scholarly journals Construction of a novel phagemid to produce custom DNA origami scaffolds

2018 ◽  
Author(s):  
Parsa M. Nafisi ◽  
Tural Aksel ◽  
Shawn M. Douglas
Keyword(s):  
Dna Sequences ◽  
Dna Origami ◽  
Single Strand ◽  
Base Pairs ◽  
Fixed Sequence ◽  
Design Processes ◽  
Low Level ◽  
Structural Details ◽  
Filamentous Bacteriophages ◽  
Fixed Region

AbstractDNA origami, a method for constructing nanoscale objects, relies on a long single strand of DNA to act as the “scaffold” to template assembly of numerous short DNA oligonucleotide “staples”. The ability to generate custom scaffold sequences can greatly benefit DNA origami design processes. Custom scaffold sequences can provide better control of the overall size of the final object and better control of low-level structural details, such as locations of specific base pairs within an object. Filamentous bacteriophages and related phagemids can work well as sources of custom scaffold DNA. However, scaffolds derived from phages require inclusion of multi-kilobase DNA sequences in order to grow in host bacteria, and thus cannot be altered or removed. These fixed-sequence regions constrain the design possibilities of DNA origami. Here we report the construction of a novel phagemid, pScaf, to produce scaffolds that have a custom sequence with a much smaller fixed region of only 381 bases. We used pScaf to generate new scaffolds ranging in size from 1,512 to 10,080 bases and demonstrated their use in various DNA origami shapes and assemblies. We anticipate our pScaf phagemid will enhance development of the DNA origami method and its future applications.

scholarly journals Construction of a novel phagemid to produce custom DNA origami scaffolds

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Parsa M Nafisi ◽  
Tural Aksel ◽  
Shawn M Douglas
Keyword(s):  
Dna Sequences ◽  
Dna Origami ◽  
Single Strand ◽  
Base Pairs ◽  
Fixed Sequence ◽  
Design Processes ◽  
Low Level ◽  
Structural Details ◽  
Filamentous Bacteriophages ◽  
Fixed Region

Abstract DNA origami, a method for constructing nanoscale objects, relies on a long single strand of DNA to act as the ‘scaffold’ to template assembly of numerous short DNA oligonucleotide ‘staples’. The ability to generate custom scaffold sequences can greatly benefit DNA origami design processes. Custom scaffold sequences can provide better control of the overall size of the final object and better control of low-level structural details, such as locations of specific base pairs within an object. Filamentous bacteriophages and related phagemids can work well as sources of custom scaffold DNA. However, scaffolds derived from phages require inclusion of multi-kilobase DNA sequences in order to grow in host bacteria, and those sequences cannot be altered or removed. These fixed-sequence regions constrain the design possibilities of DNA origami. Here, we report the construction of a novel phagemid, pScaf, to produce scaffolds that have a custom sequence with a much smaller fixed region of 393 bases. We used pScaf to generate new scaffolds ranging in size from 1512 to 10 080 bases and demonstrated their use in various DNA origami shapes and assemblies. We anticipate our pScaf phagemid will enhance development of the DNA origami method and its future applications.

The novel double-folded structure of d(GCATGCATGC): a possible model for triplet-repeat sequences

2015 ◽  
Vol 71 (10) ◽  
pp. 2119-2126 ◽  
Author(s):  
Arunachalam Thirugnanasambandam ◽  
Selvam Karthik ◽  
Pradeep Kumar Mandal ◽  
Namasivayam Gautham
Keyword(s):  
Hydrogen Bonds ◽  
Dna Sequences ◽  
Single Strand ◽  
Triplet Repeat ◽  
The Novel ◽  
Base Pairs ◽  
Purine Base ◽  
Ion Interactions ◽  
Folded Structure ◽  
Triplet Repeat Sequences

The structure of the decadeoxyribonucleotide d(GCATGCATGC) is presented at a resolution of 1.8 Å. The decamer adopts a novel double-folded structure in which the direction of progression of the backbone changes at the two thymine residues. Intra-strand stacking interactions (including an interaction between the endocylic O atom of a ribose moiety and the adjacent purine base), hydrogen bonds and cobalt-ion interactions stabilize the double-folded structure of the single strand. Two such double-folded strands come together in the crystal to form a dimer. Inter-strand Watson–Crick hydrogen bonds form four base pairs. This portion of the decamer structure is similar to that observed in other previously reported oligonucleotide structures and has been dubbed a `bi-loop'. Both the double-folded single-strand structure, as well as the dimeric bi-loop structure, serve as starting points to construct models for triplet-repeat DNA sequences, which have been implicated in many human diseases.

