High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis

Nature Biotechnology. doi:10.1038/nbt.1589

Authors: Rupali P Patwardhan, Choli Lee, Oren Litvin, David L Young, Dana Pe'er & Jay Shendure

We present a method that harnesses massively parallel DNA synthesis and sequencing for the high-throughput functional analysis of regulatory sequences at single-nucleotide resolution. As a proof of concept, we quantitatively assayed the effects of all possible single-nucleotide mutations for three bacteriophage promoters and three mammalian core promoters in a single experiment per promoter. The method may also serve as a rapid screening tool for regulatory element engineering in synthetic biology.

Nature Biotechnology. doi:10.1038/nbt.1587

Authors: Jeremy D Henson, Ying Cao, Lily I Huschtscha, Andy C Chang, Amy Y M Au, Hilda A Pickett & Roger R Reddel

Alternative lengthening of telomeres (ALT) is likely to be an important target for anticancer treatment as ∼10% of cancers depend on this telomere maintenance mechanism for continued growth, and inhibition of ALT can cause cellular senescence. However, no ALT inhibitors have been developed for therapeutic use because of the lack of a suitable ALT activity assay and of known ALT-specific target molecules. Here we show that partially single-stranded telomeric (CCCTAA)n DNA circles (C-circles) are ALT specific. We provide an assay that is rapidly and linearly responsive to ALT activity and that is suitable for screening for ALT inhibitors. We detect C-circles in blood from ALT+ osteosarcoma patients, suggesting that the C-circle assay (CC assay) may have clinical utility for diagnosis and management of ALT+ tumors.

Nature Biotechnology. doi:10.1038/nbt.1586

Authors: Shota Atsumi, Wendy Higashide & James C Liao

Global climate change has stimulated efforts to reduce CO2 emissions. One approach to addressing this problem is to recycle CO2 directly into fuels or chemicals using photosynthesis. Here we genetically engineered Synechococcus elongatus PCC7942 to produce isobutyraldehyde and isobutanol directly from CO2 and increased productivity by overexpression of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Isobutyraldehyde is a precursor for the synthesis of other chemicals, and isobutanol can be used as a gasoline substitute. The high vapor pressure of isobutyraldehyde allows in situ product recovery and reduces product toxicity. The engineered strain remained active for 8 d and produced isobutyraldehyde at a higher rate than those reported for ethanol, hydrogen or lipid production by cyanobacteria or algae. These results underscore the promise of direct bioconversion of CO2 into fuels and chemicals, which bypasses the need for deconstruction of biomass.

Nature Biotechnology. doi:10.1038/nbt.1588

Authors: Volker Schellenberger, Chia-wei Wang, Nathan C Geething, Benjamin J Spink, Andrew Campbell, Wayne To, Michael D Scholle, Yong Yin, Yi Yao, Oren Bogin, Jeffrey L Cleland, Joshua Silverman & Willem P C Stemmer

Increasing the in vivo residence times of protein therapeutics could decrease their dosing frequencies. We show that genetic fusion of an unstructured recombinant polypeptide of 864 amino acids, called XTEN, to a peptide or protein provides an apparently generic approach to extend plasma half-life. Allometric scaling suggests that a fusion of XTEN to the exenatide peptide should increase exenatide half-life in humans from 2.4 h to a projected time of 139 h. We confirmed the biological activity of the exenatide-XTEN fusion in mice. As extended stability might exacerbate undesirable side effects in some cases, we show that truncating the XTEN sequence can regulate plasma half-life. XTEN lacks hydrophobic amino acid residues that often contribute to immunogenicity and complicate manufacture. Based on data on XTEN fusions to exenatide, glucagon, GFP and human growth hormone, we expect that XTEN will enable dosing of otherwise rapidly cleared protein drugs at up to monthly intervals in humans.