Pyrrole-Imidazole Polyamides Distinguish Between Double-Helical DNA and RNA

Groove specificity: Pyrrole-imidazole polyamides are well-known for their specific interactions with the minor groove of DNA (see scheme). However, polyamides do not show similar binding to duplex RNA, and a structural rationale for the molecular-level discrimination of nucleic acid duplexes by minor-groove-binding ligands is presented

Enhancing the cellular uptake of Py–Im polyamides through next-generation aryl turns

Pyrrole–imidazole (Py–Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein–DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5′-WGWWCW-3′ (W = A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py–Im polyamides that will prove valuable for future applications in which biological potency is essential

Modulation of NF-κB-dependent gene transcription using programmable DNA minor groove binders

Nuclear factor κB (NF-κB) is a transcription factor that regulates various aspects of immune response, cell death, and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5′-WGGWWW-3′ and 5′GGGWWW-3′. The compound is capable of binding to κB sites and reducing the expression of various NF-κB–driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-κB inhibitor PS1145, identifies overlaps and differences in affected gene groups, and shows that both affect comparable numbers of TNF-α–inducible genes. Inhibition of NF-κB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists

Expanding the Repertoire of Natural Product-Inspired Ring Pairs for Molecular Recognition of DNA

A furan amino acid, inspired by the recently discovered proximicin natural products, was incorporated into the scaffold of a DNA-binding hairpin polyamide. While unpaired oligomers of 2,4-disubstituted furan amino acids show poor DNA-binding activity, furan (Fn) carboxamides paired with N-methylpyrrole (Py) and N-methylimidazole (Im) rings demonstrate excellent stabilization of duplex DNA as well as discrimination of noncognate sequences, consistent with function as a Py mimic according to the Py/Im polyamide pairing rules

Minimization of a Protein−DNA Dimerizer

A protein−DNA dimerizer constructed from a DNA-binding polyamide and the peptide FYPWMKG facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. The Exd binding domain can be reduced to a dipeptide WM attached to the polyamide through an ε-aminohexanoic acid linker with retention of protein−DNA dimerizer activity. Screening a library of analogues indicated that the tryptophan indole moiety is more important than methionine's side chain or the N-terminal acetamide. Remarkably, switching the stereochemistry of the tryptophan residue (l to d) stabilizes the dimerizer•Exd•DNA ternary complex at 37 °C. These observations provide design principles for artificial transcription factors that may function in concert with the cellular regulatory circuitry

Demonstrations of Deployable Systems for Robotic Precursor Missions

NASA is developing thin-film based, deployable propulsion, power, and communication systems for small spacecraft that serve as enabling technologies for exploration of the solar system. By leveraging recent advancements in thin films, photovoltaics, deployment systems, and miniaturized electronics, new mission-level capabilities will be demonstrated aboard small spacecraft enabling a new generation of frequent, inexpensive, and highly capable robotic precursor missions with goals extensible to future human exploration. Specifically, thin-film technologies are allowing the development and use of solar sails for propulsion, small, lightweight photovoltaics for power, and omnidirectional antennas for communication as demonstrated by recent advances on the Near Earth Asteroid (NEA) Scout and Lightweight Integrated Solar Array and anTenna (LISA-T) projects

