Mbl-aptamer

May 19, 2024 • Zhizhong Lu, Ying Ao

Description

In 2008, Goers, E. S. et al. reported the characterization of RNA binding properties of the Drosophila muscleblind protein Mbl. Systematic evolution of ligands by exponential enrichment (SELEX) was used to isolated RNA aptamers that bind Mbl with much higher affinity than CUG repeats. Characterization of the RNA binding properties of Drosophila Mbl will lead to a better understanding of its role in alternative splicing and the role of MBNL in DM^[1].

SELEX

The template for the SELEX (systematic evolution of ligands by exponential enrichment) experiment was the 80-mer DNA oligonucleotide. The initial PCRs were amplified by eight rounds of PCR and calculated to a concentration of 1×10¹⁴ molecules. For each round, the appropriate concentration of Mbl (1-105) was bound to 20 μL of glutathione-agarose beads at 4°C for 15 min, then washed three times. The DNA was amplified by 11 PCR cycles. The DNA was transcribed and the SELEX cycle repeated six times with the concentration of RNA. After the rounds were completed, individual clones were isolated by TOPO cloning and then sequenced. SELEX was performed using Mbl and a random pool of 10¹⁴ RNA oligonucleotides. Each RNA consisted of a 30-nucleotide randomized region flanked by shorter constant regions, creating an 80-nucleotide RNA. Twenty-five unique RNA sequences were identified (five sequences were found multiple times) after six rounds of SELEX^[1].
Detailed information are accessible on SELEX page.

Structure

The 2D structure of the figure is based on the article by ribodraw tool to draw^[1].

5'-GGGAAUGGAUCCACAUCUACGAAUUCACCGUAGAUGAUGGAUGUGCGGUAGAGUCUUUCACUGCAGACUUGACGAAGCUU-3'

Ligand information

SELEX ligand

Muscleblind Like Splicing Regulator 1 (MBNL1) is an RNA splicing protein that in humans is encoded by the MBNL1 gene. It has a well characterized role in Myotonic dystrophy where impaired splicing disrupts muscle development and function. In addition to regulating mRNA maturation of hundreds of genes MBNL1 (along with its paralogs MBNL2 & MBNL3) autoregulate alternative splicing of the MBNL1 pre-mRNA transcript. The founding member of the human MBNL family of proteins was the Drosophila Muscleblind protein.-----From WiKi

Name	Uniprot ID	Pfam	MW	Amino acids sequences	PDB	Gene ID
Drosophila muscleblind protein Mbl	O16011	PTHR12675	32.33 KDa	MANVVNMNSLLNGKDSRWLQLEVCREFQRNKCSRQDTECKFAHPPANVEVQNGKVTACYDSIKGRCNRDKPPCKYFHPPQHLKDQLLINGRNHLALKNALMQQMGIAPGQPVISGQVPAVATNPYLTGIPANSYSPYYTTGHLVPALLGPDPVTSQLGPVVPQTVQVAQQKIPRSDRLETSPLAAHHHQQQQQLQHQLNNINNNNNHSTAGAAATSTTATTTTNNAAAAAAAAAAAAAAAVMGHHTLEVGKKRAADTTDMFPLVFFCSFPSPCVFAFLNCSIFGFLRLWFSLFNLRH	3D2Q	36945

Some isolated sequences bind to the affinity of the protein.

Name	Sequence	Ligand	Affinity
RNA20 aptamer	5'-GGGAAUGGAUCCACAUCUACGAAUUCACCGUAGAUGAUGGAUGUGCGGUAGAGUCUUUCACUGCAGACUUGACGAAGCUU-3'	Drosophila muscleblind (Mbl)	0.23 ± 0.09 nM
RNA45 aptamer	5'-GGGAAUGGAUCCACAUCUACGAAUUCGUUUCGACGAUGUGGAUGUGCGGUAGAGUCUUCACUGCAGACUUGACGAAGCUU-3'	Drosophila muscleblind (Mbl)	1.0 ± 0.4 nM
RNA37 aptamer	5'-UUGAAUGGAUCCACAUCUACGAAUUCAAGUCUGAGCUUCUUUUCAUGUGAAAUAGCUUCACUGCAGACUUGACGAAGCUU-3'	Drosophila muscleblind (Mbl)	0.05 ± 0.007 nM
RNA50 aptamer	5'-GGGAAUGGAUCCACAUCUACGAAUUCAAGUCUGAGCCUCUGGACGUCGAUGCUUCACUGCAGACUUGACGAAGCUU-3'	Drosophila muscleblind (Mbl)	0.42 ± 0.14 nM

Similar compound

We used the Dail server website to compare the structural similarities of ligand proteins, and chose the top 10 in terms of similarity for presentation. The Dali server is a network service for comparing protein structures in 3D. Dali compares them against those in the Protein Data Bank (PDB). Z-score is a standard score that is converted from an original score. The list of neighbours is sorted by Z-score. Similarities with a Z-score lower than 2 are spurious. RMSD(Root Mean Square Deviation) value is used to measure the degree to which atoms deviate from the alignment position. We operate with human MBNL1 3D2Q as the model.

PDB	Z-socre	RMSD	Description
3D2Q-B	14.5	0.5	Muscleblind-like protein 1
3D2S-D	14.1	0.8	Muscleblind-like protein 1
3D2S-B	14.1	0.8	Muscleblind-like protein 1
3D2S-A	11.7	1.3	Muscleblind-like protein 1
3D2S-C	11.6	1.3	Muscleblind-like protein 1
3D2Q-C	11.6	1.2	Muscleblind-like protein 1
3D2Q-A	11.4	1.5	Muscleblind-like protein 1
3D2N-A	11.2	1.3	Muscleblind-like protein 1
2RPP-A	9.4	1.6	Muscleblind-like protein 2
2E5S-A	7.1	2.9	Otthump00000018578

References

[1] RNA binding specificity of Drosophila muscleblind.
Goers, E. S., Voelker, R. B., Gates, D. P., & Berglund, J. A.
Biochemistry, 47(27), 7284–7294. (2008)