An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

Kimoto, Michiko; Kawai, Rie; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, Ichiro

doi:10.1093/nar/gkn956

Cited by 174 publications

(190 citation statements)

References 31 publications

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“…S1). AFM imaging in fluid also revealed well--defined nanofibers microns in length (height 2.0 ± 0.1 nm), suggesting that CA--mediated assembly of d(A 15 ) is a solution--based phenomenon (Fig. 1c, Supplementary Fig.…”

Section: Evidence Of Ca--mediated Assembly Of D(a) 15mentioning

confidence: 87%

“…The curve revealed CA:A saturation at 1, consistent with a 1:1 CA:A ratio in the structure. A complication of the equilibrium dialysis experiment is that CA binding to d(A 15 ) is coupled to a structural change of the single--stranded DNA into a new assembly, as well as its elongation into fibers 33 . This experiment is different from binding studies of small molecules with double stranded DNA, in which the duplex structure is relatively maintained throughout the dialysis experiment 51 .…”

Section: Investigation Of Fiber Structurementioning

confidence: 99%

“…The creation of artificial nucleic acid motifs has relied on synthetic incorporation of nucleobases with altered hydrogen--bonding patterns 14,15 , or relying on hydrophobic interactions 16,17 and metal coordination 18--20 . Synthetic incorporation of unnatural bases which present more than one hydrogen--bonding 'face' in order to access higher--order DNA structures has been of great interest 21--25 .…”

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confidence: 99%

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Reprogramming the assembly of unmodified DNA with a small molecule

et al. 2016

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The information storage and encoding ability of DNA arise from a remarkably simple 4--letter -A, T, G, C nucleobase code. Expanding this DNA 'alphabet' provides information about its function and evolution, and introduces new functionalities into nucleic acids and organisms. Previous efforts relied on the synthetically demanding incorporation of non--canonical bases into nucleosides. Here we report the discovery that a small molecule, cyanuric acid, with three thymine--like faces reprograms the assembly of unmodified poly(adenine) into stable, long and abundant fibers with a unique internal structure. Poly(A) DNA, RNA and PNA all form these assemblies. Our studies are consistent with the association of adenine and cyanuric acid units into a hexameric rosette, bringing together poly(A) triplexes with subsequent cooperative polymerization. Fundamentally, this study shows that small hydrogen--bonding molecules can be used to induce the assembly of nucleic acids in water, leading to new structures from inexpensive and readily available materials.

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Section: Evidence Of Ca--mediated Assembly Of D(a) 15mentioning

confidence: 87%

Section: Investigation Of Fiber Structurementioning

confidence: 99%

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confidence: 99%

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Reprogramming the assembly of unmodified DNA with a small molecule

et al. 2016

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“…Success would represent a remarkable integration of orthogonal synthetic components into a fundamental biological system and build the foundation for a semisynthetic organism with increased potential for information storage and retrieval (1). Moreover, the constituent unnatural nucleotides could be used to site-specifically label DNA or RNA with different functionalities of interest (2-4) and potentially revolutionize the already ubiquitous in vitro applications of nucleic acids, such as aptamer and DNA/RNAzyme selections (5, 6), PCR-based diagnostics (7, 8), and DNA-based nanomaterials and devices (9).Although many candidate unnatural base pairs have been reported (10-21), only a few are actually replicable by DNA polymerases (10,11,13,16). Moreover, it is clear that most applications will require that the unnatural base pair not only be replicated with high efficiency and fidelity but also, that replication be at least approximately independent of sequence context.…”

