Selection and characterization of anti-NF-κB p65 RNA aptamers

Wurster, Susan E.; Maher, L. James

doi:10.1261/rna.878908

Cited by 44 publications

(54 citation statements)

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“…The metal-free apo-protein of pirin was isolated from iron-depleted minimum medium (42) and metal reconstitution details are given in SI Materials and Methods. p65 (19-291) was obtained from Jim Maher, III of Mayo Clinic College of Medicine (Rochester, MN) (43,44) and isolated as described in the SI Materials and Methods. The purified p65 was treated with 10 equivalents of EDTA overnight and then desalted with 20 mM Tris·HCl buffer (pH 7.4) containing 50 mM NaCl and 5% (vol/vol) glycerol.…”

Section: Methodsmentioning

confidence: 99%

Pirin is an iron-dependent redox regulator of NF-κB

Liu

Rehmani²,

Esaki³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

107

146

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Pirin is a nuclear nonheme Fe protein of unknown function present in all human tissues. Here we describe that pirin may act as a redox sensor for the nuclear factor κB (NF-κB) transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to proinflammatory signals and controls the expression of a vast array of genes involved in immune and stress responses. Pirin's regulatory effect was tested with several metals and at different oxidations states, and our spectroscopic results show that only the ferric form of pirin substantially facilitates binding of NF-κB proteins to target κB genes, a finding that suggests that pirin performs a redox-sensing role in NF-κB regulation. The molecular mechanism of such a metal identity-and redox state-dependent regulation is revealed by our structural studies of pirin. The ferrous and ferric pirin proteins differ only by one electron, yet they have distinct conformations. The Fe center is shown to play an allosteric role on an R-shaped surface area that has two distinct conformations based on the identity and the formal redox state of the metal. We show that the R-shaped area composes the interface for pirin-NF-κB binding that is responsible for modulation of NF-κB's DNAbinding properties. The nonheme Fe protein pirin is proposed to serve as a reversible functional switch that enables NF-κB to respond to changes in the redox levels of the cell nucleus.metalloprotein | coregulator | reactive oxygen species (ROS) | oxidative stress | signal transduction activation

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Section: Methodsmentioning

confidence: 99%

Pirin is an iron-dependent redox regulator of NF-κB

Liu

Rehmani²,

Esaki³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

107

146

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“…This RNA is characterized by 59 and 39 terminal sequences that are required for p65 2 binding (low nanomolar range) in vitro (Wurster and Maher 2008). Y3H analysis suggested that R1 has a high specificity for murine NF-kB p65 2 in the Y3H.…”

Section: Y3h Selections For Anti-nf-kb P65 Rna Aptamers Identify Rna mentioning

confidence: 99%

“…We transformed the Y3H assay strain with plasmids encoding the Rel homology region of NF-kB zebrafish p65 2 . We included a plasmid expressing an RNA library derived from an early round of the in vitro selection experiment that originally yielded the anti-p65 R1 aptamer (Wurster and Maher 2008). Transformants were selected for HIS3 activation.…”

Section: Y3h Selections For Anti-nf-kb P65 Rna Aptamers Identify Rna mentioning

confidence: 99%

“…Our laboratory has implemented the Y3H to confirm the ability of in vitro selected anti-NF-kB RNA aptamers to bind their transcription factor targets, and to optimize the RNA aptamers by in vivo selection Wurster and Maher 2008;Wurster et al 2009). We have routinely selected aptamer variants with improved Y3H reporter gene readout, but frequently observe that the identified RNA aptamer variants do not display increased affinity for the target protein when isolated from the display template and tested by in vitro assay.…”

Section: Introductionmentioning

confidence: 99%

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Selections that optimize RNA display in the yeast three-hybrid system

Wurster

Maher²

2009

RNA

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The yeast three-hybrid system (Y3H) is a powerful tool to select or confirm RNA-protein interactions. Target protein recognition of an RNA insert within a test transcript depends on at least three factors: intrinsic protein affinity for the properly folded insert, retention of RNA insert tertiary structure within a longer RNA transcript, and accessibility of the RNA insert to the target protein. Y3H reporter gene readout reflects the combination of these factors. Here, we discuss RNA insert tertiary structure and accessibility in the Y3H as ''RNA display.'' We review evidence that RNA display can sometimes be optimized during Y3H selections that do not increase the intrinsic affinity of an RNA insert for a target protein. This situation is more likely when a library of RNA inserts and heterogeneous flanking sequences is subjected to selection, and is less likely when point mutations are targeted to the insert in a fixed context. An RNA display vector with enhanced modularity has been developed to minimize sequence context effects in the Y3H.

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“…Chemical features of the binding sites can also affect selection: a natural nucleic acid binding site appears to be chemically more inviting than other sites for RNA aptamers. [12][13][14] Although the individual domains of a protein can be used to isolated aptamers for each of them, 15) it is difficult to isolate multiple aptamers binding to different sites on the same domain that cannot be separated physically. To address these issues, we have developed general methods of population control during in vitro selection.…”

mentioning

confidence: 99%