Structures of Artificially Designed RNA Nanoarchitectures at Near-Atomic Resolution

2020 ◽  
Author(s):  
Di Liu ◽  
Yaming Shao ◽  
Joseph A. Piccirilli ◽  
Yossi Weizmann
Keyword(s):  
Nucleic Acid ◽  
High Resolution ◽  
Single Strand ◽  
Atomic Resolution ◽  
Rna Motifs ◽  
X Ray ◽  
Structural Details ◽  
Rna Motif

<p>Though advances in nanotechnology have enabled the construction of synthetic nucleic acid based nanoarchitectures with ever-increasing complexity for various applications, high-resolution structures are lacking due to the difficulty of obtaining good diffracting crystals. Here we report the design of RNA nanostructures based on homooligomerizable tiles from an RNA single-strand for X-ray determination. Three structures are solved to near-atomic resolution: a 2D parallelogram, an unexpectedly formed 3D nanobracelet, and a 3D nanocage. Structural details of their constituent motifs—such as kissing loops, branched kissing-loops and T-junctions—that resemble natural RNA motifs and resisted X-ray determination are revealed. This work unveils the largely unexplored potential of crystallography in gaining high-resolution feedback for nanostructure design and suggests a novel route to investigate RNA motif structures by configuring them into nanoarchitectures.</p>

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced &gt;250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

scholarly journals THE DNA OF CAENORHABDITIS ELEGANS

Genetics ◽  
1974 ◽  
Vol 77 (1) ◽  
pp. 95-104
Author(s):  
J E Sulston ◽  
S Brenner
Keyword(s):  
Caenorhabditis Elegans ◽  
Chemical Analysis ◽  
Dna Sequences ◽  
Base Composition ◽  
Nematode Caenorhabditis Elegans ◽  
5S Rna ◽  
Base Pairs ◽  
Small Component ◽  
E Coli ◽  
The Mean

ABSTRACT Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x IO7 base pairs (20 times the genome of E. coli). Eighty-three percent of the DNA sequences are unique. The mean base composition is 36% GC; a small component, containing the rRNA cistrons, has a base composition of 51% GC. The haploid genome contains about 300 genes for 4s RNA, 110 for 5s RNA, and 55 for (18 + 28)S RNA.

scholarly journals Theoretical modelling of epigenetically modified DNA sequences

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 52 ◽  
Author(s):  
Alexandra Teresa Pires Carvalho ◽  
Maria Leonor Gouveia ◽  
Charan Raju Kanna ◽  
Sebastian K. T. S. Wärmländer ◽  
Jamie Platts ◽  
...  
Keyword(s):  
Molecular Mechanics ◽  
Dna Sequences ◽  
Base Pair ◽  
Density Functional ◽  
Epigenetic Modifications ◽  
Theoretical Modelling ◽  
Sugar Phosphate ◽  
Base Pairs ◽  
Phosphate Backbone ◽  
Modified Dna

We report herein a set of calculations designed to examine the effects of epigenetic modifications on the structure of DNA. The incorporation of methyl, hydroxymethyl, formyl and carboxy substituents at the 5-position of cytosine is shown to hardly affect the geometry of CG base pairs, but to result in rather larger changes to hydrogen-bond and stacking binding energies, as predicted by dispersion-corrected density functional theory (DFT) methods. The same modifications within double-stranded GCG and ACA trimers exhibit rather larger structural effects, when including the sugar-phosphate backbone as well as sodium counterions and implicit aqueous solvation. In particular, changes are observed in the buckle and propeller angles within base pairs and the slide and roll values of base pair steps, but these leave the overall helical shape of DNA essentially intact. The structures so obtained are useful as a benchmark of faster methods, including molecular mechanics (MM) and hybrid quantum mechanics/molecular mechanics (QM/MM) methods. We show that previously developed MM parameters satisfactorily reproduce the trimer structures, as do QM/MM calculations which treat bases with dispersion-corrected DFT and the sugar-phosphate backbone with AMBER. The latter are improved by inclusion of all six bases in the QM region, since a truncated model including only the central CG base pair in the QM region is considerably further from the DFT structure. This QM/MM method is then applied to a set of double-stranded DNA heptamers derived from a recent X-ray crystallographic study, whose size puts a DFT study beyond our current computational resources. These data show that still larger structural changes are observed than in base pairs or trimers, leading us to conclude that it is important to model epigenetic modifications within realistic molecular contexts.

scholarly journals High frequency repeat-induced point mutation (RIP) is not associated with efficient recombination in Neurospora.

