scholarly journals Nanopore Sequencing of an Expanded Genetic Alphabet Reveals High-Fidelity Replication of a Predominantly Hydrophobic Unnatural Base Pair

2020 ◽  
Vol 142 (5) ◽  
pp. 2110-2114 ◽  
Author(s):  
Michael P. Ledbetter ◽  
Jonathan M. Craig ◽  
Rebekah J. Karadeema ◽  
Matthew T. Noakes ◽  
Hwanhee C. Kim ◽  
...  
Keyword(s):  
Base Pair ◽  
High Fidelity ◽  
Nanopore Sequencing ◽  
Genetic Alphabet

scholarly journals High-Fidelity Nanopore Sequencing of Ultra-Short DNA Targets

2019 ◽  
Author(s):  
Brandon D. Wilson ◽  
Michael Eisenstein ◽  
H. Tom Soh
Keyword(s):  
High Fidelity ◽  
Nanopore Sequencing

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.

scholarly journals Discovery, Characterization, and Optimization of an Unnatural Base Pair for Expansion of the Genetic Alphabet

2008 ◽  
Vol 130 (7) ◽  
pp. 2336-2343 ◽  
Author(s):  
Aaron M. Leconte ◽  
Gil Tae Hwang ◽  
Shigeo Matsuda ◽  
Petr Capek ◽  
Yoshiyuki Hari ◽  
...  
Keyword(s):  
Base Pair ◽  
Genetic Alphabet

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>

scholarly journals Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

2020 ◽  
Author(s):  
Yan Wang ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Xianyang Fang
Keyword(s):  
Structural Biology ◽  
Base Pair ◽  
Solid Phase ◽  
Site Specific ◽  
X Ray ◽  
X Ray Scattering ◽  
Rna Labeling ◽  
Virus Serotype ◽  
Genetic Alphabet ◽  
Ray Scattering

AbstractConjugation of RNAs with nanoparticles is of significant importance for its numerous applications in biology and medicine, which however remains challenging, especially for large ones. So far, the majority of RNA labeling rely on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nts. We here present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with gold nanoparticle (AuNP) empowered by expanded genetic alphabet transcription. We synthesize an amine-derivatized TPT3 (TPT3A), which are site-specifically incorporated into a 97-nt 3’SL RNA and a 719-nt mini genomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by standard in vitro transcription with expanded genetic alphabet containing the A-T, G-C natural base pairs and the TPT3-NaM unnatural base pair. TPT3 modification cause minimal structural perturbations to the RNAs by small angle X-ray scattering. The purified TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-NHS-Nanogold nanoparticles via the highly selective amine-NHS ester reaction and purified under non-denaturing conditions. We demonstrate the application of the AuNP-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The inter-nanoparticle distance distributions in the 3’SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus validate the applicability of this novel labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies, and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.Significance StatementWe present a site-specific labeling strategy for large RNAs by T7 transcription with expanded genetic alphabet containing TPT3-NaM unnatural base pair. The applicability of this labeling strategy is validated by X-ray scattering interferometry measurements on a 97-nt and a 719-nt RNAs. This strategy can be applicable to natural RNAs or artificial RNA nanostructures with sizes from tens up to thousands of nucleotides, or covalent conjugation of RNAs with other metal nanoparticles. The usage of a far upstream forward primer during PCR enables easy purification of RNA from DNA templates, the non-denaturing conditions for conjugation reactions and purification avoids potential large RNA misfolding. This labeling strategy expands our capability to site-specifically conjugate RNA with nanoparticles for many applications.

Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet

Nature ◽  
1990 ◽  
Vol 343 (6253) ◽  
pp. 33-37 ◽  
Author(s):  
Joseph A. Piccirilli ◽  
Steven A. Benner ◽  
Tilman Krauch ◽  
Simon E. Moroney ◽  
Steven A. Benner
Keyword(s):  
Base Pair ◽  
Dna And Rna ◽  
Genetic Alphabet
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