Encoding Phenotype in Bacteria with an Alternative Genetic Set

Krueger, Andrew T.; Peterson, Larryn W.; Chelliserry, Jijumon; Kleinbaum, Daniel J.; Kool, Eric T.

doi:10.1021/ja208025e

Cited by 67 publications

(54 citation statements)

References 23 publications

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“…Although the 2′-endo sugar pucker is the predominantly observed conformation, some 2′-5′-linked residues can adopt both 2′-endo and 3′-endo conformations. Similar structural flexibility has also been observed in other natural and artificial DNA and RNA duplex systems capable of accommodating various perturbations (19,(36)(37)(38)(39)(40)(41).…”

Section: Discussionsupporting

confidence: 72%

Structural insights into the effects of 2′-5′ linkages on the RNA duplex

Sheng

Engelhart

et al. 2014

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

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The mixture of 2′-5′ and 3′-5′ linkages generated during the nonenzymatic replication of RNA has long been regarded as a central problem for the origin of the RNA world. However, we recently observed that both a ribozyme and an RNA aptamer retain considerable functionality in the presence of prebiotically plausible levels of linkage heterogeneity. To better understand the RNA structure and function in the presence of backbone linkage heterogeneity, we obtained high-resolution X-ray crystal structures of a native 10-mer RNA duplex (1.32 Å) and two variants: one containing one 2′-5′ linkage per strand (1.55 Å) and one containing three such linkages per strand (1.20 Å). We found that RNA duplexes adjust their local structures to accommodate the perturbation caused by 2′-5′ linkages, with the flanking nucleotides buffering the disruptive effects of the isomeric linkage and resulting in a minimally altered global structure. Although most 2′-linked sugars were in the expected 2′-endo conformation, some were partially or fully in the 3′-endo conformation, suggesting that the energy difference between these conformations was relatively small. Our structural and molecular dynamic studies also provide insight into the diminished thermal and chemical stability of the duplex state associated with the presence of 2′-5′ linkages. Our results contribute to the view that a low level of 2′-5′ substitution would not have been fatal in an early RNA world and may in contrast have been helpful for both the emergence of nonenzymatic RNA replication and the early evolution of functional RNAs.origin of life | backbone heterogeneity | X-ray crystallography T he capacity of RNA to act as both a carrier of genetic information and as a catalyst has led many to investigate its potential role as the first biopolymer (1-4). An early stage involving nonenzymatic replication simplifies RNA-first scenarios, but known nonenzymatic copying reactions generate a mixture of 3′-5′ and 2′-5′ backbone linkages because of the similar nucleophilicity and orientation of the 2′ and 3′ hydroxyl groups on ribose (Fig. 1). Although regioselectivity for the 3′-5′ linkage can be improved by using different metal ions or activated monomers, it reaches, at most, ∼90% (5-11). This lack of regiospecificity has been regarded as a central problem for the emergence of the RNA world, because the resulting backbone heterogeneity was expected to disrupt the folding, molecular recognition, and catalytic properties of functional RNAs. However, we recently observed that functional nucleic acid molecules can still evolve in the presence of nonheritable mixed DNA/RNA backbone heterogeneity (12), and known functional RNAs retain catalytic and ligand binding behavior in the presence of 2′-5′/3′-5′ backbone linkage heterogeneity (13).The well-known duplex-destabilizing property of 2′-5′ linkages can enable thermal strand separation of long RNA duplexes in the presence of the high Mg 2+ concentrations required for known prebiotic copying reactions (13-16). However, the mechanism r...

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Section: Discussionsupporting

confidence: 72%

Structural insights into the effects of 2′-5′ linkages on the RNA duplex

Sheng

Engelhart

et al. 2014

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

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“…Although many candidate unnatural base pairs have been reported (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(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.

162

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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“…NMR spectra were recorded on Bruker AC-250, Bruker Avance 400 or Jeol 400 spectrometers with 1 H spectra referenced to TMS, 13 C spectra referenced to CDCl3 or DMSO-d6, and 31 P spectra referenced to 85% H3PO4 (aq). Mass spectra were recorded in chemical ionisation (CI) or electrospray ionisation (ES) mode with a Hewlett-Packard HP 5989B MS Engine apparatus using a HP 59987A API-electrospray LC/MS interface.…”

Section: Methodsmentioning

confidence: 99%

“…13 Pyridine-stretched adenine is fluorescent and when incorporated into oligomers as the acyclonucleoside 6 provides a useful probe for the detection of single nucleotide polymorphisms in representative DNA and RNA sequences. [14][15][16] …”

mentioning

confidence: 99%

Preparation of pyridine-stretched 2'-deoxyhypoxanthosine phosphoramidite

Clayton¹,

Davis

et al. 2017

Arkivoc

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Pyridine-stretched 2′-deoxyhypoxanthosine (strH) phosphoramidite was prepared in eight steps from Hoffer's sugar (2′-deoxy-3,5-di-O-(p-toluoyl)--D-erythro-pentofuranosyl chloride). Improved synthesis of the Hoffer sugar was achieved without need for distillation or chromatographic separation of intermediates, or use of gaseous HCl. Conditions were optimised to provide a key nitrile intermediate for the preparation of strH whereby the cesium salt of 4(5)-nitroimidazole was glycosylated using Hoffer's sugar. The nitrile intermediate was also used to prepare pyridine-stretched 2′-deoxyadenosine (strA) and pyridine-stretched 2′-deoxy-2,6-diaminonebulamine (strD). Preliminary studies indicate that strH forms a stronger, size-expanded base pair with adenine compared with the Watson-Crick thymine-adenine base pair.

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Encoding Phenotype in Bacteria with an Alternative Genetic Set

Cited by 67 publications

References 23 publications

Structural insights into the effects of 2′-5′ linkages on the RNA duplex

Structural insights into the effects of 2′-5′ linkages on the RNA duplex

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

Preparation of pyridine-stretched 2'-deoxyhypoxanthosine phosphoramidite

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