Genetics ◽  
1994 ◽  
Vol 138 (4) ◽  
pp. 1093-1103 ◽  
Author(s):  
J T Irelan ◽  
A T Hagemann ◽  
E U Selker
Keyword(s):  
Point Mutation ◽  
Dna Sequences ◽  
High Frequency ◽  
Recombination Frequency ◽  
Single Copy ◽  
Sexual Cycle ◽  
Base Pairs ◽  
Copy Gene ◽  
The Relationship ◽  
Repeat Induced Point Mutation

Abstract Duplicated DNA sequences in Neurospora crassa are efficiently detected and mutated during the sexual cycle by a process named repeat-induced point mutation (RIP). Linked, direct duplications have previously been shown to undergo both RIP and deletion at high frequency during premeiosis, suggesting a relationship between RIP and homologous recombination. We have investigated the relationship between RIP and recombination for an unlinked duplication and for both inverted and direct, linked duplications. RIP occurred at high frequency (42-100%) with all three types of duplications used in this study, yet recombination was infrequent. For both inverted and direct, linked duplications, recombination was observed, but at frequencies one to two orders of magnitude lower than RIP. For the unlinked duplication, no recombinants were seen in 900 progeny, indicating, at most, a recombination frequency nearly three orders of magnitude lower than the frequency of RIP. In a direct duplication, RIP and recombination were correlated, suggesting that these two processes are mechanistically associated or that one process provokes the other. Mutations due to RIP have previously been shown to occur outside the boundary of a linked, direct duplication, indicating that RIP might be able to inactivate genes located in single-copy sequences adjacent to a duplicated sequence. In this study, a single-copy gene located between elements of linked duplications was inactivated at moderate frequencies (12-14%). Sequence analysis demonstrated that RIP mutations had spread into these single-copy sequences at least 930 base pairs from the boundary of the duplication, and Southern analysis indicated that mutations had occurred at least 4 kilobases from the duplication boundary.

scholarly journals DNA SEQUENCES OF FRAMESHIFT AND OTHER MUTATIONS INDUCED BY ICR-170 IN YEAST

Genetics ◽  
1985 ◽  
Vol 111 (2) ◽  
pp. 233-241
Author(s):  
Joachim F Ernst ◽  
D Michael Hampsey ◽  
Fred Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Genetic Analysis ◽  
Dna Sequences ◽  
Induced Mutations ◽  
Sequence Analyses ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Frameshift Mutations

ABSTRACT ICR-170-induced mutations in the CYC1 gene of the yeast Saccharomyces cerevisiae were investigated by genetic and DNA sequence analyses. Genetic analysis of 33 cyc1 mutations induced by ICR-170 and sequence analysis of eight representatives demonstrated that over one-third were frameshift mutations that occurred at one site corresponding to amino acid positions 29-30, whereas the remaining mutations were distributed more-or-less randomly, and a few of these were not frameshift mutations. The sequence results indicate that ICR-170 primarily induces G·C additions at sites containing monotonous runs of three G·C base pairs. However, some (see PDF) sites within the CYC1 gene were not mutated by ICR-170. Thus, ICR-170 is a relatively specific mutagen that preferentially acts on certain sites with monotonous runs of G·C base pairs.

scholarly journals Dynamic DNA Origami Devices: from Strand-Displacement Reactions to External-Stimuli Responsive Systems

2018 ◽  
Vol 19 (7) ◽  
pp. 2114 ◽  
Author(s):  
Heini Ijäs ◽  
Sami Nummelin ◽  
Boxuan Shen ◽  
Mauri Kostiainen ◽  
Veikko Linko
Keyword(s):  
Dna Sequences ◽  
Rapid Evolution ◽  
Dna Nanotechnology ◽  
Dna Origami ◽  
Dna Assembly ◽  
Stimuli Responsive ◽  
Nanoscale Structures ◽  
Displacement Reactions ◽  
Materials Research ◽  
External Stimuli

DNA nanotechnology provides an excellent foundation for diverse nanoscale structures that can be used in various bioapplications and materials research. Among all existing DNA assembly techniques, DNA origami proves to be the most robust one for creating custom nanoshapes. Since its invention in 2006, building from the bottom up using DNA advanced drastically, and therefore, more and more complex DNA-based systems became accessible. So far, the vast majority of the demonstrated DNA origami frameworks are static by nature; however, there also exist dynamic DNA origami devices that are increasingly coming into view. In this review, we discuss DNA origami nanostructures that exhibit controlled translational or rotational movement when triggered by predefined DNA sequences, various molecular interactions, and/or external stimuli such as light, pH, temperature, and electromagnetic fields. The rapid evolution of such dynamic DNA origami tools will undoubtedly have a significant impact on molecular-scale precision measurements, targeted drug delivery and diagnostics; however, they can also play a role in the development of optical/plasmonic sensors, nanophotonic devices, and nanorobotics for numerous different tasks.

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