WO2008139229A2 - Pores - Google Patents

Pores Download PDF

Info

Publication number
WO2008139229A2
WO2008139229A2 PCT/GB2008/050351 GB2008050351W WO2008139229A2 WO 2008139229 A2 WO2008139229 A2 WO 2008139229A2 GB 2008050351 W GB2008050351 W GB 2008050351W WO 2008139229 A2 WO2008139229 A2 WO 2008139229A2
Authority
WO
WIPO (PCT)
Prior art keywords
pore
dendrimer
membrane
species
pamam
Prior art date
Application number
PCT/GB2008/050351
Other languages
English (en)
Other versions
WO2008139229A3 (fr
Inventor
Stefan Howorka
Original Assignee
Ucl Business Plc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ucl Business Plc filed Critical Ucl Business Plc
Publication of WO2008139229A2 publication Critical patent/WO2008139229A2/fr
Publication of WO2008139229A3 publication Critical patent/WO2008139229A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/593—Polyesters, e.g. PLGA or polylactide-co-glycolide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/595—Polyamides, e.g. nylon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Definitions

  • This invention relates to pores having lumens, wherein a dendrimer is attached to the pore, and the use of such pores in separation devices, in devices for the sequencing of nucleic acids and proteins, and in pharmaceutical compositions, and also to a reagent and method for the structural analysis of proteins.
  • membranes that allow selective passage of only particular species (for example molecules or ions) has many applications in separation technologies: these could be used to separate molecules of particular size or charge from those of differing size or charge.
  • membranes comprising pores that are designed as to allow the rate at which a particular species passes through the membrane to be chosen.
  • membranes that allow passage of a particular species at a slow rate may be useful to provide analytical data about that species as it passes through the membrane.
  • a membrane developed so as to provide a constant controlled release of a species through that membrane at a given rate are also potentially useful.
  • the present invention provides a nanoscale pore having a lumen, wherein a dendrimer is attached to the pore.
  • Figure 1 shows (A) the structure of polyamido amine dendrimer of generation 3 with 50% amine and 50% hydroxyl terminal groups and (B) the cross-sectional view of the heptameric ⁇ HL pore with cysteine substitutions K46C, K8C, and S106C.
  • the model was generated using crystallographic data (Song, L.; Hobaugh, M. R.; Shustak, C;
  • the internal diameters of the channel are: 2.9 nm, cis entrance; 4.1 nm, internal cavity; 1.3 nm, inner constriction; 2 nm, trans entrance of the ⁇ -barrel.
  • Figure 2 shows RP-HPLC analysis of the derivatization of PAMAM dendrimer with the heterobifunctional cross-linker SPDP.
  • FIG. 3 MALDI-ToF analysis of SPDP-modified PAMAM dendrimers to determine the average number of coupled pyridyldisulfide groups. G3 -PAMAM before (A) and after (B) reaction with SPDP.
  • Figure 4 shows (A) quantifying the yield of a G3-P AMAM-PDP preparation using SDS gel electrophoresis and Coomassie staining. Solutions of unmodified dendrimers of known concentration and SPDP-modified dendrimers of unknown concentrations were analyzed. The resulting electropherograms were subjected to densitometric analysis to determine the unknown concentrations.
  • Lane 1 67 ⁇ M G3-PAMAM; lane 2, 33 ⁇ M G3-PAMAM; lane 3, 17 ⁇ M G3-PAMAM; lane 4, G3-PAMAM-PDP, concentration determined to be 50 ⁇ M; and (B) Sulfhydryl-reactive G3-PDP couples specifically to monomeric ⁇ HL cysteine mutant K46C and causes a gel-shift in SDS-PAGE autoradiographs.
  • Lane 1 K46C; lane 2, K46C treated with G3-PDP; lane 3, K46C treated with PEG-MAL 5 kD; lane 4, K46C with G3-PDP and excess reducing agent DTT; lane 5, K46C with PEG-MAL 5 kD and excess DTT.
  • Figure 5 shows coupling of a PAMAM-PDP dendrimer inside and outside the lumen of a protein channel.
  • Homoheptameric ⁇ HL cysteine mutants were reacted with G5-PDP, G3-PDP, and PEG-MAL and analyzed via gel electrophoresis and autoradiography.
  • K46C 7 (lanes 1 to 4) and SlOoC 7 (lanes 5 to 7) were treated with PAMAM-PDP (lanes 2 and 5), PAMAM-PDP followed by PEG-MAL (lanes 3 and 6), or PEG-MAL (lanes 4 and 7).
  • the modification reactions were performed with G5-PDP (panel A) or with G3- PDP (panel B).
  • Figure 6 shows representative single channel current traces of (A) SlOoC 7 , (B) SlOoC 7 - G3-PAMAM, (C) S106C 7 -G2-PAMAM, and (D and F) K46C 7 -G5-PAMAM.
  • E All- points histogram of K46C 7 -G5 -PAMAM (D) with a duration of 2 s.
  • the recordings were performed in 1 M KCl, 20 mM Tris ⁇ Cl pH 7.5 at a transmembrane potential of +100 mV ((A) to (D)) or -100 mV (F), with the chamber on the cis side of the protein pore grounded. The currents were filtered at 1 kHz and sampled at 5 kHz.
  • Figure 7 shows single channel current traces of (A) SlOoC 7 , (B) SlOoC 7 with 1 ⁇ M RNA oligonucleotide C 30 at the cis side, (C) S106C 7 -G3-PAMAM, and (D) SlOOC 7 - G3-PAMAM with 1 ⁇ M RNA oligonucleotide C 30 at the cis side, with a potential of +100 mV.
  • the traces were filtered at 10 kHz and sampled at 50 kHz; and
  • FIGS 8, 9 and 10 show examples of dendrimers that may be used in the invention.
  • the invention therefore, provides a nanoscale pore, wherein the pore has a lumen, and a dendrimer is bound to the pore, and wherein the dendrimer functions to modify the ability of species to pass through the pore.
  • the dendrimer may be attached to the lumen of the pore. In this position, the dendrimer has the greatest effect on the passage of a species through the pore, because the lumen comprises the part of the pore with the smallest cross sectional area.
  • the dendrimer may be attached to an exterior surface of the pore.
  • the pore is an organic pore.
  • the term organic takes its usual meaning in the art, therefore the organic pore substantially comprises carbon and hydrogen, and also other elements, especially nitrogen, oxygen, sulfur, phosphorus and halogens.
  • the pore is a protein pore.
  • a protein pore is a pore which is predominantly protein; however, other types of molecules may also be present.
  • protein pores suitable for use in the invention include alpha hemolysin, pneumolysin, outer membrane proteins such as porins, and other bacterial pore-forming toxins (Gilbert 2002) (Parker and Feil 2005) such as streptolysin O (Bhakdi, Tranum-
  • LukF Olet al. 1999
  • the latter are oligomeric assemblies of protein subunits.
  • the diameter of the lumens of protein pores depends on the type of pore and ranges from 1.2 nm for alpha hemolysin (Song, Hobaugh et al.
  • the protein pore is a ⁇ -hemolysin ( ⁇ HL) polypeptide.
  • ⁇ HL is a bacterial toxin which self-assembles to form a heptameric protein pore.
  • the X-ray structure of the ⁇ HL pore resembles a mushroom with a wide cap and a narrow stem, which spans the lipid bilayer (Fig. IB) (Song, L.; Hobaugh, M. R.; Shustak, C; Cheley,
  • the external dimensions of the heptameric ⁇ HL pore are 10 x 10 nm, while the central channel is 2.9 nm in diameter at the cis entrance and widens to 4.1 nm in the internal cavity (Fig. IB). In the transmembrane region, the channel narrows to 1.3 nm at the inner constriction and broadens to 2 nm at the trans entrance of the ⁇ -barrel.
  • the defined structure of ⁇ HL has facilitated extensive engineering studies and has led to the development of tools for the targeted permeabilization of cells (Eroglu, A.; Russo, M.
  • the invention is not limited to pores of this type.
  • the invention includes any pore to which a dendrimer can be attached, and consequently affect the passage of differing molecules through the pore.
