scholarly journals Enzymatic Formation of an Artificial Base Pair Using a Modified Purine Nucleoside Triphosphate

2020 ◽  
Vol 15 (11) ◽  
pp. 2872-2884
Author(s):  
Marie Flamme ◽  
Pascal Röthlisberger ◽  
Fabienne Levi-Acobas ◽  
Mohit Chawla ◽  
Romina Oliva ◽  
...  
Keyword(s):  
Base Pair ◽  
Purine Nucleoside ◽  
Nucleoside Triphosphate ◽  
Enzymatic Formation

scholarly journals Enzymatic Formation of an Artificial Base Pair Using a Modified Adenine Nucleoside Triphosphate

2019 ◽  
Author(s):  
Marie Flamme ◽  
Pascal Röthlisberger ◽  
Fabienne Levi-Acobas ◽  
Mohit Chawla ◽  
Romina Oliva ◽  
...  
Keyword(s):  
Base Pair ◽  
In Vitro Selection ◽  
Solid Phase ◽  
Nucleoside Triphosphate ◽  
Dna Duplexes ◽  
Base Pairs ◽  
Metal Base ◽  
Enzymatic Formation ◽  
Genetic Alphabet

The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However,the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches and very little is known on polymerase-mediated synthesis. Herein, we report on the selective and high yielding enzymatic construction of a silver-mediated base pair as well as a two-step protocol for the synthesis of DNA duplexes containing a metal UBP. Guided by DFT calculations, we also shed light into the mechanism of formation of this UBP as well as into the structural and energetic preferences. Even though this silver UBP is not directly amenable to in vitro selection experiments, the enzymatic synthesis of this UBP provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet. <br>

Enzymatic Formation of an Artificial Base Pair Using a Modified Adenine Nucleoside Triphosphate

2019 ◽  
Author(s):  
Marie Flamme ◽  
Pascal Röthlisberger ◽  
Fabienne Levi-Acobas ◽  
Mohit Chawla ◽  
Romina Oliva ◽  
...  
Keyword(s):  
Base Pair ◽  
In Vitro Selection ◽  
Solid Phase ◽  
Nucleoside Triphosphate ◽  
Dna Duplexes ◽  
Base Pairs ◽  
Metal Base ◽  
Enzymatic Formation ◽  
Genetic Alphabet

The expansion of the genetic alphabet with additional, unnatural base pairs (UBPs) is an important and long standing goal in synthetic biology. Nucleotides acting as ligands for the coordination of metal cations have advanced as promising candidates for such an expansion of the genetic alphabet. However,the inclusion of artificial metal base pairs in nucleic acids mainly relies on solid-phase synthesis approaches and very little is known on polymerase-mediated synthesis. Herein, we report on the selective and high yielding enzymatic construction of a silver-mediated base pair as well as a two-step protocol for the synthesis of DNA duplexes containing a metal UBP. Guided by DFT calculations, we also shed light into the mechanism of formation of this UBP as well as into the structural and energetic preferences. Even though this silver UBP is not directly amenable to in vitro selection experiments, the enzymatic synthesis of this UBP provides valuable insights for the design of future, more potent systems aiming at expanding the genetic alphabet. <br>

scholarly journals Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses.

1987 ◽  
Vol 7 (2) ◽  
pp. 838-846 ◽  
Author(s):  
R S McIvor ◽  
M J Johnson ◽  
A D Miller ◽  
S Pitts ◽  
S R Williams ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Adenosine Deaminase ◽  
Base Pair ◽  
Purine Nucleoside Phosphorylase ◽  
Regulatory Elements ◽  
Purine Nucleoside ◽  
Northern Analysis ◽  
Hematopoietic Stem ◽  
Nucleoside Phosphorylase

Cell lines were established which produced high titers (approximately 10(6) infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.

scholarly journals A semisynthetic organism engineered for the stable expansion of the genetic alphabet

2017 ◽  
Vol 114 (6) ◽  
pp. 1317-1322 ◽  
Author(s):  
Yorke Zhang ◽  
Brian M. Lamb ◽  
Aaron W. Feldman ◽  
Anne Xiaozhou Zhou ◽  
Thomas Lavergne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Base Pair ◽  
Genetic Information ◽  
Information Storage ◽  
Nucleoside Triphosphate ◽  
Proof Of Concept ◽  
Sequence Context ◽  
Form Of Life ◽  
Nucleoside Triphosphates ◽  
Genetic Alphabet

All natural organisms store genetic information in a four-letter, two-base-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported thatEscherichia coligrown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA containing a single dNaM-d5SICS UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used a more chemically optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet.

Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses

1987 ◽  
Vol 7 (2) ◽  
pp. 838-846
Author(s):  
R S McIvor ◽  
M J Johnson ◽  
A D Miller ◽  
S Pitts ◽  
S R Williams ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Adenosine Deaminase ◽  
Base Pair ◽  
Purine Nucleoside Phosphorylase ◽  
Regulatory Elements ◽  
Purine Nucleoside ◽  
Northern Analysis ◽  
Hematopoietic Stem ◽  
Nucleoside Phosphorylase

Cell lines were established which produced high titers (approximately 10(6) infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.

On the enzymatic incorporation of an imidazole nucleotide into DNA

2017 ◽  
Vol 15 (20) ◽  
pp. 4449-4455 ◽  
Author(s):  
Pascal Röthlisberger ◽  
Fabienne Levi-Acobas ◽  
Ivo Sarac ◽  
Philippe Marlière ◽  
Piet Herdewijn ◽  
...  
Keyword(s):  
Genetic Code ◽  
Base Pair ◽  
Nucleoside Triphosphate ◽  
Metal Base ◽  
Modified Nucleoside

We have evaluated the possibility for using an imidazole modified nucleoside triphosphate for the enzymatic construction of artificial metal base pair with view on an expansion of the genetic code.

scholarly journals In the Uncoupling Protein (UCP-1) His-214 Is Involved in the Regulation of Purine Nucleoside Triphosphate but Not Diphosphate Binding

1998 ◽  
Vol 273 (38) ◽  
pp. 24368-24374 ◽  
Author(s):  
Karim S. Echtay ◽  
Martin Bienengraeber ◽  
Edith Winkler ◽  
Martin Klingenberg
Keyword(s):  
Uncoupling Protein ◽  
Purine Nucleoside ◽  
Nucleoside Triphosphate

scholarly journals Design and synthesis of purine nucleoside analogues for the formation of stable anti-parallel-type triplex DNA with duplex DNA bearing the 5mCG base pair

RSC Advances ◽  
2021 ◽  
Vol 11 (35) ◽  
pp. 21390-21396
Author(s):  
Ryotaro Notomi ◽  
Lei Wang ◽  
Shigeki Sasaki ◽  
Yosuke Taniguchi
Keyword(s):  
Base Pair ◽  
Purine Nucleoside ◽  
Nucleoside Analogues ◽  
Triplex Dna ◽  
Duplex Dna ◽  
Design And Synthesis ◽  
Direct Recognition ◽  
First Time ◽  
Parallel Type

We herein demonstrated for the first time the direct recognition of duplex DNA bearing the 5-methyl-2′-deoxycytosine and 2′-deoxyguanosine base pair by triplex DNA formation.

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