Assay for Oligonucleotides in Serum Without Extraction or RT-PCR

Tech ID: 29566 / UC Case 2016-359-0

Background

There is increasing interest in the use of oligonucleotides for both therapeutic and diagnostic purposes for a variety of diseases, including cancer.  Current methods for their detection, necessary for monitoring treatment or disease progression are limited by several factors, most often including the need for amplification via PCR. For all samples, PCR requires extraction from biological fluids and, for therapeutics, such amplification is not compatible with oligonucleotide modifications designed to increase serum half-life and cell penetration.

Brief Description

Prof. Ameae Walker’s laboratory at the University of California, Riverside (UCR) has developed an assay to quantify oligos in sub-picomole amounts without the need for sample purification and amplification. This new competitive assay is called an ELOHA (Enzyme-Linked Oligonucleotide Hybridization Assay).

The method is illustrated in Fig. 1, below.  Capture Oligos that are to hybridize with an oligo to be measured are covalently linked to a plate (1), a Detection Oligo, with the same sequence as the oligo to be measured, has a conjugated label, such as horseradish peroxidase or biotin.  The Detection Oligo then competes with the oligo of interest for binding to the Capture Oligo (2).  Once the hybridization is complete, the unbound oligos are washed away (3).  A colorimetric readout is produced (4) to inversely quantify the oligo of interest.

ameae 1

 Fig. 1 Schematic of the ELOHA assay

ameae 2

Fig. 2 shows the use of an ELOHA for amounts of Antimaia in mouse serum. Antimaia is a splice modulating oligomer therapy for breast cancer developed in the UCR lab of Prof. Walker.


 

Advantages

  • Inexpensive, efficient, sensitive, specific, reproducible method to detect oligonucleotides directly in serum/plasma
  • Method may be used to measure morpholino oligonucleotides and oligonucleotides with 3’ cell-penetrating peptides or octaguanidine derivatizations, as well as natural sequences such as miRNA.

Applications

  • Drug development
  • Pharmacokinetics
  • Monitoring pathophysiology and treatment efficacy in a variety of diseases. 

Patent Status

Country Type Number Dated Case
United States Of America Published Application 20190136302 05/09/2019 2016-359
 

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Keywords

oligonucleotide therapy, splice modulating oligomer, antimir, diagnosis, biomarker, siRNA, miRNA, oligos, oligonucleotide, morpholinos, Vivo-Morpholinos

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