Regulation of Protein Activity and Cellular Functions Mediated by Molecularly Evolved Nucleic Acids

Abstract

Regulation of protein activity is essential for revealing the molecular mechanisms of biological processes. DNA and RNA achieve many uniquely efficient functions, such as genetic expression and regulation. The chemical capability to synthesize artificial nucleotides can expand the chemical space of nucleic acid libraries and further increase the functional diversity of nucleic acids. Herein, a versatile method has been developed for modular expansion of the chemical space of nucleic acid libraries, thus enabling the generation of aptamers able to regulate protein activity. Specifically, an aptamer that targets integrin alpha3 was identified and this aptamer can inhibit cell adhesion and migration. Overall, this chemical-design-assisted in vitro selection approach enables the generation of functional nucleic acids for elucidating the molecular basis of biological activities and uncovering a novel basis for the rational design of new protein-inhibitor pharmaceuticals.

Keywords: aptamers; cell adhesion; inhibitors; membrane proteins; molecular evolution.

Figure 1.

Characterization of ZAP-1. (a) Proposed base pairing model for Z:P pair. (b) Raman spectra of natural library (black) and ZAP-1 (red). (c) Peak-resolution of ZAP-1 Raman spectra (the dotted line in b).

Figure 2.

Analysis and verification of ZAP-1-targeted protein. (a) Flow cytometry of FAM-labelled initial library (red) and ZAP-1 (blue) with MDA-MB-231 cells treated with proteinase K (orange) and trypsin (green). (b) SDS-PAGE was used to identify ZAP-1-specific band T1, marked as asterisk. 1, marker; 2, cell lysate; 3, beads only; 4, proteins captured by initial library; 5, proteins captured by ZAP-1. (c) Fluorescence confocal images of MDA-MB-231 cells incubated with ZAP-1 and anti-ITGA3. (Scale bar, 50 μm.)

Figure 3.

Flow cytometry analysis and fluorescence confocal images of MDA-MB-231 cells incubated with LN10 (Alexa 594) and aptamer ZAP-1 (FAM). (a) Flow cytometry analysis to monitor the binding ability of LN10 and ZAP-1. Percentage of cells above the autofluorescence of cells (dash line) was listed in each sample. (b) Fluorescence confocal images of cells incubated with ZAP-1 and LN 10. Quantified results were shown in c (green channel) and d (red channel). Each bar represents the mean ± SD. (Scale bar, 50 μm.)

Figure 4.

Modulation of integrin α3β1 by ZAP-1 combination. (a) Schematic model for conformational modulation of integrin α3β1 by ZAP-1 combination. (b) MDA-MB-231 cell adhesion result. (Scale bar, 200 μm.) (c) Attached cell numbers in three separate fields expressed as percentages of the cell number after LN10-incubation. (d) Cell migration result. (e) Closure degree of the scratched area was expressed as percentages of the initial scratched area. Each bar represents the mean ± SD.

Scheme 1.

Molecular design of ANE aptamer as a protein activity regulator.