Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Jul 6;133(26):10111-8.
doi: 10.1021/ja200212b. Epub 2011 Jun 9.

Defining the RNA internal loops preferred by benzimidazole derivatives via 2D combinatorial screening and computational analysis

Sai Pradeep Velagapudi  1 , Steven J Seedhouse, Jonathan French, Matthew D Disney
Affiliations

Defining the RNA internal loops preferred by benzimidazole derivatives via 2D combinatorial screening and computational analysis

Sai Pradeep Velagapudi et al. J Am Chem Soc. .

Abstract

RNA is an important therapeutic target; however, RNA targets are generally underexploited due to a lack of understanding of the small molecules that bind RNA and the RNA motifs that bind small molecules. Herein, we describe the identification of the RNA internal loops derived from a 4096 member 3 × 3 nucleotide loop library that are the most specific and highest affinity binders to a series of four designer, druglike benzimidazoles. These studies establish a potentially general protocol to define the highest affinity and most specific RNA motif targets for heterocyclic small molecules. Such information could be used to target functionally important RNAs in genomic sequence.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The secondary structures of the RNAs used in this study. Oligonucleotide 1 is the 3×3 nucleotide internal loop library. Oligonucleotides 2-6 are competitor oligonucleotides used to constrain selected interactions to the randomized region in 1. Oligonucleotide 7 is the cassette into which the 3×3 nucleotide library was inserted.
Figure 2
Figure 2
Top, structures of the bis-benzimidazoles and benzimidazoles that were conjugated onto alkyne-agarose microarray surfaces via HDCR. Bottom, microarray surface after hybridization with internally labeled library 1 and competitor oligonucleotides 2-6. Spots of ligand-bound RNAs that were excised are indicated with a circle. The moles of ligand spotted are 1.5, 0.9, 0.5, 0.3, and 0.1 nmoles. A, array prior to excision of bound RNA. B, array after excision of bound RNA.
Figure 3
Figure 3
Venn diagram that illustrates the types of unique and overlapping RNA sequence spaces selected to bind 8, 9, and 11. The Venn diagram analysis was enabled by using the RNA-PSP v 2.0 program. Data for compound 8 are shown at the 95% confidence interval and data for compounds 9 and 11 are shown at the 99% confidence interval. Structures that are listed as “pairs” refer to two nucleotides that are across from each other in the selected structures.
Figure 4
Figure 4
The secondary structures of a subset of RNA loops selected to bind 8, 9, and 11 via 2DCS. Secondary structures were predicted by the RNAstructure program. The nomenclature for the loops refers to the ligand that the loops were selected to bind followed by an internal loop (IL) number. Values below the loop identifier are the Kd's for the RNA-ligand complex (nM). None of the compounds binds to cassette 7 or library 1, indicating that binding is specific to the selected randomized regions.
Figure 5
Figure 5
StARTS plots for 8, 9, and 11. Filled circles indicate structures to which affinities were measured (Figure 4). Open circles indicate predicted affinities for members of 1 with sum Z-score values in the top ~10%.
Figure 6
Figure 6
StARTS plots containing all library members for each ligand. Each plot depicts the sum Z-scores for each member of 1 as a function of affinity. Top, selection for 8; middle, selection for 9; and bottom, selection for 11. The open or colored circles are the RNA motif-ligand partners shown in Figure 5. A library member refers to the calculated sum Z-score for the entire 4,096-member library 1 based on the analysis of selected structures for binding a given ligand. As noted in the text, compounds that have lower relative sum Z-scores are weaker binders. Thus, by comparing the position of an RNA in each StARTS plots, the specificity of the RNA-ligand interactions can be estimated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Blount KF, Breaker RR. Nat Biotechnol. 2006;24:1558. - PubMed
    1. Calin GA, Croce CM. Oncogene. 2006;25:6202. - PubMed
    1. Poehlsgaard J, Douthwaite S. Nat Rev Microbiol. 2005;3:870. - PubMed
    1. Fedor MJ, Williamson JR. Nat Rev Mol Cell Biol. 2005;6:399. - PubMed
    1. Thomas JR, Hergenrother PJ. Chem Rev. 2008;108:1171. - PubMed

Publication types