Influence of nucleotide identity on ribose 2'-hydroxyl reactivity in RNA.

Hydroxyl-selective electrophiles, including N-methylisatoic anhydride (NMIA) and 1-methyl-7-nitroisatoic anhydride (1M7), are broadly useful for RNA structure analysis because they react preferentially with the ribose 2'-OH group at conformationally unconstrained or flexible nucleotides. Each nucleotide in an RNA has the potential to form an adduct with these reagents to yield a comprehensive, nucleotide-resolution, view of RNA structure. However, it is possible that factors other than local structure modulate reactivity. To evaluate the influence of base identity on the intrinsic reactivity of each nucleotide, we analyze NMIA and 1M7 reactivity using four distinct RNAs, under both native and denaturing conditions. We show that guanosine and adenosine residues have identical intrinsic 2'-hydroxyl reactivities at pH 8.0 and are 1.4 and 1.7 times more reactive than uridine and cytidine, respectively. These subtle, but statistically significant, differences do not impact the ability of selective 2'-hydroxyl acylation analyzed by primer extension-based (SHAPE) methods to establish an RNA secondary structure or monitor RNA folding in solution because base-specific influences are much smaller than the reactivity differences between paired and unpaired nucleotides.

ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis.

Analysis of the long-range architecture of RNA is a challenging experimental and computational problem. Local nucleotide flexibility, which directly reports underlying base pairing and tertiary interactions in an RNA, can be comprehensively assessed at single nucleotide resolution using high-throughput selective 2'-hydroxyl acylation analyzed by primer extension (hSHAPE). hSHAPE resolves structure-sensitive chemical modification information by high-resolution capillary electrophoresis and typically yields quantitative nucleotide flexibility information for 300-650 nucleotides (nt) per experiment. The electropherograms generated in hSHAPE experiments provide a wealth of structural information; however, significant algorithmic analysis steps are required to generate quantitative and interpretable data. We have developed a set of software tools called ShapeFinder to make possible rapid analysis of raw sequencer data from hSHAPE, and most other classes of nucleic acid reactivity experiments. The algorithms in ShapeFinder (1) convert measured fluorescence intensity to quantitative cDNA fragment amounts, (2) correct for signal decay over read lengths extending to 600 nts or more, (3) align reactivity data to the known RNA sequence, and (4) quantify per nucleotide reactivities using whole-channel Gaussian integration. The algorithms and user interface tools implemented in ShapeFinder create new opportunities for tackling ambitious problems involving high-throughput analysis of structure-function relationships in large RNAs.

High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.

Replication and pathogenesis of the human immunodeficiency virus (HIV) is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower (p-value < 0.0001) SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further application of this technology will make possible newly informative analysis of any RNA in a cellular transcriptome.