Capacity of N4-methyl-2′-deoxycytidine 5′-triphosphate to sustain the polymerase chain reaction using various thermostable DNA polymerases

Flores-Juárez, Cyntia R.; González-Jasso, Eva; Antaramián, Anaid; Pless, Reynaldo C.

doi:10.1016/j.ab.2013.03.025

“…Previously, we demonstrated the capacity of N 4 me-dCTP in the reaction mixture to produce specific PCR products with reduced T m values from DNA regions with moderate GC content (19). Here, GC-rich segments of different human genes were tested by PCR amplification using a reaction mixture containing the canonical set of nucleoside triphosphates at 200 M each or one in which dCTP was replaced with N 4 me-dCTP, either completely (200 M) or with various proportions of N 4 me-dCTP:dCTP [i.e., 10:1 (182 M:18 M), 5:1 (167 M:33 M), or 3:1 (150 M: 50 M)].…”

Section: Resultsmentioning

confidence: 99%

“…Primers were selected that contained the fewest possible guanines in their sequence in order to minimize the reduction of the primer hybridization temperature for the DNA template containing N 4 meC (19). Primers were also evaluated for their tendency to form homodimers, heterodimers, and hairpin structures using the OligoAnalyzer program (IDT).…”

Section: Methodsmentioning

confidence: 99%

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PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate

Flores-Juárez

¹

,

González-Jasso

²

,

Antaramián

³

et al. 2016

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GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N4me-dCTP-containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N4me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (Tm) clearly below the Tm of the same amplicon synthesized exclusively with the canonical bases.

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“…However, presence of N4C-dG base pairs induce a destabilization of duplex DNA (T m of -4.5°C per modification 5 ) because of the reduction of the hydration of the double helical structure. 6 The corresponding triphosphate (2 in Figure 2) has been synthesized and shown to be accepted by various DNA polymerases and incorporated into DNA via PCR. 6 Other dCTP analogues bearing small alkyl substituents(see reference S11 of the Further readings section)or even reactive functional groups 7 at position N4 of cytosine have been reported and shown to be accepted by polymerases even though with reduced efficiency compared to the corresponding canonical nucleotides.…”

Section: A) Epigenetic Base-modification Patterns On Dnamentioning

confidence: 99%

“…6 The corresponding triphosphate (2 in Figure 2) has been synthesized and shown to be accepted by various DNA polymerases and incorporated into DNA via PCR. 6 Other dCTP analogues bearing small alkyl substituents(see reference S11 of the Further readings section)or even reactive functional groups 7 at position N4 of cytosine have been reported and shown to be accepted by polymerases even though with reduced efficiency compared to the corresponding canonical nucleotides. Hence, the N4 position of cytosine appears to be a favorable location for the introduction of chemical modifications, both naturally occurring or synthetic, in nucleoside triphosphates without impairing their substrate capacities.…”

Section: A) Epigenetic Base-modification Patterns On Dnamentioning

confidence: 99%

Enzymatic Synthesis of Base-Modified Nucleic Acids

Hollenstein¹

2023

Handbook of Chemical Biology of Nucleic Acids

0

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Nucleic acids collectively play primordial functions, some of which are still barely understood, in all living organisms. DNA and RNA oligonucleotides have also advanced as pivotal tools in numerous applications such as therapeutic technologies, diagnostics, or material sciences. Chemical modifications, particularly at the level of the nucleobases, are ubiquitous. Indeed, base-modifications in natural DNA and RNA are nearly as important as the primary sequences and are involved in numerous biological roles and functions, mainly via epigenetic regulation mechanisms. In synthetic nucleic acids, chemical modifications provide oligonucleotides with improved binding or catalytic activities as well as exogenous reactivities, all of which are largely non-accessible to canonical DNA or RNA. Nucleobase-modifications can be incorporated into oligonucleotides either by standard solid-phase synthesis or by application of a chemoenzymatic method. In this Chapter, we will describe how the combination of polymerases and nucleoside triphosphate analogs can be harnessed to synthesize oligonucleotides containing nucleobasemodifications. We will highlight the potency of this approach through the description of relevant examples including synthesis of oligonucleotides containing naturally occurring modifications and the selection of functional nucleic acids with improved properties. In a last section of this Chapter, more recent applications of this method will be discussed with an emphasis on mRNA-based vaccines and on controlled enzymatic synthesis.

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Enzymatic Synthesis of Base-Modified Nucleic Acids

Hollenstein

¹

2023

Handbook of Chemical Biology of Nucleic Acids

3

0

View full text Add to dashboard Cite

Nucleic acids collectively play primordial functions, some of which are still barely understood, in all living organisms. DNA and RNA oligonucleotides have also advanced as pivotal tools in numerous applications such as therapeutic technologies, diagnostics, or material sciences. Chemical modifications, particularly at the level of the nucleobases, are ubiquitous. Indeed, base-modifications in natural DNA and RNA are nearly as important as the primary sequences and are involved in numerous biological roles and functions, mainly via epigenetic regulation mechanisms. In synthetic nucleic acids, chemical modifications provide oligonucleotides with improved binding or catalytic activities as well as exogenous reactivities, all of which are largely non-accessible to canonical DNA or RNA. Nucleobase-modifications can be incorporated into oligonucleotides either by standard solid-phase synthesis or by application of a chemoenzymatic method. In this Chapter, we will describe how the combination of polymerases and nucleoside triphosphate analogs can be harnessed to synthesize oligonucleotides containing nucleobasemodifications. We will highlight the potency of this approach through the description of relevant examples including synthesis of oligonucleotides containing naturally occurring modifications and the selection of functional nucleic acids with improved properties. In a last section of this Chapter, more recent applications of this method will be discussed with an emphasis on mRNA-based vaccines and on controlled enzymatic synthesis.

show abstract

Capacity of N4-methyl-2′-deoxycytidine 5′-triphosphate to sustain the polymerase chain reaction using various thermostable DNA polymerases

Cited by 5 publications

References 40 publications

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate

Enzymatic Synthesis of Base-Modified Nucleic Acids

Enzymatic Synthesis of Base-Modified Nucleic Acids

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Capacity of N4-methyl-2′-deoxycytidine 5′-triphosphate to sustain the polymerase chain reaction using various thermostable DNA polymerases

Cited by 5 publications

References 40 publications

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N 4 -Methyl-2′-Deoxycytidine 5′-Triphosphate

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N 4 -Methyl-2′-Deoxycytidine 5′-Triphosphate

Enzymatic Synthesis of Base-Modified Nucleic Acids

Enzymatic Synthesis of Base-Modified Nucleic Acids

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Product

Resources

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PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate

PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate