Peters, Justin P.

Abstract We are interested in DNA stiffness in vitro and in vivo . Classical approaches for measuring this parameter in vitro involve tedious T4 DNA ligase-mediated cyclization kinetics experiments performed for one DNA fragment length at a time to establish effective local end-end concentrations ( J -factors). Protein-mediated DNA looping is a more relevant biological reaction. An efficient approach to determine length-dependent protein-mediated DNA J - loop values in vitro has been lacking. We describe LOOP-TAG, a massively-parallel method. We leverage intramolecular DNA looping and tagmentation catalyzed by Tn5 transposase (Tnp) tethered to the terminus of a ∼1,000-bp bead-bound duplex DNA. We detected loops terminating with Tnp. Counts of the resulting DNA looping-dependent bead-bound tagmentation products from deep sequencing determine DNA loop length probabilities at single-base pair resolution for all bead-bound fragments. When normalized to DNA fragment length probabilities from tagmentation using known concentrations of free Tnp, length-dependent J - loop values are directly determined. We demonstrate LOOP-TAG for Tnp tethered in two ways and in the presence of DNA-kinking architectural DNA binding protein Nhp6A. Intermediate length J-loop values are consistent with expectations of wormlike chain theory after accounting for strong sequence-dependence of Tnp. DNA loops are shifted to smaller sizes in the presence of Nhp6A, as predicted. (200/200 words)

Abstract We are interested in artificial gene regulation by exploiting principles learned from lactose repressor (lacI) control of the Escherichia coli lac operon through the formation of tight DNA loops on the order of 100–200 base pairs. We previously showed that appropriately designed DNA target-programmable Transcription Activator-Like Effector dimer (TALED) proteins can produce DNA-looping-dependent gene repression comparable to lacI. Here, we demonstrate regulation of such switches by the principle of controlled repressor autoinhibition. Thus, TALEDs are inhibited by controlled cyclization, dependent on small molecules. Cyclized TALEDs poorly participate in DNA looping. We fit DNA loop length-dependent data with an established thermodynamic model to quantitate these effects. We further demonstrate the ability of small molecules to induce gene expression using this system in actively growing E. coli cells. The resulting designed systems demonstrate regulated gene expression by DNA looping, emulating the lac system but with entirely different molecular components.

Abstract Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes.

Abstract Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

ABSTRACT Hypoxia inducible factors (HIFs) play vital roles in cellular maintenance of oxygen homeostasis. These transcription factors are responsible for the expression of genes involved in angiogenesis, metabolism, and cell proliferation. Here, we generate a detailed mathematical model for the enzyme kinetics of α-ketoglutarate-dependent HIF prolyl 4-hydroxylase domain (PHD) dioxygenases to simulate our in vitro data showing synergistic PHD inhibition by succinate and hypoxia in experimental models of succinate dehydrogenase loss, which phenocopy familial paraganglioma. Our mathematical model confirms the inhibitory synergy of succinate and hypoxia under physiologically-relevant conditions. In agreement with our experimental data, the model predicts that HIF1α is not stabilized under atmospheric oxygen concentrations, as observed. Further, the model confirms that addition of α-ketoglutarate can reverse PHD inhibition by succinate and hypoxia in SDH-deficient cells.

DNA aptamer oligonucleotides and their protein conjugates show promise as therapeutics in animal models of diseases such as multiple sclerosis. These molecules are large and highly charged, raising questions about their biodistribution and pharmacokinetics in mammals. Here we exploit the power of quantitative polymerase chain reaction to accurately quantitate the tissue distribution of 40-nucleotide DNA aptamers and their streptavidin conjugates after intraperitoneal injection in mice. We show remarkably rapid distribution to peripheral tissues including the central nervous system. Modeling of tissue distribution data reveals the importance of DNA aptamer sequence, 3′ modification, and protein conjugation in enhancing tissue exposure. These data help to interpret the previously observed effectiveness of aptamer conjugates, as opposed to free aptamers, in stimulating central nervous system remyelination in a mouse model of multiple sclerosis.

The free solution mobilities of ssDNA and dsDNA molecules with variable charge densities have been measured by CE. DNA charge density was modified either by appending positively or negatively charged groups to the thymine residues in a 98 bp DNA molecule, or by replacing some of the negatively charged phosphate internucleoside linkers in small ssDNA or dsDNA oligomers with positively charged phosphoramidate linkers. Mobility ratios were calculated for each dataset by dividing the mobility of a charge variant by the mobility of its unmodified parent DNA. Mobility ratios essentially eliminate the effect of the BGE on the observed mobility, making it possible to compare analytes measured under different experimental conditions. Neutral moieties attached to the thymine residues in the 98‐bp DNA molecule had little or no effect on the mobility ratios, indicating that bulky substituents in the DNA major groove do not affect the mobility significantly. The mobility ratios observed for the thymine‐modified and linker‐modified DNA charge variants increased approximately linearly with the logarithm of the fractional negative charge of the DNA. Mobility ratios calculated from previous studies of linker‐modified DNA charge variants and small multicharged organic molecules also increased approximately linearly with the logarithm of the fractional negative charge of the analyte. The results do not agree with the Debye–Hückel–Onsager theory of electrophoresis, which predicts that the mobility of an analyte should depend linearly on analyte charge, not the logarithm of the charge, when the frictional coefficient is held constant.

Abstract The molecular structure of the DNA double helix has been known for 60 years, but we remain surprisingly ignorant of the balance of forces that determine its mechanical properties. The DNA double helix is among the stiffest of all biopolymers, but neither theory nor experiment has provided a coherent understanding of the relative roles of attractive base stacking forces and repulsive electrostatic forces creating this stiffness. To gain insight, we have created a family of double-helical DNA-like polymers where one of the four normal bases is replaced with various cationic, anionic or neutral analogs. We apply DNA ligase-catalyzed cyclization kinetics experiments to measure the bending and twisting flexibilities of these polymers under low salt conditions. Interestingly, we show that these modifications alter DNA bending stiffness by only 20%, but have much stronger (5-fold) effects on twist flexibility. We suggest that rather than modifying DNA stiffness through a mechanism easily interpretable as electrostatic, the more dominant effect of neutral and charged base modifications is their ability to drive transitions to helical conformations different from canonical B-form DNA.