  • Alternative embodiments of the invention exist wherein the pore is an inorganic pore.
  • the inorganic pore is composed of silica, silicon nitride, alumina, titanium, gold, platinum, zirconia, silicon nitride or a combination thereof.
  • the pore is not limited in relation to the material that it comprises, the invention is limited in relation to the size of the pore.
  • the pore must be a nanoscale pore.
  • nanoscale is meant that the pore is one wherein the lumen has a diameter of less than 1 ⁇ m.
  • the lumen has a diameter of less than 100 nm. More preferably the lumen has a diameter of 10 nm or less.
  • the pore has a diameter of at least 1 nm. References to the diameter of the pore are to be interpreted as the diameter of the pore at its minimum value. In this regard please see fig. IB which illustrates that the diameter of the lumen may vary at different positions along its length.
  • Dendrimers are highly branched (globular) nanoscale polymers
  • the IUPAC definition of a dendrimer is "A polymer having a regular branched structure" (Pure Appl. Chem., Vol. 71, No. 12, pp. 2349-2365, 1999). Their synthesis proceeds either via the divergent or the convergent route (Newkome, G. R.; Moorfield, C. N.; Vogtle, F. Dendrimers and Dendrons: Concepts, Syntheses, Applications; Wiley- VCH: Weinheim, 2001) and offers control over molecular mass, size, shape, the degree of branching, and type and number of terminal functional groups. Reflecting their special characteristics, many potential applications have been developed in materials science and nanotechnology for separation technology, surface coatings, and catalysis (Frechet, J.
  • the dendrimer is attached to the pore.
  • the role of the dendrimer is to affect the ease with which other molecules may pass though the pore.
  • the size of the dendrimer is selected by consideration of the size of pore, and the degree to which it is desired to alter the ability of other species to a pass through the pore.
  • the dendrimer is chosen by consideration of the dimensions of the lumen. Therefore the present invention is not limited in relation to type of the dendrimer. However, consideration must be given to the size of the pore to which the dendrimer is to be attached when determining the size of dendrimer to be used for a particular embodiment of the invention.
  • the dendrimer is a PAMAM dendrimer (tradename Starburst®) (Figs. IA and 8b), an aromatic polyether dendrimer (Fig. 9), a phenyl acetylene dendrimer (Fig. 10), a poly glutamic acid dendrimer (Fig. 8a), a poly (propylene imines) dendrimer (Fig. 8c), a polymelamine dendrimer (Fig. 8d), a polyester dendrimers (Fig.
  • any other dendrimer which is N-, C-, aryl-, Si-, adamantane-, N3P3-, pyridine-, ethano-, saccharide-, or cholic-acid-branched with aryl-, Si-, N-, amide-, ester-, P-,ether-, sulfone-, urea-, alkyl-, alkyne, ketone, alkene, oxazdiazole, siloxy, urethane, imide, ethyne, carbonate, thiol, phosphate, or thiourea-connectivity.or combinations thereof (see Dendrimers (Topics in Current Chemistry S.) (Hardcover) Springer- Verlag Berlin and Heidelberg GmbH & Co. K (30 JuI 1998) by Fritz Vogtle (Editor)). Methods of synthesis of these types of dendrimers are well known to the person skilled in the art.
  • PAMAM are an important sub-class of dendrimers (Figs. IA and 8b), and are particularly preferred for use in the present invention. They were historically the first dendrimers to be synthesized using the divergent strategy (Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin, S.; Roeck, J.; Ryder, J.; Smith, P. Polymer J. 1985, 17, 117-132; Matthews, O. A.; Shipway, A. N.; Stoddart, J. F. Prog Polym ScL 1998, 23, 1-56; Tomalia, D. A.; Frechet, J. M. J. JPoIy Science Part A.
  • the diameters of the dendrimer used is chosen on a case-by-case basis by consideration of the size of the pore to be used in a particular embodiment, and therefore the present invention is not limited in relation to the size of dendrimer used.
  • the dendrimer has a diameter of 20 nm or less and more preferably the dendrimer has a diameter of 10 nm or less.
  • the dendrimer has a diameter of more than 1 nm.
  • the size of a dendrimer increases as the generation of the dendrimer increases.
  • the dendrimer is a fifth-generation dendrimer, a third- generation dendrimer or a second-generation dendrimer.
  • the dendrimer is attached to the lumen of the pore, particularly preferably the dendrimer is a third-generation dendrimer or a second generation dendrimer.
  • the pore may be attached to any number of dendrimers.
  • the number of dendrimers attached to the pore may be altered for example to vary the ease with which molecules may pass through the pore. A greater number of dendrimers will hinder the passage of molecules through the pore, whereas a smaller number of pores will relatively ease the passage of molecules through the pore.
  • Embodiments of the invention are known in which one, two, three, four, five, six, seven, eight, nine or ten dendrimers are attached to the pore. More preferably from one to four dendrimers are attached to the pore. Most preferably only one dendrimer is attached to the pore.
  • the dendrimer and some part of the pore must be bound in some way.
  • the invention is not limited in respect of how this binding is achieved.
  • the dendrimer is attached to the pore by one or more linker groups.
  • the linker group may be an organic group or an inorganic group, and may comprise a single atom or multiple atoms.
  • the linker group is thiopropanoyl, wherein the sulfur atom is bound to the pore and the acyl carbon is bound to the dendrimer.
  • the linker group is attached to the pore by one or more bonds.
  • the bonds may be of any type, but examples that can be mentioned include cases in which the one or more bonds are one or more covalent bonds, electrostatic attractions, metal-chelate bonds, hydrophobic interactions or mixtures thereof.
  • steric interactions between the linker and the pore may aid the binding of the linker to the pore.
  • the linker group is attached to the pore by one or more covalent bonds, and most preferably the one or more covalent bonds are disulfide bonds or amide bonds.
  • the disulfide bond is formed by reaction of the linker group with a cysteine residue comprised within the pore.
  • the linker group may be one or more bonds, and therefore the dendrimer is bound directly to the pore.
  • the one or bonds are not limited to any particular type, but examples of the one or more bonds are one or more covalent bonds, electrostatic attractions, metal- chelate bonds, hydrophobic interactions or combinations thereof.
  • steric interactions may aid the binding of the dendrimer to the pore.
  • the pore is ⁇ HL
  • the dendrimer is a third- generation PAMAM which is attached to the lumen of the pore via a linker which is thiopropanoyl.
  • the pore is ⁇ HL
  • the dendrimer is a second- generation PAMAM which is attached to the lumen of the pore via a linker which is thiopropanoyl.
  • the invention also provides a method of attaching a dendrimer to a pore, although the invention is not limited to the pores when sythesised using this route.
  • the invention provides a method of attaching one or more dendrimers to a nanoscale pore comprising a lumen, wherein an activated dendrimer is reacted with a nucleophilic group present in the pore.
  • the dendrimer is activated by the presence of an electrophilic group on the dendrimer.
  • This electrophilic group may be already present as a natural feature of the dendrimer; alternatively, the dendrimer may activated by derivatisation via attachment of an electrophilic group to the dendrimer.
  • the method requires the presence of a nucleophilic group on the pore.
