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

Tinsley, Rebecca A.; Walter, Nils G.

doi:10.1261/rna.2165806

Cited by 75 publications

(87 citation statements)

References 46 publications

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“…Ribonucleoside 2 was then converted into the corresponding 5′-triphosphate 2TP by a conventional method, 16 using freshly distilled POCl 3 and tributyl ammonium pyrophosphate. The triphosphate was extensively purified by ion exchange chromatography and HPLC, and thoroughly characterized using mass spectrometry as well as, 1 H, 13 C and 31 P NMR spectroscopy (see Experimental Section).…”

Section: Synthesis and Photophysical Propertiesmentioning

confidence: 99%

“…13 To rectify this general deficiency in fluorescent pyrimidine analogs, we have recently initiated a program in search for families of emissive and responsive nucleobase analogs. The basic requirement that are imposed upon the new nucleoside analogs include: (a) to maintain high structural similarity to the natural nucleobases, (b) to display emission at long wavelengths, preferably in the visible range, (c) to retain sufficient emission quantum efficiency to be utilized in real time fluorescence-based assays, and, importantly, (d) to possess sensitivity to the microenvironment that is manifested in markedly different photophysical parameters (emission wavelength λ em , quantum efficiency φ f and/or excited state lifetime τ) in aqueous versus apolar environment.…”

Section: Introductionmentioning

confidence: 99%

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Fluorescent Pyrimidine Ribonucleotide: Synthesis, Enzymatic Incorporation, and Utilization

2007

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Fluorescent nucleobase analogs that respond to changes in their microenvironment are valuable for studying RNA structure, dynamics and recognition. The most commonly used fluorescent ribonucleoside is 2-aminopurine, a highly responsive purine analog. Responsive isosteric fluorescent pyrimidine analogs are, however, rare. Appending 5-membered aromatic heterocycles at the 5-position on a pyrimidine core has recently been found to provide a family of responsive fluorescent nucleoside analogs with emission in the visible range. To explore the potential utility of this chromophore for studying RNA-ligand interactions, an efficient incorporation method is necessary. Here we describe the synthesis of the furan-containing ribonucleoside and its triphosphate, as well as their basic photophysical characteristics. We demonstrate that T7 RNA polymerase accepts this fluorescent ribonucleoside triphosphate as a substrate in in vitro transcription reactions and very efficiently incorporates it into RNA oligonucleotides, generating fluorescent constructs. Furthermore, we utilize this triphosphate for the enzymatic preparation of a fluorescent bacterial Asite, an RNA construct of potential therapeutic utility. We show that the binding of this RNA target to aminoglycoside antibiotics, its cognate ligands, can be effectively monitored by fluorescence spectroscopy. These observations are significant since isosteric emissive U derivatives are scarce and the trivial synthesis and effective enzymatic incorporation of the furan-containing U triphosphate make it accessible to the biophysical community.

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Section: Synthesis and Photophysical Propertiesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Fluorescent Pyrimidine Ribonucleotide: Synthesis, Enzymatic Incorporation, and Utilization

2007

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“…In the ground state, C3 forms one hydrogen bond with C17, which is stabilized by stacking interactions, while in the active state, C3 forms a Watson-Crick base pair with G8. Since it has previously been shown that base-pairing of pyC with guanosine strongly lowers its quantum yield (Tinsley and Walter 2006), the observed decrease indicates a transition from ground to active state. For the C1.1, the base-pairing with G2.1 occurs in both ground and active states; therefore, the expected overall amplitude of signal change is small and depends only on the transition between stacking interactions with C3 in the ground state to the stacking interaction with the G8-C3 base pair in the active state.…”

