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BioAssay: AID 651821

Fluorescence-based biochemical primary high throughput screening assay to identify molecules that bind r(CAG) RNA repeats

Name: Fluorescence-based biochemical primary high throughput screening assay to identify molecules that bind r(CAG) RNA repeats. ..more
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AID: 651821
Data Source: The Scripps Research Institute Molecular Screening Center (R(CAG)_INH_FLINT_1536_1X%INH PRUN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-11-28
Modify Date: 2013-04-25

Data Table ( Complete ):           Active    All
BioActive Compounds: 4663
Depositor Specified Assays
651825Summary of the probe development effort to identify molecules that bind r(CAG) RNA repeatssummary
652065Fluorescence-based biochemical high throughput confirmation assay to identify molecules that bind r(CAG) RNA repeatsscreening
652068Counterscreen for molecules that bind rCAG RNA repeats: fluorescent based biochemical counterscreen assay for inhibitors of the DNA-based (5'CAG/3'GTC) TO-PRO-1 dye complexscreening
686959Fluorescence-based biochemical high throughput dose response assay to identify molecules that bind r(CAG) RNA repeatsconfirmatory
686960Counterscreen for molecules that bind rCAG RNA repeats: fluorescent based biochemical dose response assay for inhibitors of the DNA-based (5'CAG/3'GTC) TO-PRO-1 dye complexconfirmatory
720649On Hold
720651On Hold
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Matthew Disney, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA033977-010
Grant Proposal PI: Matthew Disney
External Assay ID: R(CAG)_INH_FLINT_1536_1X%INH PRUN

Name: Fluorescence-based biochemical primary high throughput screening assay to identify molecules that bind r(CAG) RNA repeats.


RNA plays a critical role in a host of diseases and can thus serve as an important drug target. The vast majority of RNA targets, however, have not been utilized in targeting endeavors. This is due to a lack of information on the RNA motifs that bind small molecules, such as internal and hairpin loops. Translation of mutant mRNA into protein causes disease. For example, translation of mutant Huntington mRNA, which has expanded r(CAG) repeats, produces a polyQ (glutamine)-containing form of Huntington protein that aggregates in cells and causes toxicity (1, 2). Aggregates form structures that cause cellular dysfunction, mitochondrial impairment, and cell death, resulting in Huntington's Disease (HD) (3). Both mutant and wild type alleles that produce Huntington mRNA are present in individuals affected with HD. An ideal therapeutic for treating HD, therefore, would inhibit translation of mutant but not wild type Huntington. Small molecules that target the specific structure of expanded r(CAG) repeats (4), not just the sequence, and knock down translation of mutant Huntington could provide more potent and specific HD therapies.

RNAs can also contribute to disease directly without being translated into protein. For example, expanded triplet-repeating transcripts cause a toxic RNA gain-of-function. This mechanism has been most thoroughly documented in myotonic dystrophy type 1 (DM1) (5, 6), where expansions of r(CUG) in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) transcript fold into an RNA hairpin and bind the pre-mRNA splicing regulator muscleblind-like 1 protein (MBNL1) (6-8), leading to pre-mRNA splicing defects that lead to muscle weakness, insulin insensitivity, and cardiac arrhythmia associated with DM1. Gain-of-function by r(CAG) repeats has also been observed in models of spinocerebellar ataxia type 3 (SCA3) (5). Another goal of this proposal is to identify r(CAG) repeat binders that displace MBNL1 to correct splicing defects associated with long r(CAG) repeats in HD. These compounds would serve as chemical probes to further define pre-mRNA splicing defects and the pathology of HD and SCA3.


