Genetic Alphabet Expansion Provides Versatile Specificities and Activities of Unnatural-Base DNA Aptamers Targeting Cancer Cells

Kazunobu Futami,Michiko Kimoto,Yun Wei Shermane Lim,Ichiro Hirao

doi:10.1016/j.omtn.2018.11.011

Abstract

The potential of genetic alphabet expansion technologies using artificial extra base pairs (unnatural base pairs) has been rapidly expanding and increasing. We present that the hydrophobic unnatural base, 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), which acts as a fifth letter in a DNA library, provides a series of high-affinity DNA aptamers with versatile binding specificities and activities to cancer cells. These Ds-containing DNA aptamers were generated by a method called cell-ExSELEX to target three breast cancer cell lines: MCF7, MDA-MB-231, and T-47D. Aptamer 14A-MCF7, which targets MCF7 cells, specifically binds to MCF7 cells, but not other cancer cell lines. Aptamer 07-MB231, which targets MDA-MB-231 cells, binds to a series of metastatic bone and lung cancer cell lines. Aptamer 05-MB231 targets MDA-MB-231 cells, but it also binds to all of the cancer and leukemia cell lines that we examined. None of these aptamers bind to normal cell lines, such as MCF10A and HUVEC. In addition, aptamers 14A-MCF7 and 05-MB231 are internalized within the cancer cells, and aptamer 05-MB231 possesses anti-proliferative properties against most cancer cell lines that we examined. These aptamers and the generation method are broadly applicable to cancer cell imaging, biomarker discovery, cancer cell profiling, anti-cancer therapies, and drug delivery systems.

Highlights

The creation of unnatural base pairs (UB pairs or UBPs) that exhibit high fidelity in replication as a third base pair has led to novel biotechnologies
Cell-ExSELEX Targeting Breast Cancer Cells In this cell-ExSELEX (Figure 1B), we used a Ds-predetermined DNA library,[3,28] which was a mixture of 24 different sub-libraries contain
After repetitive rounds of cellExSELEX, each DNA sequence in the enriched libraries was determined with an Ion PGM sequencing system (Life Technologies)

Summary

Introduction

The creation of unnatural base pairs (UB pairs or UBPs) that exhibit high fidelity in replication as a third base pair has led to novel biotechnologies. The UBP technologies provide new material production platforms, such as semisynthetic organisms that generate novel proteins containing unnatural amino acids[1,2] and high-affinity DNA aptamers that bind to target proteins and cells.[3,4] In particular, hydrophobic UBs as a fifth letter greatly increase the chemical and physical diversities of functional DNA molecules.[3,5] To pursue further UBP technology applications toward diagnostics and therapeutics, we have developed a method, cell-ExSELEX, to generate high-affinity UB-containing DNA aptamers that bind to cancer cells. DNA aptamers are single-stranded oligonucleotides that bind to target molecules and materials of interest They are isolated from a nucleic acid library containing $1015 different sequence species by an evolutionary engineering method called SELEX (Systematic Evolution of Ligands by EXponential enrichment).[6,7] Once their sequences are determined by the SELEX procedure, chemical synthesis enables their large-scale preparation and modification for many purposes. Many modification and modified aptamer generation methods have been reported to tackle these issues.[14,15,16,17,18,19,20,21,22]

Methods

Results

Conclusion