  • a nucleophilic group may be used: for example a heteroatom such as oxygen, sulphur or phosphorus present within the pore.
  • the method is used wherein the nucleophilic group is located on an external surface of the pore. If it is desired to attach the dendrimer to the lumen of the pore, then the method is used wherein the nucleophilic group is located within the lumen.
  • the pore is a protein pore and the nucleophilic group is the sulfur of a cysteine residue.
  • the dendrimer is attached externally to the pore
  • embodiments of the invention exist wherein the dendrimer is attached to the lumen of the pore.
  • ⁇ -hemolysin proteins were generated having cysteine residues at K46C7 (positioned at the cap of the pore forming a ring surrounding the cis entrance in the assembled pore - see Fig. IB).
  • Use of pores of this type allows attachment of the dendrimer to the exterior of the pore.
  • ⁇ -hemolysin proteins were generated having cysteine residues at S106C7 (positioned within the lumen of the assembled pore - see figure IB) (Howorka, S.; Movileanu, L.; Lu, X.; Magnon, M.; Cheley, S.; Braha, O.; Bayley, H. J Am. Chem. Soc. 2000, 122, 2411-2416).
  • Use of pores of this type allows attachment of the dendrimer to the lumen of the pore.
  • ⁇ -hemolysin proteins were generated having cysteine residues positioned at K8C7 (positioned within the cis entrance of the assembled pore - see figure IB) (Howorka, S.; Movileanu, L.; Lu, X.; Magnon, M.; Cheley, S.; Braha, O.; Bayley, H. J Am. Chem. Soc. 2000, 122, 2411 -2416).
  • the dendrimer it may be necessary to activate the dendrimer so it is electrophilic.
  • the terminal groups of dendrimers are nucleophilic: for example amine and hydroxyl groups. Therefore a preferred method of synthesising the activated dendrimer is by reaction of one or more terminal amines of a dendrimer with a bis- electrophile wherein the amine reacts with a first electrophilic site of the bis- electrophile and a second electrophilic site of the bis-electrophile is unchanged.
  • bis-electrophile is meant a species with a first electrophilic site and a second electrophilic site.
  • the first electrophilic site reacts with a nucleophilic group, for example an amine, on the dendrimer.
  • the second electrophilic site is left unchanged, and is therefore free to react with a nucleophilic site within the pore. Consequently, reaction of the first electrophilic site of the bis-electrophile with the dendrimer, and then of the second electrophilic site of the bis-electrophile with a nucleophilic site on the pore, results in attachment of the dendrimer to the pore.
  • the bis-electrophile is N-succinimidyl-3-(2-pyridyldithio)-propanoate (SPDP).
  • G5 PANAM dendrimers were used having a hydrodynamic diameter of 6.2 nm and a hard-sphere diameter of 4.2 nm (value obtained from the solvent exclusion volume) (Nourse, A.; Millar, D. B.; Minton, A. P. Biopolymers. 2000, 53, 316-328; Dvornic, P. R.; Uppuluri, S. In Dendrimers and other dendritic polymers; Frechet, J. M., Tomalia, D. A., Eds.; John Wiley & Sons, 2002).
  • G3 PAMAM dendrimers were used having hydrodynamic and hard-sphere diameters of are 4.1 and 2.9 nm respectively (Nourse, A.; Millar, D. B.; Minton, A. P. Biopolymers. 2000, 53, 316-328; Dvornic, P. R.; Uppuluri, S. in Dendrimers and other dendritic polymers; Frechet, J. M., Tomalia, D. A., Eds.; John Wiley & Sons, 2002). G2 IPAMAM dendrimers were used having hydrodynamic diameter of 2.9 nm (hard- sphere diameter not available).
  • Activated dendrimers were generated using G5 PAMAM with a mixed surface of terminal -OH and -NH 2 groups at an average ratio of 90:10, G3 PAMAM with a mixed surface of 50:50 hydroxyl/amino groups, and G2 PAMAM with terminal NH 2 groups.
  • a mixed surface with hydroxyl groups avoids the formation of multimeric aggregates which can be found in purely amino-terminated but not in hydroxyl-functionalized G5 PAMAM dendrimers (Nourse, A.; Millar, D. B.; Minton, A. P. Biopolymers. 2000, 53, 316-328).
  • the relative percentages of amine groups of the G3 and G5 PAMAM dendrimers were initially chosen to yield approximately the same number of sulfhydryl-reactive groups for each generation of dendrimer (13 for G5, and 16 for G3 and G2).
  • N-succinimidyl activated ester of SPDP 1 couples to the terminal primary amines of the dendrimers 2 to yield amide-linked 2-pyridyldithiopropanoyl (PDP) groups 3.
  • Table 1 shows the properties of the activated dendrimers.
  • the number of PDP groups coupled to PAMAM dendrimers was determined by MALDI-ToF and via photometric analysis which involved treatment of samples with excess reducing agent dithiothreitol (DTT) to cleave the disulfide bond of PDP, and the than detection of the cleavage product pyridyl-2-thione at 343 nm.
  • the results of the photometric analysis yielded 5.0 ⁇ 1.5, 11 ⁇ 2, and 13 ⁇ 2 PDP groups for G2-PDP, G3-PDP, and G5-PDP, respectively, which is similar to the numbers obtained from MALDI-MS (Table 1).
  • the yield of PAMAM-PDP dendrimers preparations was estimated using sodium- dodecylsulfate gel electrophoresis (SDS-PAGE) and Coomassie staining (Fig. 4A).
  • the gel band intensities of PDP-modified dendrimers were compared with intensities of PAMAM dendrimers of known amount. Densitometric analysis indicated yields of approx. 15% for G2-PDP, 80% for G3-PDP and 50% for G5-PDP relative to the amount used as starting material (Table 1). Table 1. Chemical Characteristics and Dimensions of G2-PDP, G3-PDP, and G5-PDP
  • G2-PDP 16 5.2 ⁇ 5.0 ⁇ 1.5 2.9 N.A. + N.A. 1.0
  • SPDP-derivatized PAMAM dendrimers were, therefore, prepared in a highly efficient manner, and the product mixture did not contain any SPDP or hydrolysis product 3-(2- pyridyldithio)-propanoic acid which might block coupling to cysteines.
  • Dendrimers G5-PDP and G3-PDP were also reacted with the cysteine mutant K46C in an assembled ⁇ HL homoheptamer.
  • the seven cysteine residues at the cap of the pore form a ring surrounding the cis entrance (Fig. IB).
  • both G3-PDP and G5-PDP were expected to react with hep tamer.
  • radiolabeled homoheptamer K46C7 was generated, treated with G5- PDP, G3-PDP or, for comparison purposes, with PEG-MAL 5 kD, and analysed via SDS-PAGE and autoradiography to detect the appearance of up-shifted bands.
  • ⁇ HL heptamers are not denatured in SDS-PAGE and therefore migrated as defined bands as seen for unmodified K46C 7 (Fig. 5A & B, lane 1).
  • a major up-shifted band appeared (Fig. 5 A, lane 2).
  • This band represents the specific covalent coupling product of G5-PDP and the ⁇ HL cysteine mutant as no up- shifted band was observed with unmodified G5 (data not shown).
  • Coupling of G5-PDP to K46C7 produced one but not a second up-shifted band strongly indicating that coupling of a second G5 dendrimer was disfavored.
  • G3 dendrimers In line with the lower molecular mass of G3 (M r 6.9 kD) both G3- heptamer conjugates migrated lower than the conjugate with G5-PDP (M r 28.9 kD)
  • Reaction of G3-PDP and G5-PDP dendrimers with K46C7 heptamers provide embodiments of the invention in which the dendrimer is attached externally to the pore. Similar coupling reactions between activated dendrimers and position S106C of the heptamer which is located inside the internal cavity of the ⁇ HL pore (Fig. IB) were also carried out. Reaction of the engineered cysteine residue with PEG-MAL 5 kD was confirmed to have taken place by gel electrophoretic analysis of radiolabeled SIO6C7 heptamers revealing one up-shifted band (Fig. 5A, lane 7).
  • G3-PDP may or not be expected to couple to the cysteine residues in the internal cavity: the cis entrance is 2.9 nm wide, whereas the G3 hydrodynamic diameter is 2.9 nm and the G3 hard-sphere diameter is 4.1 nm.
