The fluorescent analog of deoxycytidine (C), pyrrole-deoxycytidine (pyrrole-dC, PdC), specifically base pairs with deoxyguanosine (G) and is a valuable tool for probing the structures of nucleic acid or protein-nucleic acid complexes.
Pyrrolo-dC and pyrrolo-C base analogs code as cytidine (C), making them ideal probes for DNA structure and dynamics studies: Pyrrolo-dC and pyrrolo-C base analogs base pair like the standard dC nucleotide. The small size of these analogs does not perturb the structure of the DNA helix and is also well tolerated by several DNA and RNA polymerases. The analogs are highly fluorescent, and their red-shifted excitation and emission spectra eliminate or reduce background fluorescence originating from proteins.
Pyrrolo-dC (PydC) and pyrrolo-C (PyC) are structurally modified fluorescent deoxycytidine and cytidine analogs that maintain a proper Watson-Crick hydrogen bonding. The fluorescence cytidine analog Pyrrolo-C (PC), or 3-[β-D-2-ribofuranosyl]-6-methylpyrrolo [2,3-d] pyrimidin-2(3H)-one, can base-pair with guanosine (G) and can be selectively excited in the presence of natural nucleosides. Figure 1 shows the chemical structure of the pyrollo-dC analog.
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| Extinction coefficients E260 (L/mol.cm) 2,410, λmax-1 (nm) 339, Emax-1 (L/mol.cm) 2,360, λmax -2 (nm) 229, Emax-2 (L/mol.cm) 17,500. Fluorescence data E260 nm 4,000, Eλ max 3,700, Excitation max 345, Emission max 470, QY 0.07/0.02. QY 0.07 single-stranded; 0.02 ds, deprotected in ammonia 55°C ON. |
Figure 1: Pyrrolo-dC [6-methyl-3-(2-deoxy-β-d-ribo-furano-syl)-3H-pyrrolo[2,3-d]pyrimidin-2-one)].
Pyrrolo-dC or PdC {(6-Methyl-3-(β-D-2-deoxyribofuranosyl) pyrrolo [2,3-d] pyrimidine-2-one, pyrrol-2-ylcarbonyl) deoxycytidine, {6-methyl-3-(2-deoxy-b-Dribofuranosyl)-3H-pyrrolo [2,3-d] pyrimidin-2-one)} is a base analog that fluoresces when not base-paired and exhibits little effect on DNA stability, structure, and O6-alkyl-guanine DNA alkyltransferase (AGT) repair. Incorporating pyrrolo-dC into oligonucleotides allows measurement of base flipping by human O6-alkylguanine DNA alkyltransferase.
Pyrrolo-dC based probes
Incorporation of the pyrrolo-cytosine analog into oligonucleotides allows the design and synthesis of site-specific probes useful for the study of RNA structure and dynamics. In a probe strand, the fluorophore is placed opposite a SNP site. Hybridization to a perfectly matched target sequence causes the fluorophore to stack between the surrounding nucleobases, resulting in a light-down or OFF response. However, if a mismatch is present, the probe sequence cannot achieve a perfectly stacked conformation, and a light-up reaction or ON is observed.
Applications
Pyrrolo-dC, a nucleoside with unique properties, is a key player in various biochemical applications, particularly in research and drug development. Its ability to synthesize nucleic acid derivatives for the study of DNA and RNA structures is unparalleled. Moreover, its potential to inhibit cancer cell growth makes it also a promising candidate for anticancer drug development. Figure 2 illustrates the chemical structures of Pyrrolo-dC, its phosphoramidite and the respective base pairs.
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| 6-methyl-3-(2-deoxy-β-d-ribo-furano-syl)-3H-pyrrolo [2,3-d] pyrimidin-2-one) (Pyrrolo-dC) | Pyrrolo-dC phosphoramidite |
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| G:pyrrolo-dC Base Pair | G:C Base pair |
Figure 2: Upper panel: Chemical structures of pyrrolo-dC and its phosphoramidite. Lower panel: Base pairing between guanine (G) and pyrrolo-deoxycytidine (pyrrolo-dC) and between G and C.
Mammalian genomes contain cytosine-guanine (CG) dinucleotide clusters, and CG dinucleotides are the target of DNA methyltransferases. After methylation, the spontaneous deamination of methylcytosine to thymine (mC to T) makes methylated cytosines unusually susceptible to mutation and consequent depletion. CpG islands are loci in which CG dinucleotides remain relatively enriched.
