METHOD AND COMPOSITION FOR BIOCATALYTIC PROTEIN-OLIGONUCLEOTIDE CONJUGATION AND PROTEIN-OLIGONUCLEOTIDE CONJUGATE

Abstract

A composition comprising a polypeptide ligated to an oligonucleotide through a sterol linker. A method of ligating a polypeptide to an oligonucleotide, comprising a polypeptide having a hedgehog steroyl transferse catalytic domain at the C-terminal of the polypeptide with an electrophilic residue, e.g., glycine, between polypeptide and the hedgehog steroyl transferse catalytic domain, and a steroylated oligonucleotide in solution, and permitting a reaction to cleave the hedgehog steroyl transferse catalytic domain from the polypeptide while ligating the steroylated oligonucleotide to the glycine at the C-terminal of the polypeptide. The oligonucleotide may be, for example, a therapeutic, diagnostic, or affinity ligand.

Claims

1. A method for generating a polypeptide-nucleic acid conjugate, comprising: providing a polypeptide; providing a steroylated-nucleic acid; providing a protein catalyst adapted to link the polypeptide to the steroylated-nucleic acid through a sterol moiety of the steroylated-nucleic acid; and reacting the steroylated-nucleic acid and the polypeptide, catalyzed by the protein catalyst, to form a covalently linked polypeptide-nucleic acid conjugate.

2. The method according to claim 1, wherein the protein catalyst comprises a hedgehog sterol transferase activity.

3. The method according to claim 2, wherein the polypeptide and the protein catalyst are provided as a fusion protein.

4. The method according to claim 3, wherein said reacting disassociates the protein catalyst from the polypeptide-nucleic acid conjugate.

5. The method according to claim 4, wherein the protein catalyst is linked to the polypeptide through an electrophilic residue.

6. The method according to claim 5, wherein the fusion protein comprises a C-terminal portion having the hedgehog sterol transferase activity, an intervening electrophilic residue, and an N-terminal portion comprising the polypeptide.

7. The method according to claim 6, wherein the steroylated-nucleic acid comprises a sterol or stanol ring system, a nucleophilic group at the 3-position of an A-ring of the fused sterol or stanol ring system, and an oligonucleotide attached through a linker to the sterol or stanol ring system.

8. The method according to claim 1, wherein the polypeptide has a length of between 2 amino acids and 500 amino acids.

9. The method according to claim 3, wherein the polypeptide has glycine as a last amino acid residue, linked to the protein catalyst having the hedgehog sterol transferase activity.

10. The method according to claim 1, wherein the steroylated-nucleic acid comprises at least one sterol molecule joined covalently to a nucleic acid polymer.

11. The method according to claim 1, wherein the protein catalyst is configured to react with a last residue of the polypeptide through translation fusion.

12. The method according to claim 1, wherein the protein catalyst is configured to chemically link a C-terminal glycine residue of the polypeptide with the steroylated-nucleic acid.

13. A polypeptide, comprising: a C-terminal steroyl transferase activity, an N-terminal peptide, and an intervening electrophilic residue.

14. The polypeptide according to claim 13, wherein the electrophilic reside comprises glycine.

15. The polypeptide according to claim 14, in combination with a steroylated-nucleic acid, the genetically engineered polypeptide and the steroylated-nucleic acid being configured to react in solution to ligate the steroylated-nucleic acid to the N-terminal peptide through the glycine, and to disassociate the C-terminal steroyl transferase activity from the ligated steroylated-nucleic acid and N-terminal peptide.

16. A composition, comprising: a polypeptide having a C-terminal electrophilic residue, a fused sterol or stanol ring system, having a nucleophilic group at the 3-position of an A-ring of the fused sterol or stanol ring system with beta or alpha stereochemistry, covalently linked to the C-terminal electrophilic residue, and an oligonucleotide attached through a linker adjacent to a D or other ring of the fused sterol or stanol ring system.

17. The composition according to claim 16, wherein the electrophilic residue comprises glycine.

18. The composition according to claim 16, wherein the oligonucleotide is RNA.

19. The composition according to claim 16, wherein the oligonucleotide is DNA.

20. The composition according to claim 16, in a pharmaceutically acceptable dosage form for administration to a human.

21. The composition according to claim 16, having at least one of therapeutic activity and diagnostic functionality, in an animal.

22. The composition according to claim 16, configured to act as a sensor to report a presence of an analyte.

23. The composition according to claim 16, wherein the polypeptide comprises a protease-sensitive domain, adapted to be cleaved by a mammalian sequence-specific protease.

Description

BRIEF DESCRIPTION OF FIGURES

[0254] FIG. 1 shows the native conjugation activity of HST-I.

[0255] FIG. 2 shows steroylated-oligonucleotides suitable for HST-I catalyzed conjugation.

