Abstract 2058: Identification of a specific RNA aptamer targeting non-small cell lung cancer by in vivo SELEX
Authors: Wang Hanlu , Zhang Yibang , Chu Chang , Dai Wei , Liu Rihe , Jiang Yongping 
Published: July 2016

Abstract
In vivo selection of the RNA aptamers by systematic evolution of ligands by exponential enrichment (SELEX) has demonstrated a great targeting tool for diagnostics and treatments in the clinic. In this study, in vivo selection of specific RNA aptamer for cells was established through the use of tumor-bearing xenograft mice injected with human non-small cell lung cancer NCI-H460 cells. Briefly, a synthetic DNA pool was first transcribed in vitro using mutant T7 RNA polymerase (Y639F), which is capable of recognizing 2’-fluoropyrimidines to produce nuclease-resistant RNA. PEGylated RNA library was then constructed to produce stable PEG-RNA molecules. A cDNA library was produced and was further transcribed for an RNA pool. The enriched nuclease-resistant PEG-RNA pool was re-injected into NCI-H460 tumor-bearing mice again. After multiple cycle selections in vivo, the cDNA pool was cloned into T-vector for sequencing analysis for enriched RNA motifs.
On round 8 of SELEX selection, an enriched RNA sequence (RA16) was identified at a frequency of 21.2%. The RA16 preparation was further enriched up to 94.7% on round 11. To characterize the specificity in vitro, NCI-H460 cells, along with additional tumor cells or normal control cells, were incubated with Cy3-labeled RA16 or the initial RNA library. The specific fluorescent binding was observed for HCI-H460 and H1299 cells, but not for HEK293T, Hela, and H226 cells. Binding affinity (Kd 9±2 nM) for RA16 was determined by flow cytometry using FITC-labeled RA16 to HCI-H460 cells. Meanwhile, cryosections from various organs of tumor-bearing mice also revealed strong binding of RA16 with HCI-H460 tumors but only slightly with lung tissue. No any significant binding was detected in other tissues of tumor-bearing mice or in any normal tissues of control mice.
Furthermore, a quantitative-RT-PCR was performed to trap specific RA16 sequence from various tissues of control or tumor-bearing mice. The selected RNA aptamer RA16 or the initial RNA library was injected into tumor-bearing(n = 4) and normal control mice (n = 4). Total RNAs were extracted from the mouse tumor or from heart, liver, spleen, lung and kidney 3.5 hrs post injection. There was an enrichment of 44.3±3.6-folds for the injected RA16 in mouse tumors treated by RA16 preparation comparing with mouse tumors treated by the initial RNA library. No significant enrichment of RA16 was detected in any other tissues of tumor-bearing mice or tissues from normal control mice. Significantly, in vivo tumor imaging study with Cy5.5-labeled RA16 showed that aptamer RA16 was gradually migrated toward tumor site and enriched with time at tumor area in mice. Our studies thus strongly suggests that RNA aptamer RA16 was specifically selected for targeting human NCI-H460 cells, as well as xenograft tumor tissues with high affinity both in vitro and in vivo.
Citation Format: Hanlu Wang, Yibang Zhang, Chang Chu, Wei Dai, Rihe Liu, Yongping Jiang. Identification of a specific RNA aptamer targeting non-small cell lung cancer by in vivo SELEX. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2058.