  • Reaction of G3-PDP dendrimer with K8C heptamers therefore provides an embodiment of the invention in which the dendrimer is attached to the lumen of the pore.
  • the dendrimer is inserted into the lumen of the pore in order to alter the ability of differing species to pass through the pore.
  • one embodiment of the invention is a membrane comprising one or more pore as defined hereinabove.
  • the membrane has a first side and a second side, and the pore provides a route, channel or path from the first side to the second side.
  • the membrane will comprise a number of pores, the lumen of which provide a number of channels between the first side of the membrane and the second side of the membrane.
  • the precise number of pores on the membrane is not critical to the invention, and would need to be calculated on a case by case basis taking into account the size of the membrane and the required rate of passage of species between the first and second sides of the membrane.
  • the membrane has a density of pores of from 1 to 100 000 pores per ⁇ m 2 .
  • the membrane, or barrier itself is also not limited in type, and would need to be chosen by consideration of species it is desired to pass through the pore or prevent from passing through the pore, and therefore between the membrane's first and second sides. For example, the membrane must be stable in the presence of this species.
  • membranes according to the invention are not limited to biological materials: biological membranes are only one example of membranes according to the invention.
  • the membrane is organic.
  • the organic membrane is an organic polymer, most preferably the organic polymer is a polycarbonate or polyterephtalate polymer.
  • the density of pores is 1 to 100 000 pores per ⁇ m 2 .
  • the organic membrane is a lipid bilayer.
  • the membrane comprising a pore is formed by allowing mutant polypeptides K46C, K8C or S106C to assemble on rabbit erythrocyte membranes to form heptameric pores.
  • the density of pores is 0.0001 to 100 pores per ⁇ m 2 .
  • the membrane is inorganic, wherein preferably the inorganic membrane is a gold-plated porous membrane prepared by the template synthesis method by electrolessly depositing gold along the pore walls of a polycarbonate template membrane (Martin, Nishizawa et al. 2001).
  • the membrane is inorganic, preferably the density of pores is 1 to 100 000 pores per ⁇ m 2 .
  • the pore is an organic pore, preferably an polymeric organic pore or a protein pore.
  • the membrane is an organic polymer the pore is an organic pore.
  • the membrane is an inorganic membrane, the pore is an inorganic pore.
  • the membrane is a lipid bilayer
  • the pore is ⁇ HL
  • the dendrimer is a third- generation PAMAM
  • the linker is thiopropanoyl.
  • the dendrimer is attached to the pore in order to affect the ability of species to pass through the pore. It does this by being attached to the lumen of the pore, and therefore creating a blockage therein, or alternatively being bound to the exterior of the pore by a linker, wherein the linker is sufficiently flexible to allow the dendrimer to move between positions which make the entrance to the pore relatively more and less accessible.
  • the pores according to the invention are an important advance in the area of filtration membranes. Regulating and controlling the flow of matter across nanoscale porous membranes is an important topic in filtration-based separation technologies such as ultrafiltration, reverse osmosis, and dialysis in laboratory and industry.
  • Existing synthetic or artificial membranes which permit the selective transport of material are composed of polymers or ceramics with nanometer-sized holes.
  • dendrimer molecules are attached to pores in organic and inorganic membranes in a new and useful way to engineer the permeation and filtration properties of semi-permeable membranes. It has been found that using dendrimers as molecular sieves in the pores of the membranes can be advantageous as the polymer is dense and thus capable of restricting the movement of small molecules. Apart from excluding the passage of molecules based on size, the dendrimer can also be tailored so the membrane acts as a filter via chemical affinity or electrostatic interactions with the species being filtered. Using dendrimers for different inorganic structures which can have different pore diameters can benefit from the availability of dendrimers in different sizes ranging form 3 to 10 nm. Finally, due to the modular character of the approach, the semi-permeability of the membrane can be changed by choosing a dendrimer of different composition while leaving the membrane composition itself unaltered.
  • another embodiment of the invention is a filtration device comprising a membrane as described hereinabove, an inlet and an outlet.
  • the inlet and the outlet are not limited in type, style or size. However, the inlet and the outlet are in fluid communication, and the inlet is positioned at the first side of the membrane and the outlet is positioned at the second side of the membrane.
  • the filtration device can be used in a method of separating a first species and a second species, wherein a fluid comprising the first species and the second species is positioned in the inlet, passed through the membrane, and collected at the outlet.
  • the principle of the device is that the dendrimer attached to the pore affects the passage of each of a first species and a second species to a greater or lesser extent. Therefore the first species is substantially prevented from passing between the first and second sides of the membrane whereas the second species is substantially allowed to pass through. Therefore the first species and the second species are separated: the first species being on the first side of the membrane, and the second species being on the second side of the membrane.
  • the dendrimer and membrane of the device must be chosen so as to achieve the separation effectively.
  • the dendrimer must be chosen so as to prevent the first species from passing through the pore and therefore the membrane, whilst allowing the second species to pass through the pore and therefore the membrane.
  • the membrane must be chosen so that it is essentially non-permeable to the first and second species it desired to separate, otherwise after selective separation of the first and second species by the pore, re-mixing of the first and second species would occur and resolution would be lost.
  • each species may be any chemical or other type of entity. Further, each species may comprise one component only or comprise multiple components. Therefore the method or device of the invention may be used to separate three components into an individual component and a mixture of two components, wherein the individual component is the first species and the two components are the second species as defined above.
  • the method may be used when the first and second species to be separated are ions or molecules or atoms, or mixtures thereof; that is the first species and second species may individually be a mixture of ions, molecules and atoms, or the first species may be, say, ions, whereas the second species is, say, molecules.
  • the dendrimer is chosen so as to affect the separation.
  • One way of achieving this is by selecting a dendrimer of an appropriate size, and thereby the first species and the second species are separated by their differing sizes. The larger first species is prevented from passing though the lumen by the dendrimer, whereas the smaller, or more flexible second species is allowed to pass through the lumen.
