# New specific and rapid assay for in situ apoptosis labeling for cancer studies

> **NIH NIH R43** · VIVID TECHNOLOGIES · 2020 · $214,214

## Abstract

New specific and rapid assay for in situ apoptosis labeling for cancer studies
Abstract
In this project we will develop and validate in the context of commercial use a new express assay for
molecular pathology of cancer and in situ studies. The assay will selectively label apoptotic, but not necrotic
cells, based on detection of characteristic double-stranded DNA breaks produced by apoptotic executioner
nucleases. At present these biomarkers are considered highly specific for programmed cell death, but their
detection takes 24 hrs. The proposed technology will perform such specific detection within minutes,
permitting cost-effective high throughput in situ assessments of anticancer drugs and therapies. The assay
will use the unique properties of viral DNA topoisomerase, we synthesized de novo, starting from its
sequence in the genome of the giant Acanthamoeba polyphaga virus. This archaic enzyme sequence was
acquired by the virus at some point in its evolution from a now extinct animal species. We found that this
recombinant protein possesses extremely fast ligation activity. The topoisomerase can rapidly ligate DNA
ends, finishing the reaction within several seconds after addition to DNA. This surpasses the speed of all
known DNA ligases by two orders of magnitude. It permits a novel ultra-fast topoligation labeling of apoptotic
cells in tissue sections. This new assay will have high commercial potential and will offer advantages of
speed and specificity over the currently available conventional in situ approaches. It will be particularly useful
in research, where large-volume quantitations of programmed cell death cells are essential, such as in anti-
cancer pro-apoptotic drug development and in molecular pathology studies of tumors.
The Specific Aims of the proposal are:
1) To develop and validate in the context of commercial use the first express assay for specific detection of
apoptosis in histological sections. The assay will operate by the extra-fast topoligation labeling of blunt-ended
DNA breaks with terminal 5’PO4 produced by apoptotic executioner nucleases. To verify the new labeling
approach using in vitro models, and to ensure its reproducible and robust labeling, essential for the ease of
use and the future competitive advantage.
2) To validate the new express assay in the context of commercial use by using apoptotic models related to
cancer. To optimize it for the ease of use by potential customers by ensuring high and consistent speed of
detection, sensitivity, and specificity. Verify its general applicability in different samples including fixed cells
and tissue sections. Ensure high reproducibility of labeling results and high signal-to-noise ratio, providing
strong and unobscured labeling of specific DNA breaks in individual apoptotic cells in different cancer
samples.

## Key facts

- **NIH application ID:** 10008420
- **Project number:** 1R43CA250805-01
- **Recipient organization:** VIVID TECHNOLOGIES
- **Principal Investigator:** Sha Wang
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $214,214
- **Award type:** 1
- **Project period:** 2020-06-01 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10008420

## Citation

> US National Institutes of Health, RePORTER application 10008420, New specific and rapid assay for in situ apoptosis labeling for cancer studies (1R43CA250805-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10008420. Licensed CC0.

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