In a groundbreaking study, Woo et al. (1996) replaced deoxyguanosine (dG) and deoxycytidine (dC) with deoxyinosine (dI) and 3-(2'-deoxy-beta-D-ribofuranosyl) pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one (dP) to synthesize modified oligodeoxyribonucleotides (ODNs). These modified ODNs demonstrated unique hybridization properties, exhibiting selective complementary binding and the ability to form stable, sequence-specific hybrids with unmodified nucleic acid strands.
Liu and Martin (2001) site-specifically introduced fluorescent probes into DNA to map melted regions of the DNA directly in a functionally paused elongation complex.
The pyrrolo-dC probe allows investigating of the transcription bubble in the T7 RNA polymerase elongation complexes. Pairing pyrrolo-dC with complementary guanine quenches the fluorescence of this base analog but enhances its fluorescence in single-stranded DNA. Hence, the melting or opening of DNA strands increases fluorescence intensity.
The fluorescence intensity of the pyrrole-dC probe, which substitutes for cytosine bases, is sensitive to its environment; the fluorescence is quenched in duplex DNA relative to its fluorescence in single-stranded DNA. This probe provides direct information on the local melting of the DNA. Placement of this new probe at specific positions in the non-template strand shows that the elongation bubble extends about eight bases upstream of the pause site.
Building on Woo et al.’s results, Berry et al. (2004) incorporated pyrrolo-dC into oligodeoxyribonucleotides using standard automated oligonucleotide synthesis, showcasing its potential as a fluorescent analog of deoxycytidine.
Dash et al. (2004) made a significant breakthrough by demonstrating the use of pyrrolo-dC in an oligonucleotide-based probe to study RNA/DNA hybrids containing the human immunodeficiency virus type-1 3′-polypurine tract. This novel application of pyrrolo-dC showcases its versatility and potential in various research areas. Additionally, Zang et al. (2005) utilized pyrrolo-dC incorporated into oligonucleotides to study base flipping by human O6-alkylguanine DNA alkyltransferase (AGT) involved in DNA repair, further expanding the scope of its applications.
Zhang and Wadkins (2009) investigated the structural effects of pyrrolo-dC incorporated in DNA hairpins. The researchers also studied how pyrrolo-dC influenced the binding of the hairpins by a fluorescent analog of the drug Actinomycin D. The study revealed that there is very little perturbation of the DNA structure upon incorporation of pyrrolo-dC and that the analog allows studying DNA secondary structures. Further, this study extended the use of pyrrolo-dC for analyzing DNA secondary structures, such as DNA hairpins. Using pyrrolo-dC in oligonucleotides enables the differentiation of paired and unpaired bases in secondary structures. Similarly, this analog is expected to enable studies of related secondary structures such as cruciforms and quadruplexes. Further, the study revealed that pyrrolo-dC can act as a fluorescence resonance energy transfer donor for the fluorescent drug 7-aminoactinomycin D.
Li et al. (2010) developed signal-on flurescent aptasensor based on unlabeled aptemers and pyrrolo-dC for selective and sensitive target detection.
Ming and Seela (2012) reported the synthesis of pyrrolo-dC click adducts useful for SNP detection in long DNA targets.
Also in 2012, Ming et al. reported the synthesis of oligonucleotides containing a G-clamp or a pyrrolo-dC. This study revealed that a pyrrolo-dC derivative behaved like dC but the a G-clamp formed a more stable base pair with 2′-deoxyisoguanosine in DNA with parallel chain orientation than with 2′-deoxyguanosine in aps DNA.
Noe et al. (2012) described the synthesis and photophysical properties of four fluorescent nucleoside analogs, related to pyrrolo-C (PyC) and pyrrolo-dC (PydC) through the conjugation or fusion of a thiophene moiety. The research group reported the results of their photophysical analysis of the nucleosides compared to PyC.
Tor Y. (2024) reported that isomorphic emissive nucleosides and nucleotides, particularly purine analogs, can serve as substrates for diverse enzymes. For example, emissive analogs interacting with metabolic and catabolic enzymes can open optical windows into the biochemistry of nucleosides and nucleotides as metabolites, coenzymes, and second messengers. Real-time fluorescence-based assays for adenosine deaminase, guanine deaminase, and cytidine deaminase have been fabricated and used for inhibitor discovery. The synthesis of emissive cofactors, coenzymes, and second messengers, together with xyNTPs and native enzymes, allows for fluorescence-based monitoring of biotransformation in real time.
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