Design and Overview of the Solar Cruiser Mission

Solar Cruiser is a Small Satellite Technology Demonstration Mission (TDM) of Opportunity to mature solar sail propulsion technology to enable near-term, high-priority breakthrough science missions as defined in the Solar and Space Physics Decadal Survey. Solar Cruiser will demonstrate a “sailcraft” platform with pointing control and attitude stability comparable to traditional platforms, upon which a new class of Heliophysics missions may fly instruments. It will show sailcraft operation (acceleration, navigation, station keeping, inclination change) immediately applicable to near-term missions, and show scalability of sail technologies such as the boom, membrane, deployer, reflectivity control devices for roll momentum management to enable more demanding missions, such as high inclination solar imaging. A team led by the NASA Marshall Space Flight Center is developing the Solar Cruiser with partners Ball Aerospace and Roccor (a Redwire company). Ball is responsible for procuring a Venus class microsat commercial bus from Blue Canyon Technologies, defining all necessary mission-specific modifications, and performing the Integration and Test of the Bus with the Solar Sail System to form the completed sailcraft. Ball will also procure the IRIS radio from Space Dynamics Laboratories and develop the adapter and harnessing that interfaces to the Launch Vehicle. Roccor will integrate the Solar Sail System (SSS), including the sail membrane from their Subcontractor NeXolve, the Triangular, Rollable and Collapsible (TRACTM) Boom, the LISAs (Lightweight Integrated Solar Arrays) and momentum management Reflective Control Devices (RCDs), before providing it to Ball for Integration and Test. Roccor will also build the Active Mass Translator (AMT), which moves the Sail relative to the Bus to control momentum in the pitch/yaw directions, while the RCDs provide roll control. MSFC manages the overall mission and provide the specialized solar sail attitude determination and control system (SSADCS) algorithms and software necessary to fly the sailcraft. The SSADCS software created for this mission will autonomously operate the AMT and RCDs to provide complete momentum control of the sailcraft. Bus-mounted Electric Propulsion thrusters are included to provide auxiliary momentum management, if required. Solar Cruiser will launch as a secondary payload with NASA’s Interstellar Mapping and Acceleration Probe (IMAP)in early 2025. The sailcraft will separate from the launch vehicle on a near-L1 trajectory (Sun-Earth Lagrangian Point 1; sunward of L1 along the Sun-Earth Line) and complete its primary mission in 11 months or less. During this time, Solar Cruiser will complete and fully characterize a large solar sail deployment (1,653 square meters/17,793 square feet), sail operation, station keeping in a sub-L1 halo orbit, inclination changes, and a roll demonstration. This paper provides a mission and sailcraft design overview, including objectives and planned operations of the technology demonstration mission. It presents the latest findings from technology maturation efforts, major program design reviews, and initial launch integration planning

The reach of linear protein-DNA dimerizers

A protein-DNA dimerizer constructed from a DNA-binding pyrrole-imidazole polyamide and the peptide FYPWMK facilitates binding of the natural transcription factor Exd to an adjacent DNA site. Previous dimerizers have been constructed with the peptide attached to an internal pyrrole monomer in an overall branched oligomer. Linear oligomers constructed by attaching the peptide to the polyamide C-terminus expand the range of protein-DNA dimerization to six additional DNA sites. Replacing the FYPWMK hexapeptide with a WM dipeptide, which was previously functional in branched compounds, does not lead to a functional linear dimerizer. Instead, inserting an additional lysine generates a minimal, linear WMK tripeptide conjugate that maintains the activity of the larger FYPWMK dimerizers in a single DNA-binding site orientation. These studies provide insight into the importance of linker length and composition, binding site spacing and orientation, and the protein-binding domain content that are important for the optimization of protein-DNA dimerizers suitable for biological experiments

Quantitative microarray profiling of DNA-binding molecules

A high-throughput Cognate Site Identity (CSI) microarray platform interrogating all 524 800 10-base pair variable sites is correlated to quantitative DNase I footprinting data of DNA binding pyrrole-imidazole polyamides. An eight-ring hairpin polyamide programmed to target the 5 bp sequence 5'-TACGT-3' within the hypoxia response element (HRE) yielded a CSI microarray-derived sequence motif of 5'-WWACGT-3' (W = A,T). A linear beta-linked polyamide programmed to target a (GAA)_3 repeat yielded a CSI microarray-derived sequence motif of 5'-AARAARWWG-3' (R = G,A). Quantitative DNase I footprinting of selected sequences from each microarray experiment enabled quantitative prediction of K_a values across the microarray intensity spectrum

Ranking ligand affinity for the DNA minor groove by experiment and simulation

The structural and thermodynamic basis for the strength and selectivity of the interactions of minor-groove binders (MGBs) with DNA is not fully understood. In 2003 we reported the first example of a thiazole containing MGB that bound in a phase shifted pattern that spanned 6 base-pairs rather than the usual 4 (for tricyclic distamycin-like compounds). Since then, using DNA footprinting, nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and molecular dynamics, we have established that the flanking bases around the central 4 being read by the ligand have subtle effects on recognition. We have investigated the effect of these flanking sequences on binding and the reasons for the differences and established a computational method to rank ligand affinity against varying DNA sequences