200-300 bp of DNA into a loop. Here, we look at folding of intermediate DNA lengths (L = 639-3003 bp) that are 2-10 loops long. We observe two folded structures besides loops that we hypothesize are early intermediates in the toroid formation pathway. At low protamine concentrations (∼0.2 µM), we see that the DNA folds into flowers (structures with multiple loops that are positioned so they look like the petals of a flower). Folding at these concentrations condenses the DNA to 25% of its original length, takes seconds, and is made up of many small bending steps. At higher protamine concentrations (≥2 µM), we observe a second folded structure-the loop stack-where loops are stacked vertically one on top of another. These results lead us to propose a two-step process for folding at this length scale: 1) protamine binds to DNA, bending it into loops and flowers, and 2) flowers collapse into loop stacks. These results highlight how protamine uses a bind-and-bend mechanism to rapidly fold DNA, which may be why protamine can fold the entire sperm genome in minutes.

Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse protamine 1.

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. Phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues and their post-translational modifications are poorly understood. Here, we investigated the role of K49, a rodent-specific lysine residue in protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In sperm, alanine substitution (P1(K49A)) decreases sperm motility and male fertility-defects that are not rescued by arginine substitution (P1(K49R)). In zygotes, P1(K49A) leads to premature male pronuclear decompaction, altered DNA replication, and embryonic arrest. In vitro, P1(K49A) decreases protamine-DNA binding and alters DNA compaction and decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.

End-on PEGylation of heparin: Effect on anticoagulant activity and complexation with protamine.

Heparin is the most common anticoagulant used in clinical practice but shows some downsides such as short half-life (for the high molecular weight heparin) and secondary effects. On the other hand, its low molecular weight analogue cannot be neutralized with protamine, and therefore cannot be used in some treatments. To address these issues, we conjugated polyethylene glycol (PEG) to heparin reducing end (end-on) via oxime ligation and studied the interactions of the conjugate (Hep-b-PEG) with antithrombin III (AT) and protamine. Isothermal titration calorimetry showed that Hep-b-PEG maintains the affinity to AT. Dynamic light scattering demonstrated that the Hep-b-PEG formed colloidal stable nanocomplexes with protamine instead of large multi-molecular aggregates, associated with heparin side effects. The in vitro (human plasma) and in vivo experiments (Sprague Dawley rats) evidenced an extended half-life and higher anticoagulant activity of the conjugate when compared to unmodified heparin.

Arginine-rich peptides as crystallization inhibitors for sodium urate.

Inhibiting the formation of urate crystals is the key to prevent hyperuricemia from developing into gout. Although many studies have focused on the influence of biomacromolecules in the crystallization behavior of sodium urate, the role of peptides with specific structures may contribute to unprecedented regulatory effects. Here, for the first time, we studied the effects of cationic peptides on the phase behavior, crystallization kinetics, and size/morphology of urate crystals. The addition of protamine (PRTM, a typical natural arginine-rich peptide) prolongs the nucleation induction time of sodium urate and inhibits crystal nucleation effectively. PRTM binds to the surface of amorphous sodium urate (ASU) through the hydrogen bond and electrostatic attraction between guanidine groups and urate anions, which is conducive to maintaining the state of ASU and inhibiting crystal nucleation. Moreover, PRTM preferentially binds to the MSUM plane and leads to a significant reduction in the aspect ratio of MSUM filamentous crystals. Further studies showed that there are significant differences in the inhibiting effects of arginine-rich peptides with different chain lengths on the crystallization behavior of sodium urate. Both guanidine functional groups and peptide chain length determine the crystallization inhibiting effect of peptides simultaneously. The present work highlights the potential role of arginine peptides in inhibiting the crystallization of urate and provides new insights into the inhibition mechanism in the pathological biomineralization of sodium urate, demonstrating the possibility of using cationic peptides to treat gout.

A ready-to-use dry powder formulation based on protamine nanocarriers for pulmonary drug delivery.

The use of oral antibiotic therapy for the treatment of respiratory diseases as tuberculosis has promoted the appearance of side effects as well as resistance to these treatments. The low solubility, high metabolism, and degradation of drugs as rifabutin, have led to the use of combined and prolonged therapies, which difficult patient compliance. In this work, we develop inhalable formulations from biomaterials such as protamine to improve the therapeutic effect. Rifabutin-loaded protamine nanocapsules (NCs) were prepared by solvent displacement method and were physico-chemically characterized and evaluated for their dissolution, permeability, stability, cytotoxicity, hemocompatibility, internalization, and aerodynamic characteristics after a spray-drying procedure. Protamine NCs presented a size of around 200 nm, positive surface charge, and drug association up to 54%. They were stable as suspension under storage, as well as in biological media and as a dry powder after lyophilization in the presence of mannitol. Nanocapsules showed a good safety profile and cellular uptake with no tolerogenic effect on macrophages and showed good compatibility with red blood cells. Moreover, the aerodynamic evaluation showed a fine particle fraction deposition up to 30% and a mass median aerodynamic diameter of about 5 µm, suitable for the pulmonary delivery of therapeutics.

Effect of phosphorylation of protamine-like cationic peptide on the binding affinity to DNA.

Protamines are more arginine-rich and more basic than histones and are responsible for providing a highly compacted shape to the sperm heads in the testis. Phosphorylation and dephosphorylation are two events that occur in the late phase of spermatogenesis before the maturation of sperms. In this work, we have studied the effect of phosphorylation of protamine-like cationic peptides using all-atom molecular dynamics simulations. Through thermodynamic analyses, we found that phosphorylation reduces the binding efficiency of such cationic peptides on DNA duplexes. Peptide phosphorylation leads to a less efficient DNA condensation, due to a competition between DNA-peptide and peptide-peptide interactions. We hypothesize that the decrease of peptide bonds between DNA together with peptide self-assembly might allow an optimal re-organization of chromatin and an efficient condensation through subsequent peptide dephosphorylation. Based on the globular and compact conformations of phosphorylated peptides mediated by arginine-phosphoserine H-bonding, we furthermore postulate that phosphorylated protamines could more easily intrude into chromatin and participate to histone release through disruption of histone-histone and histone-DNA binding during spermatogenesis.