[0256] FIGS. 3A and 3B show results of a study which conjugates proteins to oligonucleotides using HST-I.

[0257] FIG. 4 shows that, compared to other biocatalytic conjugations, HST-I leaves the smallest residual sequence scar.

[0258] FIGS. 5A-5C show, respectively, conjugation of an oligonucleotide (or other ligand) to a bead, an antibody, and to a cell surface.

[0259] FIG. 6A shows a general scheme for creating enzyme-aptamer conjugates.

[0260] FIG. 6B shows a strand-to-quadraplex sensor for bioluminescence resonance energy transfer (BRET), for lead sensing.

[0261] FIG. 6C shows a hairpin-to-rod sensor for pathogen DNA with accompanying fluorescent enhancement.

[0262] FIG. 7 shows receptor specific binding of protein-oligonucleotide conjugates.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0263] According to one embodiment, the technology provides a method of conjugating a protein to a steroylated-oligonucleotide.

[0264] FIG. 1 shows the native conjugation activity of HST-I. A hedgehog precursor protein comprised of HhN linked to the HST-I polypeptide, associates with cholesterol in a cellular membrane; membrane bound HST-I generates an internal thioester by rearranging the peptide bond at the HhN/HST-I junction; HST-I then activates a molecule of cholesterol to attack the internal thioester, resulting in the departure of HhN and the linking of HhN to cholesterol as a carboxyl ester.

[0265] Chemical Synthesis of Steroylated-Oligonucleotide

[0266] A nominal 2-step protocol is provided to synthesize sterol-oligonucleotides via oxime chemistry, as shown schematically in FIG. 2.

[0267] The protocol takes advantage of the fact that aminooxy groups react to form oximes in buffered aqueous conditions with equilibrium constants, typically in the range of >10.sup.8 M.sup.?1. Sterol-oligonucleotides compatible with HST-I catalyzed conjugation generally have the same general structure as (II) shown in FIG. 2, namely, a canonical fused sterol or stanol ring system, a nucleophilic group at the 3-position of the A-ring with beta stereochemistry, and an oligonucleotide attached through a linker adjacent to the D ring.

[0268] FIG. 2 shows that steroylated-oligonucleotides suitable for HST-I catalyzed conjugation can be synthesized by sequential oxime formation chemistry. The reaction sequence begins with the reaction of pregnenolone and bis(aminoxy)PEG3 to form intermediate (I); followed by reaction of I with benzaldehyde modified oligonucleotide to form II.

[0269] The reaction proceeds as follows: Pregnenolone-16-ene oxime (PEG3) aminoxy (I): In a total volume of 1 ml consisting of 900 ?l MeOH/100 ?l triethanolamine acetate buffer (1 M, pH 7), dissolve 0.2 mmoles of Bis-(Aminooxy)-PEG3 with 0.1 mmoles of pregnenolone. Solution starts out cloudy, then turns clear after overnight mixing on a vortex. Oxime formation is monitored by TLC (95% Dichloromethane/5% methanol). Product, pregnonlone is isolated by organic extraction (3?) using 6 mls ethylacetate/6 mls water, followed by drying under nitrogen stream to a white solid. Typical yield is 70-85%.

[0270] Pregnenolone-16-ene bisoxime(PEG3)-oligo-dT20 (II): In microscale reaction, the product from step (I) is joined using the same oxime chemistry to an oligonucleotide equipped with a (4-formylbenzamide) group, obtained commercially (Solulink/TriLink Inc.). The solvent is 90/10, methanol/triethanolmamine acetate buffer (pH 7), with I at 0.02 M and the oligonucleotide 0.0002 M. Following overnight incubation, the sterol-oligonucleotide (II) is purified using microspin oligo clean and concentrator column (Zymogen Inc.), and eluted with water.

[0271] Fusion Protein

[0272] Create and Clone a Synthetic Gene Encoding POI Fused to HST-I.

[0273] The gene encoding the protein of interest (POI) is cloned into an expression plasmid, creating an in-frame translational fusion with HST-I. If the last amino acid of the target protein is not glycine, a glycine codon is added at the 3 of the POI gene. This step involves standard molecular biology techniques.

[0274] Express POI-HST-I Fusion Protein.

[0275] Recombinant vector encoding POI-HST-I fusion protein is transformed into a suitable expression host, e.g., E. coli, strain BL21 DE3.

[0276] Alternative hosts, ideally organisms that do not contain endogenous sterols, may be employed. Endogenous sterols may react with the POI-HST-I precursor protein, resulting in the release of POI.

