METHODS FOR PREPARING MODIFIED BIOMOLECULES, MODIFIED BIOMOLECULES AND METHODS FOR USING SAME

• US 20090186343A1
• Filed: 02/21/2008
• Published: 07/23/2009
• Est. Priority Date: 02/21/2007
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: VisiGen Biotechnologies, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Stevens, Benjamin, Wang, Hongyi, Bryant, Amy, Lincecum, Tommie JR., Williams, Amy, Hardin, Susan H., Belosludtsev, Yuri, Deluge, Norha, Kincaid, Kristi, Fa, Ming
• Application Number:
  US12/035,352
• Publication Number:
  US 20090186343A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

A novel and efficient single pot synthetic schemes are disclosed for preparing modified nucleotides, nucleotide analogs, nucleotide polyphosphates, and nucleotide polyphosphate analogs. The novel method is used to prepare nucleotides, nucleotide analogs, nucleotide polyphosphates, and nucleotide polyphosphate analogs having non-persistent or persistent and non-persistent modifications. Novel biomolecular reactions are also disclosed using the novel modified biomolecules disclosed herein.

Claims

1. A method for preparing a modified nucleotide comprising the step of:

contacting a biomolecule having a phosphate group and a modifying agent having a leaving group capable of being displaced by the phosphate group under displacement reaction conditions to form a phosphate modified biomolecule.

2. The method of claim 1, wherein the nucleotides, phosphorylated polypeptides, phosphorylated proteins, phosphorylated sugars or sacchrides, phosphorylated carbohydrates, phosphorylated enzymes, phosphorylated membranes, phosphorylated cells, phosphorylated tissues, phospholipids or any other bio-material or organized structure bearing at least one phosphate group or mixtures or combinations thereof.

3. The method of claim 1, wherein the modifying agent comprises:

a molecule having one attachment site or a plurality of attachment sites, each site bearing a leaving group.

4. The method of claim 3, wherein the molecule comprises a molecular core and the site or sites extend out from the core.

5. The method of claim 3, wherein:

the biomolecule comprises a nucleotide comprising a nucleoside or nucleoside analog having at least one phosphate group attached at its 5′

hydroxy group, and

the core comprises (a) one quencher or a plurality of quenchers, or (b) an acceptor fluorophore or a plurality of acceptor fluorophore, or (c) an acceptor fluorophore or a plurality of acceptor fluorophore and a donor fluorophore or a plurality of donor fluorophore for the acceptor or acceptors.

6. The method of claim 5, wherein the core is selected from the group consisting of bi-functional molecules, polyfunctional molecules, polyfunctional boron-nitride nanostructures, polyfunctional carbon nanostructures, polyfunctional dendrimers, polyfunctional oligomers, polyfunctional polymers, polyfunctional metal oxide nanostructures, polyfunctional quantum dots, polyfunctional metal clusters, polyfunctional nanoshells, polyfunctional liposomes, or any other structure that can support attachment of a plurality of nucleotides through their terminal phosphate or mixtures of combinations thereof.

7. The method of claim 5, wherein the quenchers are the same or different, the acceptor fluorophores are the same or different or the acceptor fluorophores are the same or different and the donor fluorophores for the acceptors are the same of different.

8. A method for preparing a modified nucleotide comprising the steps of:

contacting a biomolecule having a phosphate group and a linker having a reactive site protected by a protecting group and a leaving group capable of being displaced by the phosphate group under displacement reaction conditions to form a phosphate protected linker modified biomolecule, and

de-protecting the terminal phosphate protected linker modified biomolecule to form a terminal phosphate reactive linker modified biomolecule.

9. The method of claim 8, further comprising the step of:

contacting the terminal phosphate reactive linker modified biomolecule with a modifying agent to form biomolecule having a linker attached to its terminal phosphate group and a modifying agent attached to the reactive site of the linker,

where the modifying agent has a detectable property or is capable of interfering with, quenching, augmenting, reducing or enhancing a detectable property of an external biomolecule, biomolecular complex or biomolecular assembly.

10. The method of claim 9, wherein the nucleotides, phosphorylated polypeptides, phosphorylated proteins, phosphorylated sugars or sacchrides, phosphorylated carbohydrates, phosphorylated enzymes, phosphorylated membranes, phosphorylated cells, phosphorylated tissues, phospholipids or any other bio-material or organized structure bearing at least one phosphate group or mixtures or combinations thereof and wherein the linker is a compound having a formula Q-E-R-E′

-Q′

, where Q is a leaving group, E and E′

are B, C, Si, Ge, N, P, As, O, S, and/or Se atom-containing moieties, Q′

is a leaving group or a protecting or blocking group, and R is an alkenyl group, an arenyl group, an aralkenyl group and/or a alkarenyl group.

11. The method of claim 10, wherein the modifying agent comprises:

a molecule having one attachment site or a plurality of attachment sites, each site bearing a leaving group.

12. The method of claim 11, wherein the molecule comprises a molecular core and the site or sites extend out from the core.

13. The method of claim 11, wherein:

the biomolecule comprises a nucleotide comprising a nucleoside or nucleoside analog having at least one phosphate group attached at its 5′

hydroxy group, and

the core comprises (a) one quencher or a plurality of quenchers, or (b) an acceptor fluorophore or a plurality of acceptor fluorophore, or (c) an acceptor fluorophore or a plurality of acceptor fluorophore and a donor fluorophore or a plurality of donor fluorophore for the acceptor or acceptors.