1. Orr, H. T., and Zoghbi, H. Y. (2007) Trinucleotide repeat disorders, Annu Rev Neurosci 30, 575-621
2. MacDonald, M. E., and Gusella, J. F. (1996) Huntington's disease: translating a CAG repeat into a pathogenic mechanism, Curr Opin Neurobiol 6, 638-643.
3. Bates, G. (2003) Huntingtin aggregation and toxicity in Huntington's disease, Lancet 361, 1642-1644
4. Michlewski, G., and Krzyzosiak, W. J. (2004) Molecular architecture of CAG repeats in human disease related transcripts, J Mol Biol 340, 665-679.
5. Caskey, C. T., Pizzuti, A., Fu, Y. H., Fenwick, R. G., Jr., Nelson, D. L., and Kuhl, D. P. (1992) Triplet repeat mutations in human disease, Science 256, 784-789.
6. Mankodi, A., Logigian, E., Callahan, L., McClain, C., White, R., Henderson, D., Krym, M., and Thornton, C. A. (2000) Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat, Science 289, 1769-1773.
7. Kanadia, R. N., Johnstone, K. A., Mankodi, A., Lungu, C., Thornton, C. A., Esson, D., Timmers, A. M., Hauswirth, W. W., and Swanson, M. S. (2003) A muscleblind knockout model for myotonic dystrophy, Science 302, 1978-1980.
8. Wheeler, T. M., Sobczak, K., Lueck, J. D., Osborne, R. J., Lin, X., Dirksen, R. T., and Thornton, C. A. (2009) Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA, Science 325, 336-339


Primary, primary screen, RNA, r(CAG), repeats, nucleotide, fluorescence, TO-PRO-1, dye, binding, loops, HD, SCA3, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Assay Overview:

The purpose of this assay is to identify compounds that target 1x1 nucleotide AA internal loops in an RNA oligonucleotide. This assay utilizes the fluorescent dye TO-PRO-1 that has increased fluorescence emission upon binding RNA. In this assay, RNA nucleotide containing 1x1 AA internal loops is incubated with TO-PRO-1 and test compounds. After incubation, well fluorescence intensity is measured. As designed, test compounds that bind to the 1x1 AA loops and displace the dye will result in an increase in free (unbound) TO-PRO-1 in the well, leading to lower fluorescence intensity. Compounds are tested in singlicate at a final nominal concentration of 3 uM.

Protocol Summary:

Prior to the start of the assay, prepare AA-RNA (rCAG) in assay buffer (8 mM Na2PO4 pH 7.0, 18 5mM NaCl, 0.1 mM EDTA, and 5% DMSO) at a final concentration of 1.5 uM. Heat at 60 C for 5 minutes. Allow to cool down for 15 minutes at room temperature. Add TO-PRO-1 to a final concentration of 100 nM in the rCAG solution. Plate 5 ul of rCAG/TO-PRO-1 solution to 1536 well plates a briefly spin down.

The assay was started by dispensing 15 nL of test compound solubilized in DMSO or DMSO alone (5.3% final concentration) to the appropriate wells. Incubate at room temperature for 15 minutes. Read fluorescence on Envision.

Fluorescence was read on the Envision (Perkin Elmer) using a filter set (Excitation = 485 nm, Emission = 535 nm).

The percent inhibition for each compound was calculated as follows:

%_Inhibition = ( Test_Compound_RFU - Median_Low_Control_RFU ) / ( Median_High_Control_RFU - Median_Low_Control_RFU ) * 100


Test_Compound is defined as wells containing rCAG/TO-PRO-1 mix with test compound in DMSO.
Low_Control is defined as wells containing rCAG/TO-PRO-1 mix in the presence of DMSO.
High_Control is defined as wells containing rCAG/TO-PRO-1 mix + 100uM Mitoxantrone in DMSO.

PubChem Activity Outcome and Score:

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-34, for inactive 34-0.

List of Reagents:

AARNA (rCAG) - C,G,C,A,G,C,G,G,A,A,A,C,G,C,A,G,C,G Sequence (Dharmacon FERVC-000001)
TO-PRO-1 (Invitrogen, part T3602)
Sodium phosphate dibasic (Sigma, part S3397)
EDTA (Fisher, part BP121-500)
NaCl (Fisher, part 7647-15-5)
1536-well plates (Greiner, part 789176)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 3 uM (3μM**)Normalized percent inhibition of the primary screen at a compound concentration of 3.00 micromolar.Float%

** Test Concentration.
Additional Information
Grant Number: 1 R03 DA033977-010

Data Table (Concise)