  • the first species and the second species are separated by their differing charges or polarities. This can be achieved, for example, by synthesis of a positively charged dendrimer, which would prevent the passage through the lumen of a more positively charged first species and allow the passage through the lumen of a less positively charged second species.
  • the altered channel properties could be seen to be specific to the presence of the dendrimer because by addition of excess reducing agent DTT, which cleaved the disulfide bond between the cysteine and the PAMAM polymer, the conductance rose to that of the open pore.
  • the invention provides a method of altering the ability of pores to pass an ionic current by positioning a dendrimer within the pore.
  • a trace with a conductance of 880 pS was decorated with fast downward current fluctuations (Fig. 6D).
  • An all-points-current histogram generated from a trace of K46C 7 -G5 -PAMAM with a duration of 2 s displays the current levels for the fluctuations (Fig. 6E).
  • the peak at 88 pA corresponds to the open channel, while the peak at 68 pA and a minor peak at 53 pA reflect blockade level 1 and blockade level 2 respectively. Blockades to level 2 were not investigated further due to their low frequency.
  • the dominating current blockades from the open channel to level 1 were characterized by an amplitude of 210 ⁇ 23 pS and an average duration of 2.2 ⁇ 0.4 ms.
  • the current fluctuations of K46C 7 -G5 -PAMAM only occur at a potential of +100 mV (chamber on the cis side of the protein grounded) but not at -100 mV (Fig. 6F - chamber on the trans side of the protein grounded) suggesting that the events were dependent on the movement of the charged PAMAM relative to polarity of the potential.
  • the positive PAMAM ball would be expected to move towards the negative pole at the trans side and remain at the pore entrance without subsequent movement out of the channel.
  • the voltage-dependent dynamic behavior of the tethered PAMAM dendrimer indicates that it could function as a voltage sensing molecular-ball valve.
  • the invention provides a way to regulate the flow a matter across the membrane in response to an external stimulus such as voltage.
  • the ability of the pore comprising a dendrimer to act as an ion sieve was further investigated by determination of the permeability ratio Pci-/P ⁇ + for heptamers SIO6C7, SIO6C 7 -G2-PAMAM, and S106C 7 -G3-PAMAM: that is pores wherein the .
  • I-V curves were constructed for currents recorded under both cisltrans and translcis KCl gradients and charge selectivities were calculated from the reversal potential, V r (Table 2).
  • the permeability ratio of S106C 7 -G2-PAMAM of 2.41 ⁇ 0.14 is further enhanced by increasing the number of positive charges.
  • Preferred embodiments of the invention relate to dendrimers with high numbers of surface primary amines, which lead to a bigger preference for anions and dendrimers derivatives which terminate with quaternary amines and are therefore positively charged (Lee, J. H.; Lim, Y. B.; Choi, J. S.; Lee, Y.; Kim, T. L; Kim, H. J.; Yoon, J. K.; Kim, K.; Park, J. S. Bioconjug. Chem. 2003, 14, 1214-1221)
  • the preferred passage of anions over cations can also be enhanced by changing the size of the dendrimer. Therefore in order to gain increased selectivity, embodiments are preferred in which the space between the dendrimer and the sides of the pore are minimised, so that the anion is forced to pass through, rather than around, the dendrimer.
  • the pores according to the invention are used for stochastic sensing, wherein individual molecules are detected by their ability to modulate ionic current flowing through a single pore.
  • This approach has been used for analytes such as toxic metal ions (Braha, O.; Walker, B.; Cheley, S.; Kasianowicz, J. J.; Song, L.; Gouaux, J. E.; Bayley, H. Chem Biol. 1997, 4, 497-505), drugs, (Gu, L. Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Nature. 1999, 398, 686-690), enantiomers (Kang, X.
  • An essential component in these sensors has been the covalent attachment of small molecules and linear polymers within the pore.
  • the tethering of DNA oligonucleotides to engineered pores enabled the sequence-specific detection of individual free DNA strands (Howorka, S.; Cheley, S.; Bayley, H. Nat Biotechnol. 2001, 19, 636-639; Howorka, S.; Movileanu, L.; Braha, O.; Bayley, H. Proc Nat Acad Sci USA. 2001, 98, 12996-13001; Howorka, S.; Bayley, H. Biophys J. 2002, 53, 3202- 3210).
  • Organic polymers such as polyethylene glycol (PEG) were also tethered to pores via engineered cysteines. Single channel current recordings of these pores demonstrated that a single PEG chain modulated the ionic current passing through the pore. Based on the characteristic current modulations, differences in the conformational dynamics of individual linear polymers of different chain length could be observed (Howorka, S.; Movileanu, L.; Lu, X.; Magnon, M.; Cheley, S.; Braha, O.; Bayley, H. J Am. Chem. Soc. 2000, 122, 2411-2416).
  • PEG polyethylene glycol
  • PEG-modified pores also led to the development of biosensor elements capable of detecting protein analytes at the single-molecule level (Movileanu, L.; Howorka, S.; Braha, O.; Bayley, H. Nat Biotechnol. 2000, 18, 1091- 1095).
  • nanoscale pores attached to dendrimers is in polypeptide and nucleic acid sequencing.
  • a transmembrane potential drives individual DNA and RNA strands through a pore thereby causing characteristic blockades in ionic current.
  • nanopore recordings successfully identified nucleic acid homopolymers of different composition and block- copolymers.
  • the nanopore sequencing may read base-sequences from individual translocating DNA or RNA strands.
  • one embodiment of the invention is a nucleic acid or polypeptide sequencing device comprising a membrane as described hereinabove, an inlet and an outlet.
  • the inlet and the outlet are not limited in type, style or size. However, the inlet and the outlet are in fluid communication, and the inlet is positioned at the first side of the membrane and the outlet is positioned at the second side of the membrane.
  • the device according to the invention can be used in a method of sequencing a nucleic acid or a polypeptide in which a fluid comprising the nucleic acid or a polypeptide and an ionic salt is positioned on the first side of a membrane as described hereinabove, a potential difference is applied across the membrane and a resulting current is measured over time.
  • the purpose of measuring the resulting current over time is to infer the identity of the translocating DNA strand.
  • the method of the invention is used when the nucleic acid is DNA or RNA.
  • the dendrimer positioned within the pore is not limited in type, but must be chosen so as to decelerate the passage of DNA to an appropriate rate to achieve resolution of individual bases.
  • a nanopore filled with a positively charged and hyperbranched polymer such as PAMAM may be used to decelerate the passage of DNA via electrostatic interactions and/or steric factors.
  • PAMAM dendrimers are particularly appropriate for several reasons.
  • DNA and RNA have a high affinity to positively charged dendrimer molecules, which is exploited in gene delivery carriers.
  • the forces of attraction between DNA and dendrimer have been characterized at the single molecule level for example with optical tweezers.
  • the affinity of DNA to PAMAM can be finely tuned by varying the chemical composition and charge of the dendrimer.
  • spherical dendrimers can be confined inside the inner cavity of the ⁇ HL pore thus leaving the barrel and inner constriction largely free of polymer. This narrow part of the pore is usually used as sensor region to detect bases using e.g. circular cyclodextrin molecules.
