GL56 aptamer

Jun 7, 2024 • Zhizhong Lu, Ying Ao

Description

In 2016, Shigdar, S. et al. address the characterization of an aptamer, named GL56 that binds the human IR, but not the IGF-1R. The insulin receptor (IR) is a transmembrane receptor that is activated by insulin. The GL56 aptamer was generated by adopting a variant of the cell-based SELEX method that allows enrichment for cell-specific internalizing RNA aptamers. Our results show that the treatment of IR expressing glioma cancer cells with GL56 results in the inhibition of IR dependent signaling and in the reduction of cell viability. Further, upon binding the IR, GL56 rapidly internalize into target cells^[1].

SELEX

The pool enriched from 13 rounds of differential cell-SELEX consisting of selection onto U87MG cells preceded by a counter selection on T98G, 27 was incubated onto U87MG target cells at 37°C for 30 minutes in the first internalization round and for 15 minutes in the second internalization round. After five washes with DMEM serum free medium to remove unbound aptamers, cells were treated with proteinase K (Roche Diagnostics, Indianapolis, IN) for 30 minutes at 0.5 μg/μl final concentration. Cells were then washed with DMEM serum free and internalized RNA aptamers were recovered by RNA extraction and RT-PCR, as previously described. 44 Cloning was performed by using TOPO-TA cloning kit (Invitrogen, Carlsbad, CA) as recommended. For HTS, sequences recovered from each internalization round were sequenced following the Illumina MySeq sequence preparation with MySeq Illumina sequencer^[1].
Detailed information are accessible on SELEX page.

Structure

The 2D structure of the figure is based on the prediction results of the RNA fold website by ribodraw tool to draw^[1].

5'-GGAGACAAGAAUAAACGCUCAAUGAUUUUGCAGCACUUCUUGUUAUCUUAACGAACUGUUGAUGAUUCGACAGGAGGCUCACAACAGGC-3'

Ligand information

SELEX ligand

The insulin receptor (IR) is a transmembrane receptor that is activated by insulin, IGF-I, IGF-II and belongs to the large class of receptor tyrosine kinase. Metabolically, the insulin receptor plays a key role in the regulation of glucose homeostasis; a functional process that under degenerate conditions may result in a range of clinical manifestations including diabetes and cancer. Insulin signalling controls access to blood glucose in body cells. When insulin falls, especially in those with high insulin sensitivity, body cells begin only to have access to lipids that do not require transport across the membrane. So, in this way, insulin is the key regulator of fat metabolism as well. Biochemically, the insulin receptor is encoded by a single gene INSR, from which alternate splicing during transcription results in either IR-A or IR-B isoforms.-----from Wiki

Name	Uniprot ID	Pfam	MW	Amino acids sequences	PDB	Gene ID
insulin receptor(IR)	P06213	IPR040969	156.33 KDa	MATGGRRGAAAAPLLVAVAALLLGAAGHLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDSVEDNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCTPCLGPCPKVCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIRTSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCLKGLKLPSRTWSPPFESEDSQKHNQSEYEDSAGECCSCPKTDSQILKELEESSFRKTFEDYLHNVVFVPRKTSSGTGAEDPRPSRKRRSLGDVGNVTVAVPTVAAFPNTSSTSVPTSPEEHRPFEKVVNKESLVISGLRHFTGYRIELQACNQDTPEERCSVAAYVSARTMPEAKADDIVGPVTHEIFENNVVHLMWQEPKEPNGLIVLYEVSYRRYGDEELHLCVSRKHFALERGCRLRGLSPGNYSVRIRATSLAGNGSWTEPTYFYVTDYLDVPSNIAKIIIGPLIFVFLFSVVIGSIYLFLRKRQPDGPLGPLYASSNPEYLSASDVFPCSVYVPDEWEVSREKITLLRELGQGSFGMVYEGNARDIIKGEAETRVAVKTVNESASLRERIEFLNEASVMKGFTCHHVVRLLGVVSKGQPTLVVMELMAHGDLKSYLRSLRPEAENNPGRPPPTLQEMIQMAAEIADGMAYLNAKKFVHRDLAARNCMVAHDFTVKIGDFGMTRDIYETDYYRKGGKGLLPVRWMAPESLKDGVFTTSSDMWSFGVVLWEITSLAEQPYQGLSNEQVLKFVMDGGYLDQPDNCPERVTDLMRMCWQFNPKMRPTFLEIVNLLKDDLHPSFPEVSFFHSEENKAPESEELEMEFEDMENVPLDRSSHCQREEAGGRDGGSSLGFKRSYEEHIPYTHMNGGKKNGRILTLPRSNPS	7PG0	IPR040969

Some isolated sequences bind to the affinity of the protein.

Name	Sequence	Ligand	Affinity
GL56 aptamer	5'-GGAGACAAGAAUAAACGCUCAAUGAUUUUGCAGCACUUCUUGUUAUCUUAACGAACUGUUGAUGAUUCGACAGGAGGCUCACAACAGGC-3'	CD133 (in Hep3B cell)	6 nM

References

[1] Targeting Insulin Receptor with a Novel Internalizing Aptamer.
Iaboni, M., Fontanella, R., Rienzo, A., Capuozzo, M., Nuzzo, S., Santamaria, G., Catuogno, S., Condorelli, G., de Franciscis, V., & Esposito, C. L.
Molecular therapy. Nucleic acids, 5(9), e365. (2016)