mentioning

confidence: 99%

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

Malyshev

Dhami²,

Quach³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

159

130

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The natural four-letter genetic alphabet, comprised of just two base pairs (dA-dT and dG-dC), is conserved throughout all life, and its expansion by the development of a third, unnatural base pair has emerged as a central goal of chemical and synthetic biology. We recently developed a class of candidate unnatural base pairs, exemplified by the pair formed between d5SICS and dNaM. Here, we examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs in a wide variety of sequence contexts. Under standard conditions, we show that this DNA may be amplified with high efficiency and greater than 99.9% fidelity. To more rigorously explore potential sequence effects, we used deep sequencing to characterize a library of templates containing the unnatural base pair as a function of amplification. We found that the unnatural base pair is efficiently replicated with high fidelity in virtually all sequence contexts. The results show that, for PCR and PCR-based applications, d5SICS-dNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, it provides a fully functional six-letter genetic alphabet.expanded genetic alphabet | hydrophobic | artificial DNA | unnatural nucleotides | bioinformatics E xpansion of the genetic alphabet to include an unnatural base pair has emerged as a central goal of chemical and synthetic biology. Success would represent a remarkable integration of orthogonal synthetic components into a fundamental biological system and build the foundation for a semisynthetic organism with increased potential for information storage and retrieval (1). Moreover, the constituent unnatural nucleotides could be used to site-specifically label DNA or RNA with different functionalities of interest (2-4) and potentially revolutionize the already ubiquitous in vitro applications of nucleic acids, such as aptamer and DNA/RNAzyme selections (5, 6), PCR-based diagnostics (7, 8), and DNA-based nanomaterials and devices (9).Although many candidate unnatural base pairs have been reported (10-21), only a few are actually replicable by DNA polymerases (10,11,13,16). Moreover, it is clear that most applications will require that the unnatural base pair not only be replicated with high efficiency and fidelity but also, that replication be at least approximately independent of sequence context. Sequence dependencies would cause biased amplification and effectively preclude many uses of the unnatural base pair. No candidate unnatural base pair has been shown to be replicated without sequence bias, and thus, none can yet claim functional equivalence to a natural base pair.In general, the most promising unnatural base pair candidates currently available have been developed by pursuing one of two different strategies. The first strategy, pioneered in the work by Benner and coworkers (22), relies on the use of nucleotide analogs bearing nucleobases that pair through complementary hydrogen bonding (H-bonding) patterns that are orthogonal to those patterns of the natural pairs. Early ef...

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“…Site-specific internal modification of nucleic acids is a considerable challenge, and new approaches for this purpose would be valuable. [33] In the current experiments, G1 was the only nucleotide of the RNA substrate that was not designed to engage in Watson-Crick base pairing (Fig. 1b), which presumably suppressed nucleophilic reactivity of the nucleobase side chain of G2 or any other nucleobase within the RNA.…”

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confidence: 79%

DNA‐Catalyzed Reductive Amination

Wong¹,

Mulcrone²,

Silverman³

2011

Angewandte Chemie

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Keywordsdeoxyribozymes; DNA; in vitro selection; reductive amination Deoxyribozymes are particular DNA sequences that have catalytic ability, analogous to protein enzymes as functional amino acid sequences. [1,2] The discovery of natural RNA enzymes (ribozymes) spurred the search for artificial deoxyribozymes, [3] which are identified by in vitro selection. [4] Most deoxyribozyme-catalyzed reactions involve phosphodiester bond cleavage or ligation, [5][6][7] although other reactions such as Diels-Alder reaction [8] and thymine dimer photoreversion [9] have also been reported.We have initiated a comprehensive effort to identify deoxyribozymes that catalyze reactions of amino acid side chains, initially focusing on small peptide substrates and with the longerterm goal of covalently modifying large proteins. [10] Towards this goal, as part of a recent study we examined the ability of several new deoxyribozymes to modify tyrosine (or serine) side chains of tripeptide substrates that are not covalently tethered to the deoxyribozyme, although the tether was required to enable the selection process. [11] Deoxyribozymes identified using tethered peptide substrates were also active with untethered peptide substrates, but with substantially lower rate and yield. Therefore, in new experiments, here we performed in vitro selection directly using untethered peptide substrates. This effort required a modified selection procedure that was intended to involve reductive amination as a step merely to "capture" catalytically active DNA sequences. Surprisingly, this selection process provided deoxyribozymes that operate entirely independently of the tripeptide substrate and instead catalyze a Ni 2+ -dependent reductive amination involving the N 2 -amine of a guanosine nucleobase on an RNA substrate, which reacts with an oligonucleotidedialdehyde substrate. Reductive amination is a key biochemical process, e.g. for amino acid biosynthesis and catabolism using amino acid dehydrogenases (oxidases) [12] and transaminases (aminotransferases). [13] In vitro, enzymatic reductive amination is important in both laboratory-scale [14] and industrial-scale organic synthesis. [15] Reductive amination may also have been important in RNA World scenarios. [16,17] Our unexpected discovery of DNA-catalyzed reductive amination suggests further exploration of the abilities of nucleic acid enzymes to catalyze this interesting and potentially useful class of reaction.For any in vitro selection strategy that does not involve physical compartmentalization or segregation of individual candidate sequences, [18] the selection design must include a mechanism by which individual, functional sequences become separable from the vast excess of nonfunctional sequences. Importantly, this mechanism must operate in parallel fashion without requiring independent interrogation (i.e., screening) of each candidate [5] A key advantage of the gel-shift approach is that functional sequences are separated largely on the basis of oligonucleotide length (size), which has incr...

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An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

Cited by 174 publications

References 31 publications

Reprogramming the assembly of unmodified DNA with a small molecule

Reprogramming the assembly of unmodified DNA with a small molecule

Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

DNA‐Catalyzed Reductive Amination

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