  • RNA oligonucleotide C30 added to the cis side of SIO6C7 with a potential of +100 mV at the trans side, resulted in frequent short current deflections (Fig. 7B) which were, however, absent in recordings without RNA (Fig. 7A).
  • the high-amplitude events represent the translocation of nucleic acids strand from the cis to the trans side of the pore which temporarily block the movement of ions through the lumen of the pore (Kasianowicz, J. J. Nature Materials. 2004, 3, 355-356; Akeson, M.; Branton, D.; Kasianowicz, J. J.; Brandin, E.; Deamer, D. W. Biophys J. 1999, 77, 3227- 3233).
  • the present invention also provides a method of determining the structure of a pore, comprising attaching a dendrimer to the pore and measuring variations of an ionic current through the pore.
  • the dendimer is attached to the lumen of the pore.
  • the information relates to the dimensions of the pore.
  • the invention also relates to a sulfhydryl-reactive dendrimer according to formula I:
  • D represents a dendrimer
  • the present invention also provides use of the above sulfhydryl-reactive dendrimer in the substituted-cysteine accessibility method (SCAM).
  • the sulfhydryl-reactive dendrimers can be used in the substituted-cysteine accessibility method (SCAM) (Karlin, A.; Akabas, M. H. Methods Enzymol. 1998, 293, 123-145) which infers the surface accessibility of residues by determining how fast sulfhydryl- reactive reagents couple to single-cysteine mutants of a protein.
  • SCAM substituted-cysteine accessibility method
  • SCAM substituted-cysteine accessibility method
  • dendrimers In calibration experiments with the structurally defined membrane pore ⁇ HL, dendrimers exhibited a sharp size-dependent permeation cut-off, and readily identified a 2.9 nm wide pore entrance.
  • PAMAM-PDP dendrimers with diameters from 2 to 10 nm are well suited to probe various nanometer-sized pore-forming proteins with important biomedical roles such as bacterial pore-forming toxins (Parker, M. W.; Feil, S. C. Prog Biophys MoI Biol. 2005, 88, 91-142; Menestrina, G.
  • the approach can also be applied to explore the molecular structure of interesting biological nanomaterials such as porous S-layer proteins (Howorka, S.; Sara, M.; Wang, Y.; Kuen, B.; Sleytr, U. B.; Lubitz, W.; Bayley, H. J Biol Chem. 2000, 48, 37876-37886; Sleytr, U. B.; Messner, P.; Pum, D.; Sara, M. Angew Chem Int Edit. 1999, 35, 1035-1054).
  • the invention also relates to new method to alter the properties of proteins via targeted chemical modification by attachment of a dendrimer.
  • the dendrimer is attached using a method as described by the present invention.
  • Coupling of non- branched linear organic polymers or biopolymers has been used in the past to modify or expand the natural characteristics of proteins such as in pharmacology to increase the half-life of therapeutic proteins via PEGylation (Veronese, F. M.; Pasut, G. Drug Discov Today. 2005, 10, 1451-1458), in molecular biology to introduce sequence specificity into nucleases via attachment of a DNA oligonucleotides (Corey, D. R.; Schultz, P. G. Science.
  • Another embodiment of the invention provides a pharmaceutical composition comprising an active ingredient and one or more excipients wherein the active ingredient and the one or more excipient are enclosed by a membrane as described hereinabove.
  • the invention also provides a pharmaceutical composition that provides a controlled release of the active ingredient.
  • the active ingredient is enclosed behind the membrane according to the invention.
  • the membrane is not limited, other than it must be pharmaceutically acceptable.
  • the pore and the dendrimer are selected using the size and charge based criteria outlined hereinabove for filtration devices, so as to allow the passage of the active ingredient from the first side of the membrane to the second side of the membrane at an appropriate rate, at which second side the active ingredient may be absorbed by the subject who has been administered the active ingredient.
  • the rate of release of the active ingredient through the membrane is dependent on the pH of the solution exterior to the membrane. Therefore, for example, the rate of release may be such that an active ingredient may not be released when the pharmaceutical is in a low pH environment, such as the stomach, and then subsequently released when the pharmaceutical passes into the small intestine.
  • Active ingredients favoured for use in this embodiment of the invention include active ingredients that are unstable in the stomach, for example bisphosphonates.
  • the present invention alters pore permeation properties by placing a spherical dendrimer inside the lumen.
  • hyperbranched dendrimers has specific advantages over other spherical materials such as quantum dots of similar size or functionality.
  • compact dendrimers have some residual degree of structural flexibility which can help overcome small steric permeation barriers. Indeed, G3 PAMAM with a diameter of 2.9 nm successfully passed the 2.9 nm wide opening of the ⁇ HL pore.
  • the hyperbranched character of the dendrimer is important in controlling and tuning the flow or matter through the engineered pore while solid impermeable spheres would likely lead to much more drastic and less tuneable changes in the permeation properties.
  • placing dendrimers into a pore lumen is a unique approach to introduce ion selectivity filters or molecular sieves.
  • the approach is not only restricted to protein pores but can be applied to engineer the permeation properties of inorganic or metallic porous structures for the separation of biopolymers or linear polymers for purification or sensing purposes. Examples
  • Methoxy-polyethyleneglycol- ethylmaleimide 10 kD was obtained from Apollo Scientific (Stockport, UK).
  • Qiaprep kits for the purification of plasmid DNA were purchased from Qiagen (Crawley, UK).
  • E. coli T7 S30 extract for coupled in vitro transcription/translation from circular DNA was from Promega (Southampton, UK).
  • [ 35 S]Methionine (1,200 Ci/mmol) was supplied by GE Healthcare.
  • RNA oligonucleotides were obtained from Dharmacon.
  • 3-(2-Pyridyldithio)-propanoic acid was synthesized as a precursor for the generation of ⁇ /-succinimidyl 3-(2-pyridyldithio)-propanoate (SPDP) (Carlsson, J.; Drevin, H.; Axen, R. Biochem J. 1978, 173, 723-737).
  • SPDP ⁇ /-succinimidyl 3-(2-pyridyldithio)-propanoate
  • DPDS 2,2'-Dipyridyldisulfide
  • DPDS 2,2'-Dipyridyldisulfide
  • SPDP 50 mg, 0.16 mmol in DMSO (100 ⁇ l) was added to a solution of 0.3 M MOPS buffer pH 7.4 (2 ml) and G5 dendrimer (10% NH 2 surface groups, 3.78% w/w in MeOH) (2.4 ml, 2.3 ⁇ mol).
  • G3-P AMAM-PDP SPDP (5 mg, 16 ⁇ mol) in DMSO (100 ⁇ l) was mixed with a solution of 0.3 M MOPS buffer pH 7.4 (2 ml) and G3 dendrimer (50% NH 2 surface groups, 32.7% w/w in MeOH) (62 ⁇ l, 2.5 ⁇ mol).
  • the chemical derivatization of PAMAM dendrimers with SPDP was analyzed via reversed phase HPLC using a Varian ProStar system with a Module 410 autosampler, Model 210 solvent delivery module, and a Model 320 UV detector.
  • a Discovery Bio Widepore C5 column (250 x 4.6 mm, 5 ⁇ m beads) was loaded with 10 ⁇ l of a 10 mg/ml solution of PAMAM dendrimers at a flow rate of 1 ml/min.
  • the mobile phase was a linear gradient beginning with 90:10 water (0.1% TFA) /acetonitrile to either 50:50 or 70:30 water (0.1% TFA) /acetonitrile over 30 min followed by 2 min at the final gradient concentration. Analysis was conducted using Star Chromatography Workstation software Version 5.51.
