Enzymatic incorporation of chemically-modified nucleotides into DNAs

Kuwahara, Masayasu; Ohbayashi, Tsutomu; Hanawa, Kazuo; Shoji, Atsushi; Ozaki, Akiko; Ozaki, Hiroaki; Sawai, Hiroaki

doi:10.1093/nass/2.1.83

Cited by 6 publications

(2 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, immunohistochemical staining for VEGFα was performed in the aortic rings of α-GAL-Tg/KO and WT mice, which showed a higher degree of staining of VEGFα in α-GAL-Tg/KO mice. Although smooth muscle cells have been shown to be the major source of VEGFα expression and secretion ( 26 ), and accordingly in a model of hypertensive rats, VEGFα expression was mainly distributed in the outer to middle layers of the media ( 27 ), our data indicate that the VEGFα signal in mice aortic tissue was mainly increased in the adventitial layer. Whether this is a species-specific effect is not known.…”

Section: Discussionmentioning

confidence: 61%

Overexpression of VEGFα as a biomarker of endothelial dysfunction in aortic tissue of α-GAL-Tg/KO mice and its upregulation in the serum of patients with Fabry’s disease

Lund,

Wieboldt,

Fischer

et al. 2024

Front. Cardiovasc. Med.

View full text Add to dashboard Cite

IntroductionFabry's disease is an X-linked lysosomal storage disorder caused by reduced activity of α-galactosidase A (GAL), leading to premature death on account of renal, cardiac, and vascular organ failure. Accumulation of the GAL substrate globotriaosylceramide (Gb3) in endothelial and smooth muscle cells is associated with early vascular cell damage, suggesting endothelial dysfunction as a driver of cardiorenal organ failure. Here, we studied the vascular expression of the key angiogenic factors, VEGFα and its antagonist angiostatin, in Fabry α-GAL-Tg/KO mice and determined circulating VEGFα and angiostatin serum levels in patients with Fabry’s disease and healthy controls.MethodsCryopreserved aortic vessels from six α-GAL-Tg/KO and six wild-type (WT) mice were obtained and VEGFα and angiostatin levels were determined by performing Western blot analysis. VEGFα expression was visualized by an immunohistochemical staining of paraffin aortic rings. In addition, VEGFα and angiostatin serum levels were measured by using an enzyme-linked immunosorbent assay in 48 patients with genetically verified Fabry's disease (50% male) and 22 healthy controls and correlated with disease severity markers such as lyso-Gb3, albuminuria, NTproBNP, high-sensitive troponin T (hsTNT), and myocardial wall thickness.ResultsIt was found that there was a significant increase in VEGFα protein expression (1.66 ± 0.35 vs. 0.62 ± 0.16, p = 0.0009) and a decrease in angiostatin expression (0.024 ± 0.007 vs. 0.053 ± 0.02, p = 0.038) in aortic lysates from α-GAL-Tg/KO compared with that from WT mice. Immunohistochemical staining revealed an adventitial VEGFα signal in α-GAL-Tg/KO mice, whereas no VEGFα signal could be detected in WT mice aortas. No differences in aortic angiostatin expression between α-GAL-Tg/KO- and WT mice could be visualized. The serum levels of VEGFα were significantly upregulated in patients with Fabry’s disease compared with that in healthy controls (708.5 ± 426.3 vs. 458.5 ± 181.5 pg/ml, p = 0.048) and positively associated with albuminuria (r = 0.82, p < 0.0001) and elevated NTproBNP (r = 0.87, p < 0.0001) and hsTNT values (r = 0.41, p = 0.048) in male patients with Fabry’s disease. For angiostatin, no significant difference was found between patients with Fabry’s disease and healthy controls (747.6 ± 390.3 vs. 858.8 ± 599.3 pg/ml).DiscussionIn conclusion, an overexpression of VEGFα and downregulation of its counter player angiostatin in aortic tissue of α-GAL-Tg/KO mice support the hypothesis of an underlying vasculopathy in Fabry's disease. Elevated VEGFα serum levels were also observed in patients with Fabry’s disease and were positively associated with elevated markers of organ manifestation in males. These findings suggest that angiogenetic markers, such as VEGFα, may be potentially useful biomarkers for the detection of endothelial dysfunction in classical Fabry's disease.

show abstract

Section: Discussionmentioning

confidence: 61%

Overexpression of VEGFα as a biomarker of endothelial dysfunction in aortic tissue of α-GAL-Tg/KO mice and its upregulation in the serum of patients with Fabry’s disease

Lund,

Wieboldt,

Fischer

et al. 2024

Front. Cardiovasc. Med.

