Generation of long, fully modified, and serum-resistant oligonucleotides by rolling circle amplification

Hollenstein, Marcel

doi:10.1039/c5ob01540e

Cited by 18 publications

(17 citation statements)

References 55 publications

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“…Nevertheless, supplementing with all the necessary nucleotides would easily solve such an insignificant misincorporation process and offer an opportunity for the application of N 4 -modified nucleotides for the isothermal amplification during SELEX. Although phi29 DNA polymerase has been applied in a variety of DNA amplification procedures, only few cases have been reported regarding the use of non-natural substrates ( 56 , 57 ). Here, we demonstrate that phi29 DNA polymerase is able to incorporate N 4 -acylated cytidine analogues resulting in the synthesis of functionalized DNA.…”

Section: Discussionmentioning

confidence: 99%

N 4-acyl-2′-deoxycytidine-5′-triphosphates for the enzymatic synthesis of modified DNA

Jakubovska

Tauraitė

Birštonas

et al. 2018

Nucleic Acids Research

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A huge diversity of modified nucleobases is used as a tool for studying DNA and RNA. Due to practical reasons, the most suitable positions for modifications are C5 of pyrimidines and C7 of purines. Unfortunately, by using these two positions only, one cannot expand a repertoire of modified nucleotides to a maximum. Here, we demonstrate the synthesis and enzymatic incorporation of novel N4-acylated 2′-deoxycytidine nucleotides (dCAcyl). We find that a variety of family A and B DNA polymerases efficiently use dCAcylTPs as substrates. In addition to the formation of complementary CAcyl•G pair, a strong base-pairing between N4-acyl-cytosine and adenine takes place when Taq, Klenow fragment (exo–), Bsm and KOD XL DNA polymerases are used for the primer extension reactions. In contrast, a proofreading phi29 DNA polymerase successfully utilizes dCAcylTPs but is prone to form CAcyl•A base pair under the same conditions. Moreover, we show that terminal deoxynucleotidyl transferase is able to incorporate as many as several hundred N4-acylated-deoxycytidine nucleotides. These data reveal novel N4-acylated deoxycytidine nucleotides as beneficial substrates for the enzymatic synthesis of modified DNA, which can be further applied for specific labelling of DNA fragments, selection of aptamers or photoimmobilization.

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Section: Discussionmentioning

confidence: 99%

N 4-acyl-2′-deoxycytidine-5′-triphosphates for the enzymatic synthesis of modified DNA

Jakubovska

Tauraitė

Birštonas

et al. 2018

Nucleic Acids Research

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“…An RCA reaction requires four factors: A DNA polymerase, a corresponding DNA primer, a template, and deoxynucleotide triphosphates (dNTPs) [84]. In RCA reaction, nucleotides are added continuously to the annealed primer by the polymerase, which generates a long ssDNA with a repeating sequence [85]. RCA is a powerful induction system because it can produce large amounts of ssDNA at the scale of a micron and a detectable amplification of a single molecule [84].…”

Section: Rolling Circle Amplificationmentioning

confidence: 99%

Current and Emerging Methods for the Synthesis of Single-Stranded DNA

Hao

Qiao

2020

Genes

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Methods for synthesizing arbitrary single-strand DNA (ssDNA) fragments are rapidly becoming fundamental tools for gene editing, DNA origami, DNA storage, and other applications. To meet the rising application requirements, numerous methods have been developed to produce ssDNA. Some approaches allow the synthesis of freely chosen user-defined ssDNA sequences to overcome the restrictions and limitations of different length, purity, and yield. In this perspective, we provide an overview of the representative ssDNA production strategies and their most significant challenges to enable the readers to make informed choices of synthesis methods and enhance the availability of increasingly inexpensive synthetic ssDNA. We also aim to stimulate a broader interest in the continued development of efficient ssDNA synthesis techniques and improve their applications in future research.

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“…12,13 Recent examples include incorporation of horseradish peroxidase (HRP) conjugated to dTTP by a linker at the C5 position 14 and nucleotides conjugated to side chains of the amino acids His, Ser, and Asp. 15 SomaLogic has systematically exploited this principle to create Slow-Off-Rate Modified Aptamers (SOMAmers), in which the C5 of dU has been modified to bear a range of hydrophobic substituents ( 9 – 11 ). 16 Recent crystal structures of SOMAmers bound to their targets show a remarkable expansion in aptamer functionality, including folding motifs not previously seen in DNA.…”

Section: Replication Of Modified Nucleotides By Natural Polymerasesmentioning

confidence: 99%

Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering

2017

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ConspectusNucleic acids are a distinct form of sequence-defined biopolymer. What sets them apart from other biopolymers such as polypeptides or polysaccharides is their unique capacity to encode, store, and propagate genetic information (molecular heredity). In nature, just two closely related nucleic acids, DNA and RNA, function as repositories and carriers of genetic information. They therefore are the molecular embodiment of biological information. This naturally leads to questions regarding the degree of variation from this seemingly ideal “Goldilocks” chemistry that would still be compatible with the fundamental property of molecular heredity.To address this question, chemists have created a panoply of synthetic nucleic acids comprising unnatural sugar ring congeners, backbone linkages, and nucleobases in order to establish the molecular parameters for encoding genetic information and its emergence at the origin of life. A deeper analysis of the potential of these synthetic genetic polymers for molecular heredity requires a means of replication and a determination of the fidelity of information transfer. While non-enzymatic synthesis is an increasingly powerful method, it currently remains restricted to short polymers. Here we discuss efforts toward establishing enzymatic synthesis, replication, and evolution of synthetic genetic polymers through the engineering of polymerase enzymes found in nature.To endow natural polymerases with the ability to efficiently utilize non-cognate nucleotide substrates, novel strategies for the screening and directed evolution of polymerase function have been realized. High throughput plate-based screens, phage display, and water-in-oil emulsion technology based methods have yielded a number of engineered polymerases, some of which can synthesize and reverse transcribe synthetic genetic polymers with good efficiency and fidelity.The inception of such polymerases demonstrates that, at a basic level at least, molecular heredity is not restricted to the natural nucleic acids DNA and RNA, but may be found in a large (if finite) number of synthetic genetic polymers. And it has opened up these novel sequence spaces for investigation. Although largely unexplored, first tentative forays have yielded ligands (aptamers) against a range of targets and several catalysts elaborated in a range of different chemistries. Finally, taking the lead from established DNA designs, simple polyhedron nanostructures have been described.We anticipate that further progress in this area will expand the range of synthetic genetic polymers that can be synthesized, replicated, and evolved providing access to a rich sequence, structure, and phenotypic space. “Synthetic genetics”, that is, the exploration of these spaces, will illuminate the chemical parameter range for en- and decoding information, 3D folding, and catalysis and yield novel ligands, catalysts, and nanostructures and devices for applications in biotechnology and medicine.

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Generation of long, fully modified, and serum-resistant oligonucleotides by rolling circle amplification

Cited by 18 publications

References 55 publications

N 4-acyl-2′-deoxycytidine-5′-triphosphates for the enzymatic synthesis of modified DNA

N 4-acyl-2′-deoxycytidine-5′-triphosphates for the enzymatic synthesis of modified DNA

Current and Emerging Methods for the Synthesis of Single-Stranded DNA

Exploring the Chemistry of Genetic Information Storage and Propagation through Polymerase Engineering

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