  • Dendrimers were analyzed via SDS-polyacrylamide gel electrophoresis (20% acrylamide gels for G2 and G3, and 12% acrylamide gels for G5) and Coomassie Blue Staining. The intensities of gel bands were determined using Scion Image (Scion Imaging, Frederick, MA).
  • mutants K8C and S106C of the semisynthetic gene ⁇ HL-RL2-D8 has been published elsewhere (Howorka, S.; Movileanu, L.; Lu, X.; Magnon, M.; Cheley, S.; Braha, O.; Bayley, H. J Am. Chem. Soc. 2000, 122, 2411-2416).
  • ⁇ HL-RL2-D8 contains the conservative replacements Val-124 ⁇ Leu, Gly-130 ⁇ Ser, Asn-139 ⁇ GIn, He- 142 ⁇ Leu, which were introduced to facilitate cassette mutagenesis.
  • Lys at position 8 was replaced by Ala to prevent adventitious proteolysis.
  • D8 encodes for a C-terminal extension of eight aspartates.
  • the mutant K46C was obtained by site-directed mutagenesis using in vivo recombination PCR 31 of the gene ⁇ HL-RL2-D8. None of the mutagenic changes alter the electrical properties of the pore as shown by single channel current recordings.
  • 35 S-labeled ⁇ HL polypeptides were generated from plasmid DNA harboring mutant ⁇ HL genes by coupled in vitro transcription/translation (IVTT) using the E. coli S30 T7 IVTT kit (Promega number Ll 130) (Cheley, S.; Braha, O.; Lu, X.; Conlan, S.; Bayley, H. Protein Sci. 1999, 8, 1257-1267).
  • the reaction volume contained the amino acid mixture minus methionine (1.25 ⁇ l), premix (5 ⁇ l), and S30 mixture (3.75 ⁇ l) supplemented with 1 ⁇ g/ml rifampicin, and 7 ⁇ Ci of [ 35 S] methionine (GE Healthcare, 1,200 Ci/mmol; corresponding to a 15 mM solution) (1 ⁇ l).
  • Supercoiled plasmid DNA 500 ng was added to give a final reaction volume of 12.5 ⁇ l.
  • the reactions were incubated for 1 h at 37 0 C and then centrifuged for 5 min at 21,000 x g. The concentration of free thiols in the IVTT mix was 10 mM.
  • Translation mix of K46C ⁇ HL monomer (0.25 ⁇ l; 10 mM thiol groups) was diluted into buffer containing 10 mM MOPS-NaOH, pH 7.4, 150 mM NaCl, 0.5 mM EDTA (ME buffer) (3 ⁇ l), and immediately reacted with 2 mM G3 -PAMAM-PDP or G5 -PAMAM- PDP (2 ⁇ l), or 2 mM G2-P AMAM-PDP (4 ⁇ l) for 20 min at 20 0 C.
  • the diluted ⁇ HL translation mix was also reacted with 50 mM PEG-MAL with a MW 5,000 (2 ⁇ l).
  • ⁇ HL polypeptides K46C, K8C, and S106C were allowed to assemble on rabbit erythrocyte membranes to form heptameric pores as described (Walker, B.; Krishnasastry, M.; Zorn, L.; Kasianowicz, J.; Bayley, H. J Biol Chem. 1992, 267, 10902-10909). Briefly, translation mix (1 ⁇ l) was diluted in ME buffer (5 ⁇ l) supplemented with 10 mM DTT (3 ⁇ l). Assembly was initiated by adding a suspension of rabbit erythrocyte membranes (1 mg protein /ml, 2.5 ⁇ l).
  • the mixture was incubated for 1 hour at 20 0 C with gentle resuspension of the membranes every 10 minutes.
  • the suspension was centrifuged and the supernatant discarded.
  • the pellet was washed with 1 x ME buffer (50 ⁇ l) and taken up in 1 x ME buffer (10 ⁇ l).
  • ⁇ HL pores were then subjected to chemical modification by mixing the suspended membranes containing heptamers with 2 mM PAMAM-PDP (2 ⁇ l). After incubation for 10 minutes, unreacted cysteine residues were quenched by the addition 1 M N-maleoyl- ⁇ -alanine (2 ⁇ l).
  • Heptamers were also treated with PAMAM-PDP followed by the addition of 50 mM PEG-MAL 5 kD (2 ⁇ l), solely by reaction with 50 mM PEG-MAL 5 kD (2 ⁇ l) without dendrimers, or with 50 mM PEG-MAL 10 kD (2 ⁇ l). After 10 minutes, the suspensions were centrifuged for 5 minutes at 21,000 g. Supernatants were removed, pellets containing the heptamers were resuspended in ME buffer (10 ⁇ l), mixed with 2x Laemmli buffer and analyzed with SDS-PAGE at 100 mV followed by autoradiography.
  • modified heptamers were eluted from SDS-PAGE gels as described (Howorka, S.; Movileanu, L.; Lu, X.; Magnon, M.; Cheley, S.; Braha, O.; Bayley, H. J Am. Chem. Soc. 2000, 122, 2411- 2416).
  • Disulfide-derivatized PAMAM dendrimers are coupled inside the pore lumen of gold- plated porous membranes.
  • Disulfide-derivatized PAMAM dendrimers are prepared by coupling 7V-Succinimidyl 3-(2-pyridyldithio)-propanoate (SPDP) to amino-terminated PAMAM dendrimers of generation 5.
  • SPDP 50 mg, 0.16 mmol
  • DMSO 100 ⁇ l
  • G5 dendrimer 10% NH 2 surface groups, 3.78% w/w in MeOH
  • the dendrimer is precipitated by the addition of ice-cold acetone (20 ml) and centrifugation at 21,000 g for 1 minute. The supernatant is removed and the resulting pellet re-suspended in ddH 2 O (1.5 ml). Extraction of unreacted SPDP and its hydrolysis product 3-(2-pyridyldithio)-propanoic acid present in solution and possibly encapsulated (Beezer, King et al. 2003) within the dendrimer core is conducted using DCM.
  • the extraction is repeated 8 times with each 2 ml to remove SPDP and 3-(2- pyridyldithio)-propanoic acid as judged by TLC analysis (1:4 MeOH: DCM; stained with KMnO 4 ; Rf for 3-(2-pyridyldithio)-propanoic acid, 0.64; Rf for SPDP, 0.88).
  • the extracted dendrimer solution is precipitated by the addition of ice-cold acetone (10 ml) and centrifugation at 21,000 g. The supernatant is removed and the pellet re-suspended in ddH 2 O.
  • the gold-plated porous membranes are prepared with the template synthesis method by electrolessly depositing gold along the pore walls of a polycarbonate template membrane (Martin, Nishizawa et al. 2001).
  • the template is a commercially available filter, 6 ⁇ m thick, with cylindrical, 30-nm-diameter pores and 6 x 10 s pores per cm 2 of membrane surface area.
  • the inside diameters of the gold pores deposited within the holes of the template are controlled by varying the deposition time.
  • the gold pores of membranes used for the PAMAM coupling have an inside diameter of 10 nm, as determined by electron microscopy and ion flux measurement (Martin, Nishizawa et al. 2001).
  • PA separating the cis and trans chambers of the apparatus.
  • Each compartment contained 1.0 ml of 1 M KCl, 20 mM Tris ⁇ Cl pH 7.5.
  • Gel-purified heptameric ⁇ HL protein (final concentration 0.01-0.1 ng/ml) was added to the cis compartment, and the electrolyte in the cis chamber was stirred until a single channel inserted into the bilayer.
  • Transmembrane currents were recorded at a holding potential of +100 mV (with the cis side grounded) by using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Union City, CA). For analysis, currents were low-pass filtered at 1 kHz and sampled at
  • Ion selectivity measurements were performed using asymmetrical conditions with one chamber (cis or trans) containing 300 mM KCl and the other chamber containing 100 mM KCl, supplemented each with 10 mM Tris-HCl pH 7.5. After the measurements, the lipid bilayer membrane was broken to determine the value of electrode junction potentials (normally around 0.5 mV). The permeability ratios (P ⁇ +/P ⁇ -) were calculated from reversal potentials by using the Goldman-Hodgkin-Katz equation.
  • V r is the reversal potential (i.e., the electrical potential giving zero current)
  • ax is the activity of ion X
  • subscripts c and t represent the cis and trans compartments
  • R is the gas constant
  • F the Faraday constant.
  • the temperature was 23 ⁇ 1 0 C.
  • V r was obtained by a polynomial fit of the current- voltage (I- V) data near zero current.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention porte sur un pore à l'échelle nanométrique ayant une lumière, un dendrimère étant attaché au pore, et également sur une membrane comprenant le pore. L'invention a des applications dans des dispositifs de filtration, le séquençage d'acides nucléiques ou de polypeptides, et les enrobages de compositions pharmaceutiques.
PCT/GB2008/050351 2007-05-14 2008-05-14 Pores WO2008139229A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB0709259.6A GB0709259D0 (en) 2007-05-14 2007-05-14 Pores
GB0709259.6 2007-05-14

