ENCODED SELF-ASSEMBLING CHEMICAL LIBRARIES (ESACHEL)

• US 20100184611A1
• Filed: 09/08/2009
• Published: 07/22/2010
• Est. Priority Date: 03/08/2002
• Status: Active Grant

• Current Assignee: Eidgenoessische Technische Hochschule Zurich
• Original Assignee: Eidgenoessische Technische Hochschule Zurich
• Inventors: Melkko, Samu, Neri, Dario
• Granted Patent: US 8,642,514 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/10 involving encoding steps
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

The invention concerns a chemical compound comprising a chemical moiety (p) capable of performing a binding interaction with a target molecule (e.g. a biological target) and further comprising an oligonucleotide (b) or functional analogue thereof. In a first embodiment according to the invention, the chemical compound is characterized in that the oligonucleotide (b) or functional analogue comprises at least one self-assembly sequence (b1) capable of performing a combination reaction with at least one self-assembly sequence (b1′) of a complementary oligonucleotide or functional analogue bound to another chemical compound comprising a chemical moiety (q). In a second embodiment according to the invention, the chemical compound which comprises a coding sequence (b1) coding for the identification of the chemical moiety (p) is characterized in that the chemical compound further comprises at least one self-assembly moiety (m) capable of performing a combination reaction with at least one self-assembly moiety (m′) of a similar chemical compound comprising a chemical moiety (q). The invention comprises corresponding libraries of chemical compounds as well as methods of biopanning of target molecules and of identifying such targets.

Claims

1-32. -32. (canceled)

33. A method of biopanning ligands specific for target molecule(s), said method comprising:

incubating a mixture of a sub-library A of chemical compounds and a sub-library B of chemical compounds with target molecule(s), wherein the sub-library A comprises a first number of chemical compounds each comprising (i) a different chemical moiety (p) than other members of sub-library A and (ii) an oligonucleotide (b), or functional analogue thereof, which oligonucleotide (b) or functional analogue comprises a coding sequence (b2) coding for the identification of the chemical moiety (p) of its compound and a self-assembly sequence (b1) capable of performing a combination reaction and of building a stable combination reaction product with a self-assembly sequence (b1′

) of at least one chemical compound of the sub-library B, wherein the sub-library B comprises a second number of chemical compounds each comprising (i) a different chemical moiety (q) than other members of sub-library B and (ii) an oligonucleotide (b′

), or functional analogue thereof, which oligonucleotide (b′

) or functional analogue comprises a coding sequence (b2′

) coding for the identification of the chemical moiety (q) of its compound, and a self-assembly sequence (b1′

);

allowing a stable combination reaction product of at least one chemical compound of sub-library A and of at least one chemical compound of sub-library B to form;

allowing complex(es) of said target molecule(s) and one or both chemical moieties (p, q) of the stable combination reaction product to form;

physically separating the complex(es) from members of sub-library A and sub-library B and their stable combination reaction products which are not bound to target molecule(s); and

determining the coding sequences (b2, b2′

), respectively, of the sub-library A and sub-library B members bound to the complex(es), thereby identifying the chemical moiety (p) and/or the chemical moiety (q) of the stable combination reaction product(s) bound to the complex(es).

34. The method of claim 33, wherein said stable combination reaction products of the chemical compounds of sub-library A and sub-library B are formed in the absence of said target molecule(s).

35. The method of claim 33, wherein each of sub-library A and sub-library B comprises at least 4 members.

36. The method of claim 33, wherein the chemical moieties (p) of sub-library A are coupled to a 3′

extremity of the oligonucleotide (b1), and wherein the chemical moieties (q) of sub-library B are coupled to a 5′

extremity of the corresponding oligonucleotide (b1′

).

37. The method of claim 33, wherein said stable combination product is derived by forming oligomers selected from a group comprising dimers, trimers, and tetramers, by hetero-oligomerization of the self-assembly sequences (b1, b1′

) of the oligonucleotides (b, b′

), thereby forming heteroduplexes, heterotriplexes or heteroquadruplexes.

38. The method of claim 33, wherein in sub-library A or in sub-library B, iodoacetamido- or maleimido-derivatives of the respective chemical moieties have been coupled to individual DNA oligonucleotides which carry a thiol group at the 3′

or 5′

extremity.

39. The method of claim 33, wherein in sub-library A or in sub-library B, amide derivatives—

forming chemical structures according to the formula —

O—

P(O)₂—

O—

(CH₂)n-NH—

CO—

R, where R corresponds to a number of different chemical entities, and n ranges between 1 and 10—

have been coupled to the oligonucleotides carrying a phosphodiester bond at one extremity.

