Synthesis and Properties of Size-Expanded DNAs:  Toward Designed, Functional Genetic Systems

Krueger, Andrew T.; Lu, Haige; Lee, and Alex H. F.; Kool, Eric T.

doi:10.1021/ar068200o

Cited by 138 publications

(113 citation statements)

References 73 publications

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“…It is also possible that the DNA plays a more active role in overcoming the substantial activation barrier against fusion. To shed more light on the fusion mechanism, we are continuing to explore the possibility of varying DNA sequence using both natural and artificial bases (34) to tailor hybridization energetics and to add dye-labels to allow for the quantification of hybridized DNA by FRET. Single vesicle assays with tethered vesicles are expected to be particularly useful to distinguish the progression in the stages of fusion more clearly than experiments performed in bulk.…”

Section: Discussionmentioning

confidence: 99%

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Chan

Lengerich

Boxer

2009

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

278

287

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Synthetic lipid-oligonucleotide conjugates inserted into lipid vesicles mediate fusion when one population of vesicles displays the 5 -coupled conjugate and the other the 3 -coupled conjugate, so that anti-parallel hybridization allows the membrane surfaces to come into close proximity. Improved assays show that lipid mixing proceeds more quickly and to a much greater extent than content mixing, suggesting the latter is rate limiting. To test the effect of membrane-membrane spacing on fusion, a series of conjugates was constructed by adding 2-24 noncomplementary bases at the membrane-proximal ends of two complementary sequences. Increasing linker lengths generally resulted in progressively reduced rates and extents of lipid and content mixing, in contrast to higher vesicle docking rates. The relatively flexible, single-stranded DNA linker facilitates docking but allows greater spacing between the vesicles after docking, thus making the transition into fusion less probable, but not preventing it altogether. These experiments demonstrate the utility of DNA as a model system for fusion proteins, where sequence can easily be modified to systematically probe the effect of distance between bilayers in the fusion reaction.DNA machine ͉ synthetic biology F usion of lipid membranes plays a central role in many biological processes. For example, in the case of synaptic transmission, the reaction is tightly regulated by a number of proteins of which the SNAREs [soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptors] have been shown to play a crucial role (1-3). In vitro studies have demonstrated the sufficiency of SNAREs to mediate fusion when reconstituted into a number of model systems, including freestanding lipid vesicles (4-7), supported lipid bilayers (8-10), and tethered vesicles (11). Generally these studies suggest that SNAREs are able to bring two membranes into close apposition by forming a trans complex in a docking reaction which is highly energetically favorable (12). This is illustrated in Fig. 1A, which is based on the x-ray structures of the soluble domains of SNARE proteins in complex (13). Several steps along the mechanism to achieve membrane fusion following this basic docking step have been proposed, and some of these are illustrated in Fig. 2, which also serves to illustrate assays commonly used to assess their role. In this step-wise mechanism, the bilayers first dock, then adopt an intermediate, hemifused state in which the outer leaflets have merged, but the inner leaflets and contents remain distinct. A transition into full fusion then involves mixing of both leaflets and content exchange.Despite substantial research on the role of SNARE proteins in the fusion process, many questions remain regarding the physical mechanism. Studies using simpler model systems can provide insight into the fundamental mechanisms of the rearrangement of bilayers and lead to a better understanding of the biological process (14, 15). We have prepared a series of DNA-lipid conjugates in which DNA o...

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Section: Discussionmentioning

confidence: 99%

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Chan

Lengerich

Boxer

2009

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

278

287

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“…Most of these investigations were driven by the search for additional base pairs to be used for the extension of the genetic alphabet, [1][2][3][4][5][6][7][8] as tools in biotechnology, [9][10][11] for probing recognition, fidelity, and nucleotide processing by DNA polymerases, [12][13][14][15] or for designing novel genetic systems. [16,17] Of special interest amongst these artificial constructs are aromatic base replacements that interact with each other, specifically without the formation of hydrogen bonds, merely on the basis of edge-on or face-on hydrophobic or stacking interactions. Interesting examples are shape mimics of the natural base pairs as in the case of, for example, the difluorotoluene/methylbenzimidazole pair, [18] or aromatic units that recognize each other and stabilize duplexes through interstrand stacking interactions as, for example, the propynylcarbostyryl pair.…”

