Molecular library encoding system and methods

A binary encoding system for identification of carrier beads, the system comprising:

• providing a number N of different labelling bead types, the N different labelling bead types being used to prepare an exponentially large number, 2N, of carrier bead types, each labelling bead type being adapted to attach to the surface of a carrier bead or be captured in the bulk of a carrier bead, each labelling bead type having unique fluorescent label which is optically resolvable from the unique fluorescent label of other labelling bead type when attached to the surface of a carrier bead or captured in the bulk of a carrier bead;
• wherein:
• the unique fluorescent label on each labelling bead type comprises one, two or more fluorescence emission bands and one, two or more fluorescence excitation bands, each fluorescence emission band being distinguishable by one, two or more of:
• (i) the representative wavelength of emission, (ii) the intensity of emission, (iii) the characteristic wavelength of the excitation band exciting the emission, and (iv) the efficiency of excitation in this excitation band exciting the emission;
• and
• the unique fluorescent label on each labelling bead type is spectrally distinct from the unique fluorescent label on the other labelling bead types in the set of labelling beads, such that the plurality of labelling bead types, in combination, are each uniquely labelled, and the unique fluorescent label on each labelling bead type is identifiable with an optical interrogation technique;
• and
• optically interrogating the carrier beads with three-dimensional (3D) resolution and the diameter of the smallest optically resolved 3D region is smaller than the distance between the carrier beads, wherein
• the presence or the absence of a labelling bead type on the surface or in the bulk of the carrier bead provides 1 bit of encoding for the carrier bead type, and
• wherein all possible combinations of beads of N different types being present or absent on the surface or in the bulk of the carrier bead provide N bits of encoding and 2N unique fluorescent encodings, and
• wherein the availability of 10, 20, and 30 different types of labelling beads makes it possible to prepare carrier beads with, respectively, 210≈103, 220≈106, and 230≈109 unique fluorescent encodings.

A system for preparing a combinatorial library with fluorescent encoding in a combinatorial synthesis, the system comprising:

• a) providing a set of labelling beads comprised of a plurality of labelling bead types, each labelling bead type being adapted to attach to the surface of a carrier bead or be captured in the bulk of a carrier bead, each labelling bead type having unique fluorescent label which is optically resolvable from the unique fluorescent label of other labelling bead type when attached to the surface of a carrier bead or captured in the bulk of a carrier bead;
• wherein:
• the unique fluorescent label on each labelling bead type comprises one, two or more fluorescence emission bands and one, two or more fluorescence excitation bands, each fluorescence emission band being distinguishable by one, two or more of:
• (i) the representative wavelength of emission, (ii) the intensity of emission, (iii) the characteristic wavelength of the excitation band exciting the emission, and (iv) the efficiency of excitation in this excitation band exciting the emission;
• and
• the unique fluorescent label on each labelling bead type is spectrally distinct from the unique fluorescent label on the other labelling bead types in the set of labelling beads, such that the plurality of labelling bead types, in combination, are each uniquely labelled;
• b) providing a plurality of carrier beads;
• c) providing a set of molecular building blocks for a combinatorial synthesis, the combinatorial synthesis having a plurality of steps, and each step of the combinatorial synthesis having a molecular building block, each building block being the same or different;
• d) performing a sequence of steps of the combinatorial synthesis on a group of carrier beads, the sequence of steps comprising adding a new molecular building block to a group of carrier beads at each step of the combinatorial synthesis to form the combinatorial library, each group of carrier beads in the library having a unique synthetic compound;
• e) matching each molecular building block bound to the group carrier beads in a given sequence step of the combinatorial synthesis with a labelling bead type, thereby forming a population of different carrier bead groups, each carrier bead group having a unique synthetic molecule and corresponding unique fluorescent label encoded on the carrier bead, the unique fluorescent label being produced by the plurality of different labelling bead types, each with unique fluorescent labels, attached to the surface of the carrier beads in consecutive steps of the combinatorial synthesis;
• f) identifying the labelling bead types attached to each carrier bead by an optical interrogation technique, wherein the unique fluorescent label on at least one labelling bead type is identifiable with an optical interrogation technique, and wherein the carrier beads are optically interrogated with three-dimensional (3D) resolution and the diameter of the smallest optically resolved 3D region is smaller than the diameter of the carrier beads.

A combinatorial library established on a population of fluorescently encoded carrier beads, the fluorescently encoded carrier beads comprising:

• a plurality of groups of carrier beads, each group of carrier beads comprising:
• a synthetic molecule, the synthetic molecule having a plurality of molecular building blocks added at N consecutive steps in a combinatorial synthesis;
• and
• a plurality of different labelling bead types attached to the surface of the carrier bead
• wherein each labelling bead type has a unique fluorescent label which is optically resolvable from the unique fluorescent label of other labelling bead types on the surface of the carrier bead, and
• wherein the labelling bead type attached to the carrier bead at a given step of the combinatorial synthesis uniquely matches the type of the molecular building block added to the carrier bead at the same step of the combinatorial synthesis, such that the set of labelling bead types attached to a given carrier bead uniquely fluorescently encodes the set of the molecular building blocks in the synthetic molecules on the carrier bead, and
• wherein all carrier beads belonging to a given group in the population of the carrier beads carry the same synthetic molecules comprised of the same sequence of molecular building blocks and have the same fluorescent encoding, whereas beads belonging to different groups carry different synthetic molecules and have different fluorescent encodings that are optically resolvable on the carrier beads with the optical interrogation technique wherein the unique fluorescent label on at least one labelling bead type is identifiable with the optical interrogation technique, and wherein the carrier beads are optically interrogated with three-dimensional (3D) resolution and the diameter of the smallest optically resolved 3D region is smaller than the diameter of the carrier beads.

A method of labelling carrier beads in a solid phase combinatorial synthesis, the method comprising:

• a) providing a carrier bead having a primary compound attached thereto;
• b) providing a set of molecular building blocks for each step of the synthesis;
• c) combining the carrier bead having the primary compound with a molecular building block to form synthetic molecule M1 that is comprised of the primary compound and the first added building block;
• d) attaching to the carrier bead with synthetic molecule M1 a plurality of first labelling bead types, the first labelling bead type being correlated to the first molecular building block to form a carrier bead having molecule M1 and label L1;
• e) adding molecular building blocks M2-M4 to the synthetic molecule M1 on the surface of the carrier bead and attaching a second labelling bead type, L2-L4 to the surface of the carrier bead to form carrier beads having synthetic molecules M1,M2,L1,L2;
• M1,M3,L1,L3;
• and M1,M4,L1,L4;
• f) adding subsequent building blocks Mx and correlating labelling bead types Lx to the molecules of step e) to form carrier beads with synthetic molecules M1,M2,Mx,L1,L2;
• M1,M3,Mx,L1,L3Lx;
• and M1,M4,Mx,L1,L4,Lx, where Mx represents the set of building blocks, and Lx represents the corresponding labelling bead type;
• f) obtaining, as an end product, carrier beads compatible with an optical interrogation technique, making it possible to detect the types of labelling beads attached to the surface of each carrier bead and identify the corresponding synthetic compound.