Fluorescence-based biochemical high throughput confirmation assay to identify molecules that bind r(CAG) RNA repeats
Name: Fluorescence-based biochemical high throughput confirmation assay to identify molecules that bind r(CAG) RNA repeats. ..more
BioActive Compounds: 2966
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_3X%INH CRUN
Name: Fluorescence-based biochemical high throughput confirmation assay to identify molecules that bind r(CAG) RNA repeats.
Post-transcriptional control of gene expression, such as alternative splicing and tissue-specific silencing, allow for great protein diversity (1). The elements in the mRNA 3' untranslated regions (UTRs) influence the expression of genes involved in proliferation and differentiation of stem cells and germ cells (2). These elements are critical during spermatogenesis and oogenesis in hermaphrodite Caenorhabditis elegans nematodes (3). GLD-1 (defective in Germ Line Development ) is a germline-specific protein and a member of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins (4, 5), which are thought to link RNA metabolism to signal transduction pathways in invertebrates and vertebrates (4, 6). GLD-1 is found exclusively in the cytoplasm of germ cells (7), where it binds specifically to tra-2 mRNA GLI elements (TGEs) in the 3' UTRs of the mRNA produced by the sex determination gene, tra-2. Studies showing that gld-1 loss-of-function mutants make few or no sperm (8, 9), and that worm hermaphrodites with null mutations develop germ line tumors resulting from the inappropriate exit of germ cells from meiosis (7, 10), support a role for GLD-1 as a tumor suppressor, translational repressor, and important regulator of germ cell fate.
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
9. Clemons, P. A., Bodycombe, N. E., Carrinski, H. A., Wilson, J. A., Shamji, A. F., Wagner, B. K., Koehler, A. N., and Schreiber, S. L. (2010) Small molecules of different origins have distinct distributions of structural complexity that correlate with protein-binding profiles, Proc Natl Acad Sci U S A 107, 18787-18792.
10. Lee, M. M., Childs-Disney, J. L., Pushechnikov, A., French, J. M., Sobczak, K., Thornton, C. A., and Disney, M. D. (2009) Controlling the specificity of modularly assembled small molecules for RNA via ligand module spacing: targeting the RNAs that cause myotonic muscular dystrophy, J Am Chem Soc 131, 17464-17472.
CRUN, confirm, confirmation, triplicate, biochemical, 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.
The purpose of this assay is to confirm compounds identified in a previous set of experiments entitled, "Fluorescence-based biochemical primary high throughput screening assay to identify molecules that bind r(CAG) RNA repeats" (AID 651821) as active inhibitors of RNA:TO-PRO complex. 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 triplicate at a final nominal concentration of 3 uM.
Prior to the start of the assay, prepare AA-RNA (rCAG) in assay buffer (8 mM Na2PO4 pH 7.0, 18 5 mM 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 + 100 uM 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. The MLSMR was not able to provide all compounds selected for testing in this assay.
** Test Concentration.
Data Table (Concise)