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1.  To DNA, all information is equal 
Artificial DNA, PNA & XNA  2012;3(3):109-111.
Information storage capabilities are key in most aspects of society and the requirement for storage capacity is rapidly expanding. In principle, DNA could be a high-density medium for information storage. Church and coworkers recently demonstrated how binary data can be encoded, stored in, and retrieved from a library of oligonucleotides, increasing by several orders of magnitude the amount and density of manmade information stored in DNA to date. The technology remains in its infancy and important hurdles have yet to be overcome in order to realize its potential. However, DNA may be particularly useful as a storage-medium over long time-scales (centuries), because data-access is compatible with any large-scale DNA-sequencing and -synthesis technology.
PMCID: PMC3581509  PMID: 23104084
DNA; information storage in DNA; bit; byte; binary encoding
2.  A DNA nanocapsule with aptamer-controlled open-closure function for targeted delivery 
Artificial DNA, PNA & XNA  2012;3(1):3-4.
A DNA capsule fitted with aptamer controlled target sensing has been “woven” using a 7308-base single-stranded DNA “thread” and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces.
PMCID: PMC3368814  PMID: 22679527
aptamer; delivery; DNA origami; nanocapsule; nanoscience
3.  RNA-DNA sequence differences spell genetic code ambiguities 
Artificial DNA, PNA & XNA  2011;2(3):69-70.
A recent paper in Science by Li et al. 20111 reports widespread sequence differences in the human transcriptome between RNAs and their encoding genes termed RNA-DNA differences (RDDs). The findings could add a new layer of complexity to gene expression but the study has been criticized. 
PMCID: PMC3324336  PMID: 22567189
gene expression; RNA editing; RNA-DNA differences; transcription; transcriptome
4.  A ribozyme transcribed by a ribozyme 
Artificial DNA, PNA & XNA  2011;2(2):40-42.
Prominent current ideas on how life emerged on Earth include an RNA world hypothesis in which RNA performed informational as well as catalytic functions in the absence of both DNA and protein. Demonstration of a self-replicative system based on ribonucleic acid polymers as both information carriers and catalysts would lend support to such a scenario. A pivotal component of this system would be an RNA dependent RNA polymerase ribozyme capable of replicating its own RNA gene. Recent work from the Holliger group at the Laboratory for Molecular Biology in Cambridge has provided synthetic ribozymes1 that just might foreshadow the future engineering of such self-replicative systems.
PMCID: PMC3166488  PMID: 21912725
ribozyme; RNA dependent RNA polymerase; In vitro evolution; RNA engineering; transcription
5.  Adding mRNA to the list of spatially organized components in bacteria 
Artificial DNA, PNA & XNA  2010;1(2):66-67.
Using LNA in situ hybridization, select mRNAs have been shown to be spatially confined to their chromosomal loci in two distantly related bacterial organisms. Translating ribosomes are diffusion limited by mRNA association.
PMCID: PMC3116576  PMID: 21686240
mRNA spatial distribution; locked nucleic acid (LNA); fluorescence in situ hybridization (FISH)
6.  Mega-cloning and the advent of synthetic genomes 
Artificial DNA, PNA & XNA  2010;1(1):54-57.
Molecular biology owes its prominent role in the biological sciences to the tools of recombinant DNA. While the foundations of recombinant DNA were laid in the 1970s with the discovery of type II restriction endonucleases,1,2 development of robust sequencing technology3 and pioneering work on gene synthesis,4,5 it was not until the turn of the new millennium before the first complete synthetic viral genomes saw the light of day including that of hepatitis C,6 poliovirus,7 and bacteriophage PhiX174.8 Recombinant DNA has come of age as entire cellular genomes are sequenced and stored as digitized information. So what's next? One novel branch of recombinant DNA, referred to as synthetic genomics,9 is occupied with (re)construction of entire cellular genomes from virtual sequence information and using chemical components. Here we look at the most recent developments in such de novo construction. For a broader and more extensive review on genome engineering, the reader is referred to the excellent paper by Carr and Church.10
PMCID: PMC3109442  PMID: 21687527
synthetic genomics; recombinant DNA; genome transplantation; whole-genome assembly; synthetic chromosome
7.  Protein Evolution via Amino Acid and Codon Elimination 
PLoS ONE  2010;5(4):e10104.
Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles.
Methodology/Principal Findings
The strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance.
The results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.
PMCID: PMC2859931  PMID: 20436666
8.  High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers 
Nucleic Acids Research  2009;37(13):4498-4507.
While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA–dsDNA triplexes—mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine substitution, in combination with (oligo)lysine or 9-aminoacridine conjugation, homopyrimidine PNA oligomers bind complementary dsDNA targets via triplex formation with (sub)nanomolar affinities (at pH 7.2, 150 mM Na+). Binding affinity can be modulated more than 1000-fold by changes in pH, PNA oligomer length, PNA net charge and/or by substitution of pseudoisocytosine for cytosine, and conjugation of the DNA intercalator 9-aminoacridine. Furthermore, 9-aminoacridine conjugation also strongly enhanced triplex invasion. Specificity for the fully matched target versus one containing single centrally located mismatches was more than 150-fold. Together the data support the use of homopyrimidine PNAs as efficient and sequence selective tools in triplex targeting strategies under physiological relevant conditions.
PMCID: PMC2715256  PMID: 19474349
10.  Structural diversity of target-specific homopyrimidine peptide nucleic acid–dsDNA complexes 
Nucleic Acids Research  2006;34(20):5790-5799.
Sequence-selective recognition of double-stranded (ds) DNA by homopyrimidine peptide nucleic acid (PNA) oligomers can occur by major groove triplex binding or by helix invasion via triplex P-loop formation. We have compared the binding of a decamer, a dodecamer and a pentadecamer thymine–cytosine homopyrimidine PNA oligomer to a sequence complementary homopurine target in duplex DNA using gel-shift and chemical probing analyses. We find that all three PNAs form stable triplex invasion complexes, and also conventional triplexes with the dsDNA target. Triplexes form with much faster kinetics than invasion complexes and prevail at lower PNA concentrations and at shorter incubation times. Furthermore, increasing the ionic strength strongly favour triplex formation over invasion as the latter is severely inhibited by cations. Whereas a single triplex invasion complex is formed with the decameric PNA, two structurally different target-specific invasion complexes were characterized for the dodecameric PNA and more than five for the pentadecameric PNA. Finally, it is shown that isolated triplex complexes can be converted to specific invasion complexes without dissociation of the Hoogsteen base-paired triplex PNA. These result demonstrate a clear example of a ‘triplex first’ mechanism for PNA helix invasion.
PMCID: PMC1635314  PMID: 17053099
11.  In vitro transcription of a torsionally constrained template 
Nucleic Acids Research  2002;30(3):803-809.
RNA polymerase (RNAP) and the DNA template must rotate relative to each other during transcription elongation. In the cell, however, the components of the transcription apparatus may be subject to rotary constraints. For instance, the DNA is divided into topological domains that are delineated by rotary locked boundaries. Furthermore, RNAPs may be located in factories or attached to matrix sites limiting or prohibiting rotation. Indeed, the nascent RNA alone has been implicated in rotary constraining RNAP. Here we have investigated the consequences of rotary constraints during transcription of torsionally constrained DNA by free RNAP. We asked whether or not a newly synthesized RNA chain would limit transcription elongation. For this purpose we developed a method to immobilize covalently closed circular DNA to streptavidin-coated beads via a peptide nucleic acid (PNA)–biotin conjugate in principle mimicking a SAR/MAR attachment. We used this construct as a torsionally constrained template for transcription of the beta-lactamase gene by Escherichia coli RNAP and found that RNA synthesis displays similar characteristics in terms of rate of elongation whether or not the template is torsionally constrained. We conclude that transcription of a natural bacterial gene may proceed with high efficiency despite the fact that newly synthesized RNA is entangled around the template in the narrow confines of torsionally constrained supercoiled DNA.
PMCID: PMC100306  PMID: 11809894

Results 1-11 (11)