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US 2020/0332441 A1Published application
Molecules for verifying oligonucleotide directed combinatorial synthesis and methods of making and using the same
AssigneeHaystack Sciences Corporation
InventorsRichard Edward Watts, Divya Kanichar
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Abstract
The present disclosure relates to multifunctional verification molecules, including molecules according to formula (I): G-L-(B)K-Q-U, wherein G, L, B, K, Q, and U are defined herein. The present disclosure also relates to methods of preparing and using such multifunctional verification molecules to remove defective multifunctional molecules and to quantify synthetic yield.
Claims20
A molecule according to formula (I),
- G-L-(B)K-Q-U (I)
- wherein
- G includes an oligonucleotide, the oligonucleotide comprising at least two coding regions, wherein the at least two coding regions are single stranded, and, optionally, G contains T, wherein T is an attachment oligonucleotide that is from 0 to about 120 bases from a 3′ end of G, wherein T is at least partially single stranded;
- L is a linker that operatively links G to (B)K;
- B is a positional building block and K represents an integer from 1 to about 20;
- Q is a non-positional building block directly attached to B at position K;
- and
- U is an oligonucleotide that is capable of hybridizing to T or U is a chromatography agent;
- wherein each positional building block B at position K is identified by one of the coding regions.
6 more elements
A method of forming a molecule of formula (I) comprising:
- reacting a molecule of formula (II),
- G-L-(B)K (II)
- with a molecule of formula (III),
- Q-U (III)
- to form a molecule of formula (I),
- G-L-(B)K-Q-U, (I)
- wherein
- G includes an oligonucleotide, the oligonucleotide comprising at least two coding regions, wherein the at least two coding regions are single stranded, and, optionally, G contains T, wherein T is an attachment oligonucleotide that is from 0 to about 120 bases from a 3′ end of G, wherein T is at least partially single stranded;
- L is a linker that operatively links G to (B)K;
- B is a positional building block and K represents an integer from 1 to about 20;
- Q is a non-positional building block directly attached to B at position K;
- and
- U is an oligonucleotide that is capable of hybridizing to T or is an affinity chromatography agent;
- wherein each positional building block B at position K is identified by one of the coding regions.
11 more elements
A method of purifying molecules comprising:
- providing a pool of molecules containing at least one molecule of formula (I),
- G-L-(B)K-Q-U (I)
- wherein
- G includes an oligonucleotide, the oligonucleotide comprising at least two coding regions, wherein the at least two coding regions are single stranded, and, optionally, G contains T, wherein T is an attachment oligonucleotide that is from 0 to about 120 bases from a 3′ end of G, wherein T is at least partially single stranded;
- L is a linker that operatively links G to (B)K;
- B is a positional building block and K represents an integer from 1 to about 20;
- Q is a non-positional building block directly attached to B at position K;
- and
- U is an oligonucleotide that is capable of hybridizing to T or U is a chromatography agent;
- wherein each positional building block B at position K is identified by one of the coding regions;
- provided that U is an oligonucleotide, removing contaminants by annealing the oligonucleotide U to T to form a loop structure, performing polymerase chain reaction (PCR) to form a double stranded pool of molecules of formula (I), wherein G is double stranded, and adding a selection agent to the double stranded pool of molecules of formula (I), or
- provided that U is a chromatography agent, removing contaminants from the molecules of formula (I) by performing at least one of affinity, immobilized metal ion, metal chelation, size exclusion, normal, and reverse phase chromatography.
9 more elements
A method for analyzing synthetic yield comprising:
- reacting a molecule of formula (II),
- G-L-(B)K (II)
- with a molecule of formula (III),
- Q-U (III)
- to form a molecule of formula (I),
- G-L-(B)K-Q-U, (I)
- wherein
- G includes an oligonucleotide, the oligonucleotide comprising at least two coding regions, wherein the at least two coding regions are single stranded, and, optionally, G contains T, wherein T is an attachment oligonucleotide that is from 0 to about 120 bases from a 3′ end of G, wherein T is at least partially single stranded;
- L is a linker that operatively links G to (B)K;
- B is a positional building block and K represents an integer from 1 to about 20;
- Q is a non-positional building block directly attached to B at position K;
- and
- U is an oligonucleotide that is capable of hybridizing to T or U is a chromatography agent;
- wherein each positional building block B at position K is identified by one of the coding regions in G;
- purifying molecules for formula (I) from unreacted molecules of formula (II) by a method comprising:
- provided that U is an oligonucleotide, annealing the oligonucleotide U to T to form a loop structure;
- performing polymerase chain reaction (PCR) to form a double stranded pool of molecules of formula (I), wherein G is double stranded;
- and adding a selection agent to the double stranded pool of molecules of formula (I);
- or
- provided that U is a chromatography agent, performing at least one of affinity, size exclusion, normal, or reverse phase chromatography configured to select for the chromatography agent;
- and
- amplifying at least one oligonucleotide G to form a pool of post-reaction copies of G.
19 more elements
Dependencies are inferred from the claim text.