View full text Add to dashboard Cite

show abstract

“…Starting over 15 years ago, we and many others have reported on the enzymatic incorporation of modified nucleoside triphosphates to produce modified DNA with additional functionalities. In many examples only one modified dXTP is incorporated [11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34], while in other cases two [35,36] and three modified nucleosides have been incorporated simultaneously in lieu of their unmodified counterparts [37,38,39,40]. In order to increase the chemical functionality of aptamers and DNAzymes, various nucleoside triphosphates must be first tested as substrates for various DNA polymerases.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates

2015

View full text Add to dashboard Cite

Abstract:To expand the chemical functionality of DNAzymes and aptamers, several new modified deoxyuridine triphosphates have been synthesized. An important precursor that enables this aim is 5-aminomethyl dUTP, whereby the pendent amine serves as a handle for further synthetic functionalization. Five functional groups were conjugated to 5-aminomethyl dUTP. Incorporation assays were performed on several templates that demand 2-5 sequential incorporation events using several commercially available DNA polymerases. It was found that Vent (exo-) DNA polymerase efficiently incorporates all five modified dUTPs. In addition, all nucleoside triphosphates were capable of supporting a double-stranded exponential PCR amplification. Modified PCR amplicons were PCR amplified into unmodified DNA and sequenced to verify that genetic information was conserved through incorporation, amplification, and reamplification. Overall these modified dUTPs represent new candidate substrates for use in selections using modified nucleotide libraries.

show abstract

Enzymatic Incorporation of Emissive Pyrimidine Ribonucleotides

Srivatsan

Tor

2009

Chemistry An Asian Journal

View full text Add to dashboard Cite

The enzymatic incorporation of a series of emissive pyrimidine analogues into RNA oligonucleotides is explored. T7 RNA polymerase is challenged with accepting three non-natural, yet related, triphosphates as substrates and incorporating them into diverse RNA transcripts. The three ribonucloside triphosphates differ only in the modification of their uracil nucleus and include a thieno[3,2-d]pyrimidine nucleoside, a thieno[3,4-d]pyrimidine derivative, and a uridine containing a thiophene ring conjugated at its 5-position. All thiophene-containing uridine triphosphates (UTPs) get incorporated into RNA oligonucleotides at positions that are remote to the promoter, although the yields of the transcripts vary compared with the transcript obtained with only native triphosphates. Among the three derivatives, the 5-modified UTP is found to be the most “polymerase-friendly” and is well accommodated by T7 RNA polymerase. Although the fused thiophene analogues cannot be incorporated next to the promoter region, the 5-modified non-natural UTP gets incorporated near the promoter (albeit in relatively low yields) and even in multiple copies. Labeling experiments shed light on the mediocre incorporation of the fused analogues, suggesting the enzyme frequently pauses at the incorporation position. When incorporation does take place, the enzyme fails to elongate the modified oligonucleotide and yields aborted transcripts. Taken together, these results highlight the versatility and robustness, as well as the scope and limitation, of T7 RNA polymerase in accepting and incorporating reporter nucleotides into modified RNA transcripts.

show abstract

Enzymatic incorporation of chemically-modified nucleotides into DNAs

Cited by 6 publications

References 0 publications

Overexpression of VEGFα as a biomarker of endothelial dysfunction in aortic tissue of α-GAL-Tg/KO mice and its upregulation in the serum of patients with Fabry’s disease

Overexpression of VEGFα as a biomarker of endothelial dysfunction in aortic tissue of α-GAL-Tg/KO mice and its upregulation in the serum of patients with Fabry’s disease

Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates

Enzymatic Incorporation of Emissive Pyrimidine Ribonucleotides

Contact Info

Product

Resources

About