Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

Bradrick, Thomas D.; Marino, John P.

doi:10.1261/rna.7620304

Cited by 73 publications

(95 citation statements)

References 48 publications

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“…Argininamide and TAR are in fast exchange on the NMR time scale; thus, collectively induced chemical shift changes of 2F-adenosines were fitted by nonlinear regression to yield a K D of 1.1 AE 0.5 mM ( Fig. 15D; Fielding, 2007), which is in excellent agreement with affinities obtained from fluorescence-based assays (Bradrick & Marino, 2004).…”

Section: Ligand-induced Chemical Shift Changessupporting

confidence: 67%

19F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR

Scott

Hennig

2016

Methods in Enzymology

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Section: Ligand-induced Chemical Shift Changessupporting

confidence: 67%

19F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR

Scott

Hennig

2016

Methods in Enzymology

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“…Binding of ligands to RNA hairpins I and II in solution was assessed using the base analog 2-ap as a fluorescent probe (Bradrick and Marino 2004). For these measurements, the fluorescent base was substituted for the adenines indicated in Figure 3, and the peptoid-dependent change in fluorescence was used to determine the dissociation constant.…”

Section: Affinities and Specificities Of 'Hit' Compoundsmentioning

confidence: 99%

Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha

Diaz

Chirayil

et al. 2014

RNA

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We have found a small molecule that specifically inhibits cleavage of a precursor to the oncogenic miRNA, miR-21, by the microprocessor complex of Drosha and DGCR8. We identified novel ligands for the apical loop of this precursor from a screen of 14,024 N-substituted oligoglycines (peptoids) in a microarray format. Eight distinct compounds with specific affinity were obtained, three having affinities for the targeted loop in the low micromolar range and greater than 15-fold discrimination against a closely related hairpin. One of these compounds completely inhibits microprocessor cleavage of a miR-21 primary transcript at concentrations at which cleavage of another miRNA primary transcript, pri-miR-16, is little affected. The apical loop of pri-miR-21, placed in the context of pri-miR-16, is sufficient for inhibition of microprocessor cleavage by the peptoid. This compound also inhibits cleavage of pri-miR-21 containing the pri-miR-16 apical loop, suggesting an additional site of association within pri-miR-21. The reported peptoid is the first example of a small molecule that inhibits microprocessor cleavage by binding to the apical loop of a pri-miRNA.

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“…One of the most popular fluorescent nucleobases is 2-aminopurine, an analog of the purines adenine and guanine (Ward et al 1969;Doudna et al 1990;Fujimoto et al 1996;Millar 1996;Rist and Marino 2002). It specifically Watson-Crick base-pairs with thymine or uracil and has proven its utility as a probe to monitor local conformational changes in nucleic acid structure, for example, in catalytic RNA and upon formation of nucleic acid-protein complexes (Millar 1996;O'Neill and Barton 2002;Walter et al 2002;Patel and Bandwar 2003;Roy 2003;Bradrick and Marino 2004;Clerte and Hall 2004). Several fluorescent pyrimidine analogs also have been developed, although they have not yet reached a similar level of popularity (Inoue et al 1985;Wu et al 1990;Godde et al 1998;Wilhelmsson et al 2001).…”

Section: Introductionmentioning

confidence: 99%

Pyrrolo-C as a fluorescent probe for monitoring RNA secondary structure formation

Tinsley¹,

Walter²

2006

RNA

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Pyrrolo-C (PC), or 3-[b-D-2-ribofuranosyl]-6-methylpyrrolo [2,3-d]pyrimidin-2(3H)-one, is a fluorescent analog of the nucleoside cytidine that retains its Watson-Crick base-pairing capacity with G. Due to its red-shifted absorbance, it can be selectively excited in the presence of natural nucleosides, making it a potential site-specific probe for RNA structure and dynamics. Similar to 2-aminopurine nucleoside, which base-pairs with uridine (or thymidine), PC's fluorescence becomes reversibly quenched upon base-pairing, most likely due to stacking interactions with neighboring bases. To test its utility as an RNA probe, we examined PC's fluorescent properties over a wide range of ionic strengths, pH, organic cosolvents, and temperatures. Incorporation of PC into a single-stranded RNA results in an $60% reduction of fluorescence intensity, while duplex formation reduces the fluorescence by $75% relative to the free ribonucleoside. We find that the fluorescence intensity of PC is only moderately affected by ionic strength, pH, and temperature, while it is slightly enhanced by organic cosolvents, making it a versatile probe for a broad range of buffer conditions. We demonstrate two applications for PC: fluorescent measurements of the kinetics of formation and dissociation of an RNA/DNA complex, and fluorescent monitoring of the thermal denaturation of the central segment of an RNA duplex. Taken together, our data showcase the potential of pyrrolo-C as an effective fluorescent probe to study RNA structure, dynamics, and function, complementary to the popular 2-aminopurine ribonucleoside.

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Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

Abstract: Replication of human immunodeficiency virus type 1 (HIV-1) is regulated in part through

Cited by 73 publications

References 48 publications

19F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR

19F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR

Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha

Pyrrolo-C as a fluorescent probe for monitoring RNA secondary structure formation

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