Multimer glycosylated nucleic acid binding protein conjugates and uses thereof

• US 8,951,736 B2
• Filed: 04/25/2012
• Issued: 02/10/2015
• Est. Priority Date: 04/29/2011
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Schmidt, Karsten
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US13/456,179
• Publication Number:
  US 20120276548A1
• Time in Patent Office: 1,021 Days
• US Class Current: 435/6.19
• CPC Class Codes: C07K 14/44 from protozoa
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

The technology relates in part to multimer conjugates comprising a scaffold linked to two or more polypeptides that specifically interact with a nucleic acid containing beta-D-glucosyl-hydroxymethylcytosine or beta-D-glucosyl-hydroxymethyluracil. The scaffold can be chosen from an antibody, an antibody fragment, a multimerized binding partner that interacts with a binding partner counterpart in each of the polypeptides, a polymer, and a polyfunctional molecule. The polypeptides can be from a kinetoplastid flagellate organism and may comprise a full-length native or modified protein or a fragment thereof that specifically interacts with the beta-D-glucosyl-hydroxymethylcytosine and/or the beta-D-glucosyl-hydroxymethyluracil in the nucleic acid. The conjugates provided herein can be used to detect the presence, absence or amount of beta-D-glucosyl-hydroxymethylcytosine and/or beta-D-glucosyl-hydroxymethyluracil-containing nucleic acid in a sample.

Claims

1. A method for detecting the presence, absence or amount of nucleic acid containing beta-D-glucosyl-hydroxymethylcytosine and/or a nucleic acid containing beta-D-glucosyl-hydroxymethyluracil in a sample, comprising:

contacting the sample that may contain nucleic acid containing beta-D-glucosyl-hydroxymethylcytosine and/or a nucleic acid containing beta-D-glucosyl-hydroxymethyluracil with a multivalent multimer conjugate comprising two or more polypeptides selected from the group consisting of a full-length native J-DNA binding protein (JBP) or substantially identical polypeptide thereof, a J-DNA binding fragment of a full-length native JBP or substantially identical polypeptide thereof and a polypeptide comprising the J-DNA binding domain of a JBP or substantially identical polypeptide thereof, wherein each polypeptide in the multimer is directly conjugated to a scaffold, wherein the scaffold is an antibody or an antibody fragment, which polypeptides specifically interact with a nucleic acid containing beta-D-glucosyl-hydroxymethylcytosine and/or a nucleic acid containing beta-D-glucosyl-hydroxymethyluracil; and

determining the presence, absence or amount of the multimer that specifically interacts with the nucleic acid in the sample, whereby the presence, absence or amount of nucleic acid containing beta-D-glucosyl-hydroxymethylcytosine and/or a nucleic acid containing beta-D-glucosyl-hydroxymethyluracil in a sample is determined.

2. The method of claim 1, which is performed in vitro.

3. The method of claim 1, wherein the two or more polypeptides specifically interact with a nucleic acid containing beta-D-glucosyl-hydroxymethylcytosine.

4. The method of claim 1, wherein the two or more polypeptides specifically interact with a nucleic acid containing beta-D-glucosyl-hydroxymethyluracil.

5. The method of claim 1, wherein the scaffold is coupled to a solid support.

6. The method of claim 1, wherein the antibody fragment is an Fc portion of an antibody.

7. The method of claim 1, wherein one or more of the two or more polypeptides comprise one or more substantially identical polypeptides, wherein the one or more substantially identical polypeptides comprise modified JBP that specifically interacts with the beta-D-glucosyl-hydroxymethylcytosine in the nucleic acid and/or the beta-D-glucosyl-hydroxymethyluracil in the nucleic acid, which modified protein comprises one or more amino acid modifications to a full-length native JBP or to a J-DNA binding fragment of a full-length native JBP.

8. The method of claim 1, wherein one or more of the two or more polypeptides comprise one or more full-length native JBP from a kinetoplastid flagellate organism, a J-DNA binding fragment of a full-length native JBP from a kinetoplastid flagellate organism, or a polypeptide comprising the J-DNA binding domain of a JBP from a kinetoplastid flagellate organism, or substantially identical polypeptides thereof.

9. The method of claim 8, wherein the kinetoplastid flagellate organism is selected from the group consisting of Trypanosoma spp. organism, Leishmania spp. organism, Crithidia spp. organism and Euglena spp. organism.

10. The method of claim 8, wherein the native JBP comprises a polypeptide sequence selected from the group consisting of SEQ ID NOs:

1 to 11 and 12.

11. The method of claim 8, wherein a fragment of the native JBP comprises amino acids 382 to 561 of SEQ ID NO:

1.

12. The method of claim 8, wherein the native JBP or a fragment of the native JBP comprises an alpha helix 4 of L. tarentolae polypeptide.

13. The method of claim 1, wherein the scaffold, or portion thereof, and the polypeptides that specifically interact with a nucleic acid containing beta-D-glucosyl-hydroxymethylcytosine and/or a nucleic acid containing beta-D-glucosyl-hydroxymethyluracil contain amino acids and are contiguous.