14. The method of claim 11, wherein the core is selected from the group consisting of bi-functional molecules, polyfunctional molecules, polyfunctional boron-nitride nanostructures, polyfunctional carbon nanostructures, polyfunctional dendrimers, polyfunctional oligomers, polyfunctional polymers, polyfunctional metal oxide nanostructures, polyfunctional quantum dots, polyfunctional metal clusters, polyfunctional nanoshells, polyfunctional liposomes, or any other structure that can support attachment of a plurality of nucleotides through their terminal phosphate or mixtures of combinations thereof.

15. The method of claim 11, wherein the quenchers are the same or different, the acceptor fluorophores are the same or different or the acceptor fluorophores are the same or different and the donor fluorophores for the acceptors are the same of different.

16. A biomolecular composition comprising:

a molecular core,

a first plurality of attachment sites extending out from the core,

a second plurality of biomolecules, each biomolecule including a phosphate-containing group,

where each biomolecule is attached to an attachment site through a direct bond to a terminal phosphate moiety of the phosphate-containing group or through a linker interposed between the site and the terminal phosphate moiety of the phosphate-containing group.

17. The composition of claim 16, wherein:

the biomolecule comprises a nucleotide comprising a nucleoside or nucleoside analog having at least one phosphate group attached at its 5′

hydroxy group, and

the core includes;

(a) one quencher or a plurality of quenchers, or (b) an acceptor fluorophore or a plurality of acceptor fluorophore, or (c) an acceptor fluorophore or a plurality of acceptor fluorophore and a donor fluorophore or a plurality of donor fluorophore for the acceptor or acceptors, where the quenchers are the same or different, the acceptor fluorophores are the same or different or the acceptor fluorophores are the same or different and the donor fluorophores for the acceptors are the same of different.

18. The composition of claim 16, wherein the core is selected from the group consisting of bi-functional molecules, polyfunctional molecules, polyfunctional boron-nitride nanostructures, polyfunctional carbon nanostructures, polyfunctional dendrimers, polyfunctional oligomers, polyfunctional polymers, polyfunctional metal oxide nanostructures, polyfunctional quantum dots, polyfunctional metal clusters, polyfunctional nanoshells, polyfunctional liposomes, or any other structure that can support attachment of a plurality of nucleotides through their terminal phosphate or mixtures of combinations thereof.

19. The composition of claim 16, wherein the biomolecules are the same or different.

20. The composition of claim 16, wherein the linkers are the same or different and are compounds having a formula Q-E-R-E′

-Q′

, where Q is a leaving group, E and E′

are B, C, Si, Ge, N, P, As, O, S, and/or Se atom-containing moieties, Q′

is a leaving group or a protecting or blocking group, and R is an alkenyl group, an arenyl group, an aralkenyl group and/or a alkarenyl group.

21. A method for sequencing comprising the steps of:

providing a biomolecular composition comprising;

a molecular core,

first plurality of attachment sites extending out from the core,

a second plurality of biomolecules, each biomolecule including a phosphate-containing group,

where each biomolecule is attached to an attachment site through a direct bond to the site an a terminal phosphate moiety of the phosphate-containing group or through a linker interposed between the site and the terminal phosphate moiety of the phosphate-containing group,

providing a sequencing solution including a polymerizing agent, a primer-template duplex, and sequencing buffer,

detecting a plurality of detectable events evidencing binding and/or nucleotide incorporation events, and

analyzing the events to determine a sequence of nucleotide incorporations complementary to a sequence on the template.

22. The method of claim 21, wherein:

the biomolecule comprises a nucleotide comprising a nucleoside or nucleoside analog having at least one phosphate group attached at its 5′

hydroxy group, and

the core includes;

(a) one quencher or a plurality of quenchers, or (b) an acceptor fluorophore or a plurality of acceptor fluorophore;

(c) an acceptor fluorophore or a plurality of acceptor fluorophore and a donor fluorophore or a plurality of donor fluorophore for the acceptor or acceptors, where the quenchers are the same or different, the acceptor fluorophores are the same or different or the acceptor fluorophores are the same or different and the donor fluorophores for the acceptors are the same of different.

23. The method of claim 21, wherein the core is selected from the group consisting of bi-functional molecules, polyfunctional molecules, polyfunctional boron-nitride nanostructures, polyfunctional carbon nanostructures, polyfunctional dendrimers, polyfunctional oligomers, polyfunctional polymers, polyfunctional metal oxide nanostructures, polyfunctional quantum dots, polyfunctional metal clusters, polyfunctional nanoshells, polyfunctional liposomes, or any other structure that can support attachment of a plurality of nucleotides through their terminal phosphate or mixtures of combinations thereof.

24. The method of claim 21, wherein the biomolecules are the same or different.

25. The composition of claim 21, wherein the linkers are the same or different and are compounds having a formula Q-E-R-E′

-Q′

, where Q is a leaving group, E and E′

are B, C, Si, Ge, N, P, As, O, S, and/or Se atom-containing moieties, Q′

is a leaving group or a protecting or blocking group, and R is an alkenyl group, an arenyl group, an aralkenyl group and/or a alkarenyl group.