Pyrrolo-dC and pyrrolo-C: fluorescent analogs of cytidine and 2′-deoxycytidine for the study of oligonucleotides

Berry, David A.; Jung, K. W.; Wise, Dean S.; Sercel, Anthony D.; Pearson, William H.; Mackie, Hugh; Randolph, John B.; Somers, Robert L.

doi:10.1016/j.tetlet.2004.01.108

Cited by 139 publications

(139 citation statements)

References 49 publications

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“…They typically do not disrupt duplex formation, making them useful probes of basepairing and base-stacking with neighboring natural bases. Fluorescent base analogs generally have large Stokes shifts and their fluorescence is sensitive to their environment (Jean and Hall 2001), making them good probes of local conformational changes that occur within nucleic acids (Millar 1996;Jameson and Eccleston 1997;Rist and Marino 2002;Walter et al 2002;Berry et al 2004). A number of fluorescent base analogs have been incorporated into nucleoside phosphoramidites and thus synthetic nucleic acids.…”

Section: Introductionmentioning

confidence: 99%

“…A recent addition to the arsenal of fluorescent pyrimidine analogs available for probing nucleic acids is 3 (Berry et al 2004;Fig. 1A).…”

Section: Introductionmentioning

confidence: 99%

“…PC is of relatively small size, which is unlikely to perturb the structure of an A-type RNA helix. In addition, it promises to have potent applications in RNA structural analysis as it shares many of the same fluorescence characteristics that make 2-aminopurine ribonucleoside a versatile probe (Berry et al 2004). PC is highly fluorescent, with excitation and emission maxima of 350 and 460 nm, respectively, relatively far from those of RNA and protein, making it a suitable probe for both RNAs and RNA-protein complexes.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Introductionmentioning

confidence: 99%

“…A recent addition to the arsenal of fluorescent pyrimidine analogs available for probing nucleic acids is 3 (Berry et al 2004;Fig. 1A).…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Tinsley¹,

Walter²

2006

RNA

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“…To date, elucidating of the folding pathways has been hampered by the lack of assays that can independently monitor the environment of nucleobases throughout the ribozyme-substrate complex in real time without structural ambiguity. 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).…”

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).…”

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-dC and pyrrolo-C: fluorescent analogs of cytidine and 2′-deoxycytidine for the study of oligonucleotides

Cited by 139 publications

References 49 publications

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

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

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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