Combinatorial Selection of RNA Ligands for Complex Cellular Targets: The RNA Liagands-Based Proteomics

Huicheng Tian

doi:10.1074/mcp.t100002-mcp200

Abstract

This study explores the selection of high affinity RNA ligands for the complex cellular targets present in crude HeLa nuclear extract through directed evolution and deconvolution. RNA ligands for the mixed nuclear targets were selected from around 6 x 10(14) RNA sequences through an iterated enrichment process. RNA ligands for various gene products of the extract were simultaneously selected and were shown to specifically interact with their target molecules. The target molecules were isolated from the nuclear extract by affinity chromatography using columns tagged with the RNA ligands, resolved on two-dimensional gels, and identified by mass spectrometry. These RNA ligands may be useful in characterizing novel functions of cellular proteins and modulating complex molecular events.

Highlights

This study explores the selection of high affinity RNA ligands for the complex cellular targets present in crude HeLa nuclear extract through directed evolution and deconvolution
Simultaneous Enrichment of RNA Ligands for Complex Nuclear Targets—The current strategy of selection for RNA ligands has been mostly limited to purified targets, in which the binding affinity of RNA ligands for target molecules can be monitored with simple assays and the selection “pressure” can be regulated by changing conditions for binding
This study shows that RNA ligands can be simultaneously selected against various targets present in crude cellular extracts and used to identify factors recognized by the ligands and that these ligands may prove useful in identifying novel functions of cellular proteins

Summary

Introduction

This study explores the selection of high affinity RNA ligands for the complex cellular targets present in crude HeLa nuclear extract through directed evolution and deconvolution. The target molecules were isolated from the nuclear extract by affinity chromatography using columns tagged with the RNA ligands, resolved on twodimensional gels, and identified by mass spectrometry These RNA ligands may be useful in characterizing novel functions of cellular proteins and modulating complex molecular events. There have been attempts to select RNA ligands for complex proteins, RNA pools tend to be quickly reduced to bind only one or a few predominant proteins after the initial rounds of selection (reviewed in Ref. 9) This present study reports that high affinity RNA ligands for numerous targets present in a crude cellular extract have been simultaneously selected and that specific cellular targets have been identified using the RNA ligands

Methods

Results

Conclusion