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Multi-epitope tag (MET)-recombinant antibody toolbox for detection and manipulation of RNA modifications

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R43DA042463-01
Agency Tracking Number: R43DA042463
Amount: $224,995.00
Phase: Phase I
Program: SBIR
Solicitation Topic Code: 43
Solicitation Number: DA16-005
Solicitation Year: 2016
Award Year: 2016
Award Start Date (Proposal Award Date): 2016-08-15
Award End Date (Contract End Date): 2017-07-31
Small Business Information
Carlsbad, CA 92008-6509
United States
DUNS: 109145701
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 (760) 431-1263
Business Contact
Phone: (760) 431-1263
Research Institution

RNA plays a central role in numerous cellular processes Over RNA modifications have been
characterized of which twelve have been identified in messenger RNA mRNA With the exception of the
mRNA cap structure little is known about their function The discovery that the FTO gene initially linked to
obesity and energy homeostasis is an oxidative demethylase eraser of methyl adeonsine m A in RNA
has spurred interest in understanding the biological function of RNA modifications and how their dysregulation
may impact disease The identification of hydroxymethyl adenine hm A and formyl adensosine f A as
demethylation intermediates with the potential for independent biological function has resulted in the coining of
the terms epitranscriptome and epi modifications drawing comparison to the dynamic regulation of
methylcytosine m C and hydroxymethylcytosine hm C in DNA The epitranscriptome field is still in its
discovery phase with an unmet need for highly sensitive and specific reagents for the visualization isolation
and analysis of the biological function of RNA epi modifications This Phase I proposal intends to develop and
validate an RNA epi modification multi epitope tag MET recombinant antibody toolbox wherein the antibody
heavy chain is engineered to contain the sequences recognized by the protein ligase SortaseA and the biotin
ligase BirA thereby enabling targeted end user customizable labeling XHis tag is also present enabling
purification of the functionalized antibody under mild conditions SortaseA can be used to attach a large
repertoire of payloads ranging from fluorescent dyes to bioactive peptides Targeted biotinylation enables
leveraging of avidin streptavidin based technologies for maximizing detection sensitivity or isolation efficiency
in enrichment based detection methods Aim efforts will develop a specificity validation pipeline using
existing RNA modification antibodies and DNA modification antibodies whose targets are also found in RNA In
Aim recombinant MET antibodies will be generated to two RNA epi modifications using the Aim specificity
pipeline to identify highly specific antibodies Aim efforts will establish proof of concept for the targeted
conjugation of MET antibodies to a fluorophore using Sortase A or to biotin using BirA Future Phase II efforts
will refine these methodologies and will be expand to include development of kits and reagents for end user
customized antibody conjugates and assay systems for the visualization isolation and analysis of RNA epi
modifications in a variety of cell and tissue types These antibodies and kits will serve as enabling tools
allowing the discovery of the fundamental mechanisms that regulate RNA epigenetics and could eventually be
used in clinical or diagnostic applications Project Narrative
While the biological significance of chemical modifications found on RNA molecules and their interacting proteins is still
in its discovery phase already it is known that the dysfunction of these proteins are involved in obesity mental
retardation and possibly substance abuse This grant proposes to develop a highly specific and sensitive tool kit of
antibodies that will allow for the visualization isolation and study of these biomolecules which will further our
understanding of their role in human diseases

* Information listed above is at the time of submission. *

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