[0277] It is also possible to produce the POI-HST-I fusion peptide by in vitro translation. See, www.neb.com/tools-and-resources/feature-articles/the-next-generation-of-cell-free-protein-synthesis; www.thermofisher.com/us/en/home/references/ambion-tech-support/large-scale-transcription/general-articles/the-basics-in-vitro-translation.html; en.wikipedia.org/wiki/Cell-free_protein_synthesis; Mikami S et al. (2006) A hybridoma-based in vitro translation system that efficiently synthesizes glycoproteins. J Biotechnol 127(1):65-78; Mikami S et al. (2006) An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr Purif 46(2):348-57.

[0278] Purify POI-HST-1 Fusion Protein.

[0279] Fusion protein is purified from the cell extract using an appropriate chromatography method. For example, immobilized metal affinity chromatography may be used (IMAC). Other purification techniques (GST-tag; chitin-tag; MBP-tag) could also be used.

[0280] Conjugation

[0281] Conjugation of POI to the sterol-oligonucleotide through the action of HST-I is initiated at room temperature by addition of a sterol-oligonucleotide to a final concentration 100-200 ?M. Progress of the reaction can be followed by a variety of analytical methods. For example, SDS-PAGE may be used to monitor the change in molecular weight as the POI is conjugated and released from HST-1.

[0282] A final chromatography step can be carried out to separate HST-I from the conjugated target protein.

Example

[0283] The feasibility of HST-I catalyzed conjugation of protein to nucleic acids has been assessed through pilot scale experiments. In one example, a chimeric gene encoding a 20 kDa POI fused to HST-1 was created. This gene product, a 46 kDa precursor polypeptide, was expressed in E. coli and purified under native conditions using immobilized metal affinity chromatography. To test conjugation activity, a 30 ?l solution of the purified protein (2 ?M, final) in BisTris buffered solution, was mixed with sterol-oligonucleotide (?35 ?M, final).

[0284] The oligonucleotide used in this experiment was chemically modified with a fluorescein group. After 3 hours at room temperature, contents of the reaction and control reactions were separated by SDS-PAGE. The gel was first imaged using UV light source to detect the fluorescent oligonucleotide, and then by Coomassie staining which detects all proteins, as shown in FIGS. 3A and 3B.

[0285] FIGS. 3A and 3B show results of a pilot study which establishes feasibility of conjugating proteins to oligonucleotides using HST-I. FIG. 3A shows a scheme for the conjugation activity of POI-HST-I precursor protein. FIG. 3B shows conjugation of 46 kDa POI-HST-I fusion protein with sterol-oligonucleotide. Images of gels following SDS-PAGE to resolve reactions of POI-HST-I in the absence (lane 1) and presence of cholesterol (lane 2), synthetic sterol (lane 3), and a synthetic sterol-oligonucleotide. The gel was imaged under UV light (right) to detect the oligonucleotide, which was equipped with a fluorescein molecule; and under white light following straining with Coomassie dye (left).

[0286] Symbols:

[0287] POI-HST-I precursor protein (top triangles);

[0288] The POI-sterol-DNA conjugate (next-to-top triangle);

[0289] HST-I protein, released by conjugation, (next-to bottom triangles);

[0290] The sterol-modified POI (bottom triangles).

[0291] FIG. 4 shows that, compared to other biocatalytic conjugations, HST-I leaves the smallest residual sequence scar.

[0292] FIGS. 5A-5C show, respectively, conjugation of an oligonucleotide (or other ligand) to a bead, an antibody, and to a cell surface.

[0293] FIG. 6A shows a general scheme for creating enzyme-aptamer conjugates.

[0294] FIG. 6B shows a strand-to-quadraplex sensor for bioluminescence resonance energy transfer (BRET), for lead sensing, which exploits the ability of certain DNA to form a quadraplex with Pb.sup.2+ ions, which brings a dye, e.g., alexfluor 610 in close proximity to a nanoluciferase peptide.

[0295] FIG. 6C shows a hairpin-to-rod sensor for pathogen DNA, which exploits the ability of DABCYL to quench nanoluciferase when in close proximity, but to permit luminescence when displaced, such as during a hairpin-to-rod transformation or DNA or RNA.

[0296] FIG. 7 shows receptor specific binding of protein-oligonucleotide conjugates. In this case, FIG. 7 proposes a toxin ligated to the oligonucleotide, which is then endocytosed, and processed with lysosomes by normal cell activity, to release the toxin.

[0297] In the sample containing the HST-I precursor protein, conjugation activity is indicated by the diminished staining of the precursor protein compared with control, as well as by the appearance of protein corresponding to the molecular weight of HST-I. Finally, in this sample, a high molecular weight product (dots) is observed that reacts with the Coomassie stain and gives off a fluorescence signal. Together, these characteristics indicate that this species is the desired protein-oligonucleotide conjugate.

[0298] Each reference cited herein is expressly incorporated herein by reference it its entirety.

[0299] It is to be understood that while a certain form of the invention is illustrated, it is not to be limited to the specific form or arrangement herein described and shown. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification and the figures included herein. The embodiments, methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims.