Oligonucleotide directed and recorded combinitorial synthesis of encoded probe molecules

A method of forming a probe molecule, wherein the probe molecule is according to formula (I), the method comprising:

• providing at least one hybridization array, the at least one hybridization array comprising at least one single stranded anti-codon oligomer immobilized on the at least one hybridization array, wherein the at least one single stranded anti-codon oligomer immobilized on the at least one hybridization array is capable of hybridizing to a coding region of a molecule of formula (II):
• ([(BI)(M−I)-D-L1]Y—HI)O-G-(H2-[L2-E-(B2)(K−1)]W)P (II)
• wherein
• G is an oligonucleotide, the oligonucleotide comprising at least two coding regions encoding positional building blocks and a first terminal coding region encoding a first building block and a second terminal coding region encoding a second building block, wherein the at least two coding regions are single stranded and the first and second terminal coding regions are single or double stranded;
• H1 is a hairpin structure comprising oligonucleotides comprising a loop portion, a stem portion, and a 5′ single stranded portion, wherein H1 comprises a 5′ end that is complementary to an end of the oligonucleotide G;
• H2 is a hairpin structure comprising oligonucleotides comprising a loop portion, a stem portion, and a 3′ single stranded portion, wherein H2 comprises a 3′ end that is complementary to an end of the oligonucleotide G;
• D is the first building block;
• E is the second building block, wherein D and E are the same or different;
• B1 is a positional building block and M represents an integer from 1 to 20;
• B2 is a positional building block and K represents an integer from 1 to 20, wherein B1 and B2 are the same or different, wherein M and K are the same or different;
• L1 is a linker that covalently bonds D to the loop portion or the stem portion of H1;
• L2 is a linker that covalently bonds E to the loop portion or the stem portion of H2;
• O is 1;
• P is 1;
• Y is an integer from 1 to 5;
• W is an integer from 1 to 5;
• and
• wherein at least one of each positional building block B1 at position M and B2 at position K is identified by one of the coding regions and wherein at least one of the first building block D and second building block E is identified by at least one of the first and second terminal coding regions;
• sorting a pool of molecules of formula (II) into sub-pools by hybridizing a coding region of a sub-pool of molecules of formula (II) to the at least one single stranded anti-codon oligomer immobilized on the at least one hybridization array;
• a step of optionally releasing the sub-pool of molecules of formula (II) from the at least one hybridization array into separate containers;
• providing at least one of positional building blocks B1 and B2;
• and
• reacting the at least one of positional building blocks B1 and B2 with the molecule of formula (II) to form a sub-pool of probe molecules of formula (I):
• ([(B1)M-D-L1]Y—H1)O-G-(H2-[L2-E-(B2)K]W)P, (I)
• wherein
• G is an oligonucleotide, the oligonucleotide comprising at least two coding regions encoding positional building blocks and a first terminal coding region encoding a first building block and a second terminal coding region encoding a second building block, wherein the at least two coding regions are single stranded and the first and second terminal coding regions are single or double stranded;
• H1 is a hairpin structure comprising oligonucleotides comprising a loop portion, a stem portion, and a 5′ single stranded portion, wherein H1 comprises a 5′ end that is complementary to an end of the oligonucleotide G;
• H2 is a hairpin structure comprising oligonucleotides comprising a loop portion, a stem portion, and a 3′ single stranded portion, wherein H2 comprises a 3′ end that is complementary to an end of the oligonucleotide G;
• D is the first building block;
• E is the second building block, wherein D and E are the same or different;
• B1 is a positional building block and M represents an integer from 1 to 20;
• B2 is a positional building block and K represents an integer from 1 to 20, wherein B1 and B2 are the same or different, wherein M and K are the same or different;
• L1 is a linker that covalently bonds D to the loop portion or the stem portion of H1;
• L2 is a linker that covalently bonds E to the loop portion or the stem portion of H2;
• O is 1;
• P is 1;
• Y is an integer from 1 to 5;
• W is an integer from 1 to 5;
• and
• wherein at least one of each positional building block B1 at position M and B2 at position K is identified by one of the coding regions, and wherein at least one of the first building block D and second building block E is identified by at least one of the first and second terminal coding regions.