Section: Distinct Folding Of Catalytic Core and Auxiliary Structural mentioning

confidence: 81%

“…Here, we describe a fast kinetic folding analysis approach using the highly sensitive fluorescent cytosine analog pyrrolo-cytosine (pyC) (Berry et al 2004). pyC forms a Watson-Crick-like base pair with guanosine and was shown to maintain all structural and thermodynamic features of a standard guanosine-cytosine base pair (Dash et al 2004; Thompson and Miyake 2005;Tinsley and Walter 2006;Zhang and Wadkins 2009). Its incorporation at multiple positions in the extended hammerhead ribozyme was previously shown not to interfere with the function of the ribozyme (Lambert et al 2006).…”

Section: +mentioning

confidence: 99%

Folding of the hammerhead ribozyme: Pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

Buskiewicz¹,

Burke²

2012

RNA

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The catalytic activity of the hammerhead ribozyme is limited by its ability to fold into the native tertiary structure. Analysis of folding has been hampered by a lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time. Here, we report the development and application of a new folding assay in which we use pyrrolo-cytosine (pyC) fluorescence to (1) probe active-site formation, (2) examine the ability of peripheral ribozyme domains to support native folding, (3) identify a pH-dependent conformational change within the ribozyme, and (4) explore its influence on the equilibrium between the folded and unfolded core of the hammerhead ribozyme. We conclude that the natural ribozyme folds in two distinct noncooperative steps and the pH-dependent correlation between core folding and activity is linked to formation of the G8-C3 base pair.

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“…The PyC analog was previously shown to be an excellent probe for proteinnucleic acid interactions (Berry et al 2004;Tinsley and Walter 2006), because it can be selectively excited at 350 nm, well separated from the absorbance at 260 nm of natural nucleotides, and has an emission maximum of 460 nm, far removed from that of protein tryptophans (emission=340 nm). In addition, its fluorescence is sensitive to local conformational changes of nucleic acids, which has been exploited to study the dynamics and stabilities of DNA helices, RNA duplexes, and a DNA/RNA hybrid (Liu and Martin 2002;Tinsley and Walter 2006). Our method of fluorescent labeling of tRNA uses the CCA enzyme to incorporate PyC and generates fluorescent products that are substrates for aminoacylation.…”

Section: Introductionmentioning

confidence: 99%

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Zhang¹,

Liu²,

Christian³

et al. 2008

RNA

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All mature tRNA molecules have the conserved CCA sequence at the 39 end with a range of dynamic conformations that are important for tRNA functions. We present here the details of a general approach to fluorescent labeling of the CCA sequence with the fluorescent base analog pyrrolo-C (PyC) at position 75 as a molecular probe for monitoring the dynamics of the tRNA 39 end. Using Escherichia coli tRNACys as an example, we achieve such labeling by first synthesizing the tRNA as a transcript up to C74 and then employing the tRNA CCA-adding enzyme to incorporate PyC75 and A76, using pyrrolo-CTP (PyCTP) and ATP as the respective substrates. PyC-labeled full-length tRNA Cys , separated from the unlabeled precursor tRNA by reverse phase highpressure liquid chromatography, is an efficient substrate for aminoacylation by E. coli cysteinyl-tRNA synthetase (CysRS). Fluorescence binding measurement of the PyC-labeled tRNACys with E. coli CysRS reveals an equilibrium K d closely similar to the value determined from the fluorescence of intrinsic enzyme tryptophans. Kinetic measurements of translocation of the PyClabeled tRNA from the ribosomal A to P sites identify a kinetic intermediate with a rate of formation and decay similar to the values reported for tRNAs labeled with the fluorescent proflavin at the tertiary core. These results highlight the potential of PyC to probe the dynamics of the tRNA CCA end in reactions ranging from aminoacylation to those on the ribosome.

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

Cited by 75 publications

References 46 publications

Fluorescent Pyrimidine Ribonucleotide: Synthesis, Enzymatic Incorporation, and Utilization

Fluorescent Pyrimidine Ribonucleotide: Synthesis, Enzymatic Incorporation, and Utilization

Folding of the hammerhead ribozyme: Pyrrolo-cytosine fluorescence separates core folding from global folding and reveals a pH-dependent conformational change

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

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