Protamine-Based Strategies for RNA Transfection

Protamine is a natural cationic peptide mixture mostly known as a drug for the neutralization of heparin and as a compound in formulations of slow-release insulin. Protamine is also used for cellular delivery of nucleic acids due to opposite charge-driven coupling. This year marks 60 years since the first use of Protamine as a transfection enhancement agent. Since then, Protamine has been broadly used as a stabilization agent for RNA delivery. It has also been involved in several compositions for RNA-based vaccinations in clinical development. Protamine stabilization of RNA shows double functionality: it not only protects RNA from degradation within biological systems, but also enhances penetration into cells. A Protamine-based RNA delivery system is a flexible and versatile platform that can be adjusted according to therapeutic goals: fused with targeting antibodies for precise delivery, digested into a cell penetrating peptide for better transfection efficiency or not-covalently mixed with functional polymers. This manuscript gives an overview of the strategies employed in protamine-based RNA delivery, including the optimization of the nucleic acid’s stability and translational efficiency, as well as the regulation of its immunostimulatory properties from early studies to recent developments.

Quantitative Heterodimerization of Aromatic Peptides through Host-Enhanced Polar–π Interactions for On-Resin Recognition

Peptide dimerization plays an important role in both natural and artificial supramolecular systems. A major challenge to date is the quantitative heterodimerization of two peptides without formation of homodimers. Here, we employ a macrocyclic host to simultaneously encapsulate a canonical aromatic peptide and a non-canonical perfluorophenylalanine-containing peptide through polar–π interactions, thus forming an unprecedented new series of heteropeptide dimers with high binding affinity. This new peptide heterodimerization was applied to on-resin recognition and separation of aromatic peptides in a peptide mixture exhibiting over 95% isolation purity. This research unveils a generic approach to exploit quantitative heteropeptide dimers for the design of supramolecular (bio)systems.

Quantitative Heterodimerization of Aromatic Peptides through Host-Enhanced Polar–π Interactions for On-Resin Recognition

Peptide dimerization plays an important role in both natural and artificial supramolecular systems. A major challenge to date is the quantitative heterodimerization of two peptides without formation of homodimers. Here, we employ a macrocyclic host to simultaneously encapsulate a canonical aromatic peptide and a non-canonical perfluorophenylalanine-containing peptide through polar–π interactions, thus forming an unprecedented new series of heteropeptide dimers with high binding affinity. This new peptide heterodimerization was applied to on-resin recognition and separation of aromatic peptides in a peptide mixture exhibiting over 95% isolation purity. This research unveils a generic approach to exploit quantitative heteropeptide dimers for the design of supramolecular (bio)systems.

Protamine for RNA Transfection: From Heparin Antagonist to RNA Delivery

Protamine is a natural cationic peptide mixture mostly known as a drug for the neutralization of heparin and as a compound in formulations of slow-release insulin. Protamine is also used for cellular delivery of nucleic acids due to opposite charge-driven coupling. This year marks60 years since the first use of Protamine as a transfection enhancement agent. Since then, Protamine has been broadly used as a stabilization agent for RNA delivery. It has also been involved in several compositions for RNA-based vaccinations in clinical development. Protamine stabilization of RNA shows double functionality: it not only protects RNA from degradation within biological systems, but also enhances penetration into cells. A Protamine-based RNA delivery system is a flexible and versatile platform that can be adjusted according to therapeutic goals: fused with targeting antibodies for precise delivery, digested into a cell penetrating peptide for better transfection efficiency or not-covalently mixed with functional polymers. This manuscript gives an overview of the strategies employed in protamine-based RNA delivery, including the optimization of the nucleic acid’s stability and translational efficiency, as well as the regulation of its immunostimulatory properties from early studies to recent developments.

CMMB (Carboxylate Modified Magnetic Bead) -based isopropanol gradient peptide fractionation (CIF) enables rapid and robust off-line peptide mixture fractionation in bottom-up proteomics

Deep proteome coverage in bottom-up proteomics requires peptide-level fractionation to simplify the complex peptide mixture before analysis by tandem mass spectrometry. By decreasing the number of co-eluting precursor peptide ions, fractionation effectively reduces the complexity of the sample leading to higher sample coverage and reduced bias towards high abundance precursors that are preferentially identified in data-dependent acquisition strategies. To achieve this goal, we report a bead-based off-line peptide fractionation method termed CIF or Carboxylate modified magnetic bead-based isopropanol gradient peptide fractionation. CIF is an extension of the SP3 (single-pot solid-phase-enhanced sample preparation) strategy and provides an effective but complementary approach to other commonly used fractionation methods including strong cation exchange (SCX) and reversed phase (RP)-based chromatography. We demonstrate that CIF is an effective offline separation strategy capable of increasing the depth of peptide analyte coverage both when used alone or as a second dimension of peptide fractionation in conjunction with high pH RP. These features make it ideally suited for a wide range of proteomic applications including the affinity purification of low abundance bait proteins.