Probing RNA Structure with Chemical Reagents and Enzymes

Ziehler, William A.; Engelke, David R.

doi:10.1002/0471142700.nc0601s00

Cited by 76 publications

(98 citation statements)

References 12 publications

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“…Our experiment outlined below is based on experiments previously described (Ziehler and Engelke 2000;Zhang et al 2002). 5Ј-end-labeled DsrA (0.1 pmole/µL and 5 × 10 4 cpm) was ethanolprecipitated and resuspended in 1.2 µL of Na 2 CO 3 /EDTA solution (50 mM Na 2 CO 3 , 1 mM EDTA).…”

Section: Minimal Binding Assay Of Dsra-hfq Complexmentioning

confidence: 99%

Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure

Brescia¹,

Mikulecky²,

Feig³

et al. 2003

RNA

138

191

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Section: Minimal Binding Assay Of Dsra-hfq Complexmentioning

confidence: 99%

Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure

Brescia¹,

Mikulecky²,

Feig³

et al. 2003

RNA

138

191

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“…The threedimensional structure of RNA can often be modeled once the secondary structure is known (Michel and Westhof 1990;Masquida and Westhof 2006), but determination of secondary structure is not trivial. Typical methods for inferring secondary structure include sequence comparison (Pace et al 1999) and chemical or enzymatic mapping (Ziehler and Engelke 2000), where free energy minimization is often combined with either or both of these sets of data (Duan et al 2006). Binding of oligonucleotides by RNA to ascertain exposed sequences can also provide insight into RNA structure (Lewis and Doty 1970;Uhlenbeck et al 1970;Sohail et al 1999;Hopkins and Woodson 2005;Kierzek et al 2006a).…”

Section: Introductionmentioning

confidence: 99%

Selective quenching of fluorescence from unbound oligonucleotides by gold nanoparticles as a probe of RNA structure

Li¹,

Liang²,

Turner³

et al. 2007

RNA

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Binding of small oligonucleotides to the periphery of folded RNA can provide insight into the secondary structure of complex RNA in solution. To discriminate between bound and unbound fluorescein-labeled 29-O-methyl RNA probes, we use ionically coated gold nanoparticles to selectively adsorb unbound probes and quench their fluorescence. The target is the 39 untranslated region of Bombyx mori R2 RNA. Fluorescence indicates that R2 sequences complementary to some of the probes are accessible for binding in the three-dimensional structure. Hybridization occurs under homogeneous conditions in the absence of the gold nanoparticles so that steric issues associated with chip-based assays are avoided. The assay is compatible with well plate formats, takes less than 5 min, and requires only 2 pmol or less of unlabeled target RNA per probe sequence tested.

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“…This was achieved by combining RNase protection and primer-extension analyses (for overview of the assay, see Fig. 2A), an approach that was originally designed to probe RNA secondary structure (Ziehler and Engelke 2001). The assay works by exploitation of RNase A and RNase T1 substrate specificities; under high-salt conditions ($300 mM NaCl), RNase A and RNase T1 only cleave single-stranded RNAs at 59-(C/U)YN, or 59-GYN, respectively, leaving perfectly base paired RNA:DNA (or RNA:RNA) hybrids intact.…”

Section: Resultsmentioning

confidence: 99%

Use of two novel approaches to discriminate between closely related host microRNAs that are manipulated by Toxoplasma gondii during infection

Zeiner

Boothroyd²

2010

RNA

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MicroRNAs (miRNAs) are a class of small, endogenously encoded regulatory RNAs that function to post-transcriptionally regulate gene expression in a wide variety of eukaryotes. Within organisms, some mature miRNAs, such as paralogous miRNAs, have nearly identical nucleotide sequences, which makes them virtually indistinguishable from one another by conventional hybridization-based approaches. Here we describe two inexpensive, sensitive methods for rapidly discriminating between paralogous miRNAs or other closely related miRNAs and for quantifying their abundance. The first approach is a sequential ribonuclease-protection and primer-extension assay; the second approach is a primer-extension assay that employs short oligonucleotide probes to exacerbate the instability of mismatched probe:miRNA hybrids. Both approaches are rapid and can be easily performed in their entirety using common laboratory equipment. As a proof of concept, we have used these methods to determine the exact identities of the human miR-17 family members that are increased by infection with the intracellular parasite Toxoplasma gondii. These methods can be used to rapidly and inexpensively discriminate between any closely related miRNAs in any organism.

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Probing RNA Structure with Chemical Reagents and Enzymes

Cited by 76 publications

References 12 publications

Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure

Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure

Selective quenching of fluorescence from unbound oligonucleotides by gold nanoparticles as a probe of RNA structure

Use of two novel approaches to discriminate between closely related host microRNAs that are manipulated by Toxoplasma gondii during infection

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