Conversion of alpha-hydroxyalkylated residues in biomolecules using methyltransferases

• US 9,505,797 B2
• Filed: 11/17/2014
• Issued: 11/29/2016
• Est. Priority Date: 04/02/2009
• Status: Active Grant

• Current Assignee: Vilnius University
• Original Assignee: Vilnius University
• Inventors: Klimasauskas, Saulius, Liutkeviciute, Zita, Kriukiene, Edita
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/542,770
• Publication Number:
  US 20150093752A1
• Time in Patent Office: 743 Days
• Field of Search: 536/23.1, 536/24.1, 435/6.1
• US Class Current: 1/1
• 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 1/48 involving transferase
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/125 Methyl transferase, i.e. methylase
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 2333/91011 Methyltransferases (general) (2.1.1.)

Abstract

The present invention relates to targeted conversion of alpha-hydroxyalkylated residues in biomolecules in the presence of a directing methyltransferase, namely to targeted removal of the alpha-hydroxyalkyl moieties to give unmodified residues, or targeted derivatization of the alpha-hydroxyalkyl groups by covalent coupling of non-cofactor compounds represented by formula HQ-LX, wherein X represents a functional group or a reporter group attached via a linker moiety L, and QH is selected from HS—, HSe—, HO—H₂N—, HN₃ or HCN in the presence of a directing methyltransferase. Further development of the method of targeted conversion comprises methods for targeted labeling a biomolecule and method for detecting hydroxymethylated target sites in a biomolecule according to the present invention.

Claims

1. A modified nucleic acid comprising a -Q-LX group in place of a hydroxy group at the 5 position of a hydroxymethylcytosine or a hydroxymethyluracil residue, where X represents a functional group and/or a reporter group attached by linker L to Q, and Q is selected from the group consisting of S, Se, O, N, and C.

2. The modified nucleic acid of claim 1 where linker L is selected from the group consisting of —

CH₂CH(CO2H)—

, (poly)ethyleneglycol chains (CH₂CH₂O)_n where n=1-100, a covalent bond, and a linear, cyclic, and/or aromatic alkyl optionally comprising one or more of NHCO, O, and S.

3. The modified nucleic acid molecule of claim 1 where X comprises at least one reactive group selected from the group consisting of a primary amino group, a thiol group, a 1,2-diol group, a haloacetamide group, a maleimide group, an aldehyde group, a ketone group, an azido group, an alkyne group, a 1,3-diene function, a dienophilic function, an arylhalide group, a terminal alkyne group, an arylboronic acid group, a terminal haloalkyne group, a terminal silylalkyne group and a protected amino, thiol, 1,2-diol, hydrazino, hydroxyamino, aldehyde, ketone, and 1,2-aminothiol group.

4. The modified nucleic acid molecule of claim 1 where X comprises at least one reporter selected from the group consisting of a heavy atom or heavy atom cluster, a radioactive or stable rare isotope, a residue of a member selected from fluorophores, fluorescence quenchers, or chromophores, an affinity label, a spin label, a group containing a radioactive or stable rare isotope, a group containing a heavy atom, a crosslinking agent, a nucleic acid cleaving group, a hapten, a nanoparticle, a bead, and combinations thereof.

5. The modified nucleic acid molecule of claim 1 where -LX is selected from the group consisting of —

CH₂CH₂OH, —

CH₂CH(CO₂H)NH₂, -5′

-deoxyadenosine, —

CH₂CH₂NH₂, —

CH₂CH(OH)CH(OH)CH₂SH, and —

OH.

6. The modified nucleic acid molecule of claim 1 where the nucleic acid is DNA, RNA, or DNA/RNA hybrids.

7. The modified nucleic acid molecule of claim 1 where the DNA is single stranded or double stranded.

8. The modified nucleic acid molecule of claim 1 where the modified nucleic acid molecule comprises a 5-(2-hydroxyethylthio)methyl-2′

-deoxycytidine, 5-(S-cysteinyl)methyl-2′

-deoxycytidine, 5-(5′

-adenosyl)thiomethyl-2′

-deoxycytidine, 5-(2-aminoethyl)thiomethyl-2′

-deoxycytidine, 5-(2,3-dihydroxy-4-mercaptobutyl)thiomethy1-2′

-deoxycytidine, 5-(2-amino-2-carboxyethyl)selenomethy1-2′

-deoxycytidine, 5-(2-aminoethyl)selenomethy1-2′

-deoxycytidine, and/or 5-hydroxylaminomethyl-2′

-deoxycytidine.

9. The modified nucleic acid molecule of claim 1 where the hydroxymethyl group of the hydroxymethylcytosine or the hydroxymethyluracil is —

CH(OH)—

R, where R is H, a C₁-C₁₂ alkyl, an alkenyl, or an alkynyl.

10. The modified nucleic acid molecule of claim 6, wherein the DNA is genomic DNA.

11. The modified nucleic acid of claim 1 where the modified nucleic acid is fragmented.

12. The modified nucleic acid of claim 11, where the fragmentation is random.

13. The modified nucleic acid of claim 11 where the fragmentation is performed by digestion with a restriction enzyme.

14. The modified nucleic acid of claim 1 in a composition further comprising a transferase.

15. The modified nucleic acid of claim 14 where the transferase is a methyltransferase.

16. The modified nucleic acid of claim 1 in a composition further comprising a solid support.

17. The modified nucleic acid of claim 16 where the solid support is a bead.

18. The modified nucleic acid of claim 1 in a composition further comprising at least one primer.

19. The modified nucleic acid of claim 1 further comprising a secondary modification as a result of

i) treatment with an UDP-glucose:

DNA D-glucosyltransferase and UDP-glucose leading to targeted incorporation of D-glucose into the modified nucleic acid;

ii) treatment with a 5-hydroxymethylcytosine-DNA deaminase that converts 5-hydroxymethylcytosine into 5-hydroxymethyluridine;

or

iii) treatment with a 5-hydroxymethylcytosine-DNA glycosylase producing an abasic site in the modified nucleic acid.

20. A modified nucleic acid comprising a hydroxymethylcytosine or hydroxymethyluracil residue where -Q-LX replaces —

OH at the 5 position, where X represents a functional group and/or a reporter group attached by linker L to Q, and Q is selected from the group consisting of S, Se, O, N, and C, and further comprising a secondary modification of -Q-LX as a result of treatment with a UDP-glucose;

DNA D-glucosyltransferase and UDP-glucose leading to targeted incorporation of D-glucose into the modified nucleic acid.