RNA Secondary Structure Prediction Using High-throughput SHAPE

Summary

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Abstract

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

Introduction

To understand the functions of catalytic and non-coding RNAs involved in regulation of splicing, translation, virus replication and cancer, a detailed knowledge of RNA structure is required^1,2. Unfortunately, accurate prediction of RNA folding presents a formidable challenge. Classical probing agents suffer from many disadvantages such as toxicity, incomplete nucleotide coverage and/or throughput limited to 100-150 nucleotides per experiment. Unaided secondary structure prediction algorithms are similarly disadvantageous, owing to inaccuracies resulting from their inability to effectively distinguish among energetically similar structures. Large RNAs in par....

Protocol

Primer design and extension of the RNA 3' terminus

To analyze long RNAs by high-throughput SHAPE, a series of primer hybridization sites should be selected such that they (i) are separated by ~300 nt, (ii) are 20-30 nt in length, and (iii) that RNA/DNA hybrids produced by annealing DNA to these sites have an expected melting temperature of >50 °C. In addition, segments of RNA that are predicted to be highly structured should be avoided, although making such a determination requires some foreknowledge of the RNA structure, which is often unavailable. DNA primers that hybridize to these sites should then be designed, taking care to ensure th....

Results

RNA containing the HIV-1 rev response element (RRE) and a 3' terminal structure cassette⁴ was prepared from a linearized plasmid by in vitro transcription, after which it was folded by heating, cooling, and incubation at 37 °C in the presence of MgCl₂. RNA was exposed to NMIA and then reverse transcribed from a 5'-end-labeled DNA primer hybridized to the 3' terminal structure cassette. The resulting SHAPE cDNA library, together with control and sequencing reactions, was then fractionate.......

Discussion

We present here a detailed protocol for high-throughput SHAPE, a technique that allows secondary structure determination to single-nucleotide resolution for RNAs of any size. Moreover, coupling experimental SHAPE data with secondary structure prediction algorithms facilitates generation of RNA 2D models with a higher degree of accuracy than is possible with either method alone. The combination of fluorescently-labeled primers and automated CE provides significant advantages over the traditional gel-based SHAPE, facilitat.......

Materials

Name	Company	Catalog Number	Comments
			REAGENTS
N-methylisatoic anhydride (NMIA)	Life technologies	M25	Dissolve in anhydrous DMSO
1-methyl-t-nitroisatoic anhydride (1M7)	see ref. 22
Superscript III Reverse Transcriptase	Life technologies	18080044	10,000 units
Thermo sequenase cycle sequencing kit	Affymetrix	78500
			Materials provided by the user
RNA of interest			6 pmol per reaction (the limit of detection will be determined by the instrument)
Sets of four 5' labeled primers (Cy5, Cy5.5, WellRed D2 and WellRed D1/Licor IR800)			Primers are complementary to the RNA and are used in reverse transcription and sequencing reactions. The listed fluorophores are optimal for the Beckman Coulter 8000 CEQ. Primers may be purchased or synthesized in house.
DNA template			DNA is used for sequencing reactions, and must contain the sequence of the RNA being studied - including any 3'terminal extension, if present. Where applicable, it is often convenient to use the RNA transcription template.
			Buffers
10x RNA renaturation buffer			100 mM Tris-HCl pH 8.0, 1 M KCl, 1 mM EDTA
5X RNA folding buffer			200 mM Tris-HCl pH 8.0, 25 mM MgCl2, 2.5 mM EDTA, 650 mM KCl. (This buffer might be changed depending on the case (e.g. pH, EDTA, Mg, RNase inhibitor)
2.5X RT mix			4 μl 5X buffer, 1 μl 100 mM DTT, 1.5 μl water,1 μl 10 mM dNTPs, 0.5 μl SuperScript III. Note that the 5X buffer and 100 mM DTT are provided with purchase of SuperScript III (Invitrogen).
GenomeLab Sample Loading Solution (Beckman Coulter)			Attention: Avoid multiple freeze-thaw cycles
			EQUIPMENT
Capillary electrophoresis	Beckman	CEQ8000
Thermocycler	varies