Section: Introductionmentioning

confidence: 99%

2‐Phenanthrenyl–DNA: Synthesis, Pairing, and Fluorescence Properties

Grigorenko

Leumann

2008

Chemistry A European J

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Three 2′‐phenanthrenyl‐C‐deoxyribonucleosides with donor (phenNH2), acceptor (phenNO2), or no (phenH) substitution on the phenanthrenyl core were synthesized and incorporated into oligodeoxyribonucleotides. Duplexes containing either one or three consecutive phenR residues, which were located opposite each other, were formed. Within these residues, the phenR residues are expected to recognize each other through interstrand stacking interactions, in much the same way as described previously for biphenyl DNA. The thermal, thermodynamic, and fluorescence properties of such duplexes were determined by UV melting analysis and fluorescence spectroscopy. Depending on the nature of the substituent, the thermal stability of single‐modified duplexes can vary between −2.7 to +11.3 °C in Tm and that of triple‐modified duplexes from +7.8 to +11.1 °C. Van′t Hoff analysis suggested that the observed higher thermodynamic stability in phenH‐ and phenNO2‐containing duplexes is of enthalpic origin. A single phenH or phenNO2 residue in a bulge position also stabilizes a corresponding duplex. If a phenNO2 residue is placed in a bulge position next to a base mismatch this can lead, in a sequence‐dependent manner, to duplex destabilization. The phenNO2 residue was found to be a highly efficient (10–100‐fold) quencher of phenH and phenNH2 fluorescence if placed in the opposite position to the fluorophores. When phenH and phenNH2 residues were placed opposite each other, efficient quenching of phenH and enhancement of phenNH2 fluorescence was found, which is an indicator for electron‐ or energy‐transfer processes between the aromatic units.

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“…information transfer ͉ polymerase N ucleic acid analogs with altered backbones or bases are of significant interest in the search for biopolymers with novel chemical and biological properties, and many such analogs have been designed and synthesized (1)(2)(3). Evaluation of the hybridization and nuclease-resistance properties of these synthetic nucleic acids has led to several nucleic acid analogues with potential biological applications (4)(5)(6).…”

mentioning

confidence: 99%

Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation between product and template

Tsai

Chen

Szostak

2007

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

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Glycerol nucleic acid (GNA) is an interesting alternative basepairing system based on an acyclic, glycerol-phosphate backbone repeat unit. The question of whether DNA polymerases can catalyze efficient template-dependent synthesis using GNA as the template is of particular interest because GNA is unable to form a stable duplex with DNA. In the present study, we screened a variety of DNA polymerases for GNA-dependent DNA synthesis. We find that Bst DNA polymerase can catalyze full-length DNA synthesis on a dodecamer GNA template. The efficiency of DNA synthesis is increased by replacing adenine with diaminopurine in both the GNA template and the DNA monomers and by the presence of manganese ions. We suggest that the BstDNA polymerase maintains a short, transient region of base-pairing between the DNA product strand and the GNA template, but that stable duplex formation between product and template strands is not required for template-dependent polymerization.information transfer ͉ polymerase N ucleic acid analogs with altered backbones or bases are of significant interest in the search for biopolymers with novel chemical and biological properties, and many such analogs have been designed and synthesized (1-3). Evaluation of the hybridization and nuclease-resistance properties of these synthetic nucleic acids has led to several nucleic acid analogues with potential biological applications (4-6). However, much less is known about the potential for information transfer between synthetic nucleic acid systems and the modern DNA/RNA system via templatedependent polymerization, a crucial aspect of the chemical etiology of nucleic acids and directed evolution of functional biopolymers based on a synthetic nucleic acid system. Recently, our group and others have studied the enzymatic synthesis of (3Ј 3 2Ј) ␣-L-threose nucleic acid (TNA; Fig. 1) (7-13). TNA was discovered by Eschenmoser and coworkers (7, 8) during a systematic evaluation of the base-pairing properties of nucleic acids containing alternative sugar-phosphate backbones. TNA was found to be capable of forming stable, antiparallel duplexes with RNA, DNA, and itself, a surprising property for a nucleic acid analog with a shorter backbone repeat unit than DNA or RNA (8,14,15). This remarkable intersystem duplex formation has been considered to be the basis of possible information transfer between TNA and DNA/RNA. Later studies by our group revealed that ␣-L-threose nucleoside 3Ј-triphosphates were substrates for efficient template-dependent enzymatic polymerization by Therminator DNA polymerase, a mutated archaeal family B DNA polymerase (10,11,16). Therminator DNA polymerase was also shown to be an efficient and accurate TNA-dependent DNA polymerase (9). The information transfer between TNA and DNA catalyzed by polymerases provides support for a possible role of TNA as a progenitor of DNA/RNA.The search for nucleic acid analogs with even simpler backbones led to studies of the glycerol nucleic acids (GNAs), which have a three-carbon, acyclic backbone (17-19) (Fi...

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Synthesis and Properties of Size-Expanded DNAs: Toward Designed, Functional Genetic Systems

Cited by 138 publications

References 73 publications

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

Effects of linker sequences on vesicle fusion mediated by lipid-anchored DNA oligonucleotides

2‐Phenanthrenyl–DNA: Synthesis, Pairing, and Fluorescence Properties

Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation between product and template

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