40. The method of claim 33, wherein in sub-library A the self-assembly sequence (b1) is interrupted by a d-spacer in opposite position to a code (B) of the chemical compounds of the sub-library B, the d-spacer preventing any undesired pairing to the bases of code (B) which encodes sub-library B, and wherein the oligonucleotide (b) of sub-library A has its distinctive code (A) towards the 5′

extremity.

41. The method of claim 33, wherein each chemical compound of sub-library A further comprises at least one chemical group that forms a covalent bond with at least one respective chemical group of a chemical compound of sub-library B with which had been formed a stable combination reaction product.

42. The method of claim 33, wherein in sub-library A, each oligonucleotide (b) or functional analogue thereof is directly and covalently linked to a chemical moiety (p) and wherein in sub-library B, each oligonucleotide (b′

) of or functional analogue thereof is directly and covalently linked to a chemical moiety (q).

43. The method of claim 33, wherein in the chemical compounds of sub-library A and sub-library B, the respective oligonucleotides (b, b′

) or functional analogues thereof further each comprise a respective linking portion which is situated between the respective self-assembly sequences (b1, b1′

) and the respective chemical moieties (p, q).

44. The method of claim 33, wherein the respective coding sequences (b2,b2′

) of the oligonucleotides (b, b′

) or the functional analogues thereof are situated between their respective chemical moieties (p, q) and the respective self-assembly sequences (b1, b1′

).

45. The method of claim 33, wherein at least one of the chemical moieties (p,q) is a polypeptide.

46. The method of claim 33, wherein the target molecule is a protein.

47. The method of claim 33, wherein the coding sequences (b2, b2′

) are determined by PCR methods.

48. The method of claim 33, wherein the coding sequences (b2, b2′

) are concatenated and the coding sequences (b2, b2′

) are then determined by sequencing the concatenamers.

49. The method of claim 33, wherein the method is repeated using those sub-library members having as their chemical moieties the identified chemical moieties of the stable combination reaction product.

50. The method of claim 33, wherein the oligonucleotides of the sub-libraries A and/or B are only partially annealing and the unpaired DNA strands of the sub-library A and sub-library B members bound to the complex are hybridized with oligonucleotides immobilized on one or more chips.

51. The method of claim 33, wherein by using a chip A or a chip B respectively, the reading of the identity and/or frequency of members of sub-library A or sub-library B respectively, rescued after a bio-panning experiment, is carried out and by decoding on chip A and B, candidate components of sub-libraries A and B, to be re-annealed and screened in a successive round of bio-panning, are suggested.

52. The method of claim 33, wherein the DNA of selected binding moieties is PCR amplified prior to chip hybridization.

53. The method of claim 33, wherein further the target molecule of the complex is identified.

54. A chemical library for use in bio-panning or identifying target molecules, the chemical library comprising stable combination reaction products between a first number of chemical compounds of a sub-library A with a sub-library B of chemical compounds, wherein the compounds of the sub-library A comprise:

(i) a chemical moiety (p) capable of performing a binding interaction with a single target molecule, and

(ii) an oligonucleotide (b) or functional analogue thereof, which comprises a variable, unique coding sequence (b2) coding for the identification of the chemical moiety (p) of its compound and a self-assembly sequence (b1) capable of performing a combination reaction and of building a stable combination reaction product with a self-assembly sequence (b1′

) of at least one chemical compound of the sub-library B, wherein the sub-library B comprises a second number of chemical compounds each comprising;

(i) a different chemical moiety (q) than other members of the sub-library B capable of performing a binding interaction with a single target molecule, and

(ii) an oligonucleotide (b′

) or functional analogue thereof, which comprises a variable, unique coding sequence (b2′

) coding for the identification of the chemical moiety (q) of its compound, and the self-assembly sequence (b1′

).

55. A chemical compound or sub-library A of chemical compounds for use in bio-panning of ligands for a target molecule, the chemical compound or each chemical compound of said sub-library A comprising:

(i) a chemical moiety (p) capable of performing a binding interaction with a single target molecule, and

(ii) an oligonucleotide (b) or functional analogue thereof, both of which comprise a variable, unique coding sequence (b2) coding for the identification of the chemical moiety (p),

wherein the chemical compound or each chemical compound of said sub-library A further comprises at least one self-assembly sequence (b1) capable of performing a combination reaction and of building, in the absence of said target molecule, a stable combination reaction product with a self-assembly sequence (b1′

) of at least one chemical compound of a sub-library B with a chemical moiety (q) and an oligonucleotide (b′

) or functional analogue thereof.