Publications (2)

Publication Number Publication Date
WO2008139229A2 true WO2008139229A2 (fr) 2008-11-20
WO2008139229A3 WO2008139229A3 (fr) 2009-03-19

Family

ID=38219386

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2008/050351 WO2008139229A2 (fr) 2007-05-14 2008-05-14 Pores

Country Status (2)

Country Link
GB (1) GB0709259D0 (fr)
WO (1) WO2008139229A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105778895A (zh) * 2016-04-08 2016-07-20 兰州文理学院 CdS/PAMAM纳米复合材料的制备及在检测Cu2+中的应用
CN111701467A (zh) * 2020-05-26 2020-09-25 浙江工业大学 一种利用共价键层层自组装提高均孔膜抗污染改性的方法
CN113735098A (zh) * 2020-05-29 2021-12-03 中国石油天然气股份有限公司 氮元素掺杂碳纳米环、其制备方法及应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106589401B (zh) * 2017-01-04 2020-07-14 安庆师范大学 一种含p硅胶负载pamam型树枝状大分子的制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6472571B1 (en) * 1999-10-01 2002-10-29 Degussa-Huls Ag Process for the production of organic compounds in a membrane reactor
US20040063200A1 (en) * 2000-07-28 2004-04-01 Elliot Chaikof Biological component comprising artificial membrane
WO2005009602A2 (fr) * 2003-07-22 2005-02-03 Iowa State University Research Foundation, Inc. Silicates mesoporeux coiffes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6472571B1 (en) * 1999-10-01 2002-10-29 Degussa-Huls Ag Process for the production of organic compounds in a membrane reactor
US20040063200A1 (en) * 2000-07-28 2004-04-01 Elliot Chaikof Biological component comprising artificial membrane
WO2005009602A2 (fr) * 2003-07-22 2005-02-03 Iowa State University Research Foundation, Inc. Silicates mesoporeux coiffes

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105778895A (zh) * 2016-04-08 2016-07-20 兰州文理学院 CdS/PAMAM纳米复合材料的制备及在检测Cu2+中的应用
CN111701467A (zh) * 2020-05-26 2020-09-25 浙江工业大学 一种利用共价键层层自组装提高均孔膜抗污染改性的方法
CN111701467B (zh) * 2020-05-26 2022-05-10 浙江工业大学 一种利用共价键层层自组装提高均孔膜抗污染改性的方法
CN113735098A (zh) * 2020-05-29 2021-12-03 中国石油天然气股份有限公司 氮元素掺杂碳纳米环、其制备方法及应用
CN113735098B (zh) * 2020-05-29 2023-08-22 中国石油天然气股份有限公司 氮元素掺杂碳纳米环、其制备方法及应用

Also Published As

Publication number Publication date
WO2008139229A3 (fr) 2009-03-19
GB0709259D0 (en) 2007-06-20

Similar Documents

Publication Publication Date Title
US11761956B2 (en) Nanopore biosensors for detection of proteins and nucleic acids
US11946102B2 (en) Coupling method
JP7555987B2 (ja) 変異体ポア
EP2836506B1 (fr) Pores de mutants de lysenin
WO2009007743A1 (fr) Procédé de détection d'acides nucléiques
GB2453377A (en) Transmembrane protein pores and molecular adapters therefore.
WO2001059453A2 (fr) Compositions de biocapteurs et procedes d'utilisation associes
JP2023503514A (ja) ナノポアを使用する標的ペプチドの特徴付けの方法
TW202140798A (zh) 具有用於模板多核苷酸接附的單一位置的奈米顆粒
Martin et al. Nanoscale protein pores modified with PAMAM dendrimers
Tang et al. The coiled coils in the design of protein-based constructs: hybrid hydrogels and epitope displays
WO2008139229A2 (fr) Pores
WO2020074399A1 (fr) Ajustement à base d'oligonucléotides de peptides formant des pores pour augmenter la taille des pores, l'affinité membranaire, la stabilité et l'activité antimicrobienne
ES2911439T3 (es) Procedimientos y composiciones de bioconjugación específica de sitio útiles para sistemas de nanoporos
AU2021356235A1 (en) Modification of a nanopore forming protein oligomer
Krishnan et al. Designed alpha-helical barrels for charge-selective peptide translocation
JP2010122071A (ja) 物質を固定化した物質固定化担体および物質固定化担体を作製する方法
CN118475593A (zh) 孔
WO2015011675A1 (fr) Complexes nano-assemblés d'acides nucléiques, d'avidine et de composés biotinylés utilisables en tant que supports d'administration intracellulaire
CN106883158B (zh) 生物可降解的氨基脂质类化合物及其制备方法和应用
CN111205350A (zh) 作为组蛋白脱乙酰酶抑制剂的大环硫代酸酯前体药物
AU2022246740A1 (en) Polypeptide nanopores synthetically functionalized with positively charged species, and methods of making and using the same
Mereuta et al. Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection
WO2003040094A9 (fr) Procedes de preparation de molecules macrocycliques, molecules macrocycliques preparees a l'aide de ces procedes, ainsi que substrats et supports solides mis en oeuvre dans ces procedes.
ES2310969B1 (es) Biotina-soporte poroso, metodos de obtencion y usos.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08750750

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08750750

Country of ref document: EP

Kind code of ref document: A2