Modular linkers for conjugation of organic substances to substantially inorganic substances and methods of manufacture and use thereof
Inventors
Medintz, Igor L. • Berti, Lorenzo • Facci, Paolo • Mattoussi, Hedi M.
Assignees
Publication Number
US-9139614-B2
Publication Date
2015-09-22
Expiration Date
2027-04-25
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Abstract
A modular linker includes an inorganic binding entity having an affinity for a substantially inorganic substance, and an organic binding entity capable of binding with an organic substance covalently bonded thereto. The modular linker is capable of being stored in a stable condition for later use. The modular linker may be synthesized by modifying the inorganic binding entity to be covalently bonded to an organic binding entity and storing the modular linker in an inert environment from about a day up to at least 1 week. The modular linker may be conjugated to an organic substance and to a substantially inorganic substance in substantially a 1:1 ratio. The modular linker may have more than one organic binding entity covalently bonded to an inorganic binding entity or vice-versa. Also, a particular modular linker may have an organic binding entity capable of binding with a nucleic acid sequence.
Core Innovation
The present invention is directed to modular linkers designed for the conjugation of organic substances to substantially inorganic substances, with an emphasis on stability and storability. The modular linker includes an inorganic binding entity that has an affinity for a substantially inorganic substance and an organic binding entity capable of binding with an organic substance, wherein the organic binding entity is covalently bonded to the inorganic binding entity. These modular linkers may be synthesized and stored for a substantial period, such as from about a day up to at least one week, enabling their availability for immediate and controlled conjugation.
The problem addressed by this invention arises from the difficulty in conjugating organic substances, including biomolecules, to substantially inorganic substances like nanoparticles and quantum dots using current methods. Existing methods are often multi-step, cumbersome, produce heterogeneous or aggregated products, and lack control over stoichiometry and stability. Furthermore, these methods require immediate conjugation before degradation, involve several purification steps, and are limited to specific applications and pairs of organic and inorganic substances.
The modular linker invention provides a general solution by creating stable, storable, and modular molecules that systematically link organic and inorganic substances in a controllable and predictable manner, typically in a 1:1 ratio, reducing the need for excess reagents and minimizing aggregation and cross-reactivity. The modular design allows customization of binding entities to target a variety of substances and enables simple, scalable synthesis methods such as solid phase peptide synthesis. This approach facilitates stoichiometric control and enables production and commercial distribution as research tools or kits for diverse conjugation needs.
Claims Coverage
The patent includes multiple independent claims covering various modular linker configurations and methods related to synthesis and conjugation. The main inventive features identified pertain to the structure of modular linkers, their stability, storability, specific binding entities, and methods for controlled conjugation.
Modular linker structure with covalently bonded inorganic and organic binding entities
A modular linker comprising an inorganic binding entity having an affinity for a substantially inorganic substance, which is a polypeptide sequence selected from poly-histidine, poly-cysteine, and SEQ ID NOS 1-28, and an organic binding entity capable of binding with an organic substance, covalently bonded to the inorganic binding entity; the modular linker is capable of being stored in a stable condition for later use.
Stable storage of modular linkers in inert environments
The modular linker can be stored in a stable condition in an inert environment at ambient temperature for about a day up to at least one week, or at about −20° C. for about a day up to at least one year, without compromising activity of the organic binding entity.
Organic binding entities with diverse specificities and reactivities
The organic binding entity may include a functional chemical group selective to particular biomolecule regions, aptamers, biotin, sugars, antibodies, nucleotides, and other biomolecular selective groups enabling covalent or reactive binding to organic substances.
Modular linkers with spacers and solubility-modulating entities
The modular linker may include spacers covalently bonded between the inorganic and organic binding entities, which can be peptide sequences, nucleotides, alkane chains, polymers, or lipids, and are optionally susceptible to chemical cleavage under predetermined conditions. Additionally, solubility-modulating entities like hydroxylated compounds or charged peptidyl residues may be included.
Conjugation methods enabling controlled 1:1 ratio linking
Methods for synthesizing the stable modular linker by covalent bonding of inorganic and organic binding entities and storing in inert environments; and methods for linking substantially inorganic substances to organic substances by reacting modular linkers with the organic substances and subsequently conjugating to inorganic substances in suitable buffers, both in substantially 1:1 ratios.
Modular linker variants with multiple organic or inorganic binding entities
Modular linkers comprising one inorganic binding entity and multiple organic binding entities capable of binding different organic substances, or one organic binding entity and multiple inorganic binding entities capable of binding different substantially inorganic substances, each covalently bonded accordingly.
Modular linkers capable of binding nucleic acid sequences
A modular linker wherein the organic binding entity is specifically capable of binding with a nucleic acid sequence, with the inorganic binding entity selected from poly-histidine, poly-cysteine, or SEQ ID NOS 1-28.
Overall, the claims cover modular linkers incorporating polypeptide inorganic binding entities with covalently bonded organic binding entities, stable storage conditions, diverse organic binding specificity and reactivity, inclusion of spacers and solubility modifiers, controlled conjugation methods, modular variants with multiple binding entities, and application to nucleic acid binding, along with conjugation kits comprising such modular linkers.
Stated Advantages
The modular linker can be stored in a stable and reactive condition for periods ranging from about a day to at least one week at ambient temperature and up to at least one year at −20° C., facilitating commercial availability and ease of use.
The modular linker allows simple, efficient, and rapid conjugation of organic substances to substantially inorganic substances in a controlled manner, typically achieving about a 1:1 ratio, reducing the need for excess reagents and minimizing aggregation and cross-reactivity.
The modular design enables customization of binding entities and inclusion of spacers or solubility-modulating entities to optimize conjugation properties and adapt to various organic and inorganic substances.
The linkers simplify synthesis, for example via standard solid phase peptide synthesis methods, allowing scalable production and functional versatility.
Stoichiometric control is imparted by the modular linker, enabling homogenous conjugates with precise ratios of organic to inorganic components.
Documented Applications
Use as tools or probes in biosensing, medical diagnostics, drug detection, drug candidate screening, and both qualitative and quantitative bio-analysis in tethered and soluble assay formats.
Facilitation of conjugation of biomolecules such as peptides, proteins, nucleotides, enzymes, antibodies, carbohydrates, sugars, and lipids to nanoparticles, nanocrystals, quantum dots, and other substantially inorganic substances.
Particularly for constructing fluorescent inorganic-biomaterial constructs, such as quantum dot-linker-oligonucleotide conjugates, useful for fluorescence resonance energy transfer (FRET) applications.
Provision as commercially available reactive linkers in conjugation kits to facilitate immediate and controlled conjugation reactions.
Potential therapeutic delivery applications, for example using cleavable linkers to release therapeutic components from magnetic nanoparticles in targeted tissues.
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