Lynn M. McGregor

Minimally cationic, cell-permeable miniature proteins via alpha-helical arginine display

Protein therapeutics are a blossoming industry, with revenues exceeding

A DNA-based molecular probe for optically reporting cellular traction forces

51 billion in 2005 and a growth rate nearly three times that of the overall pharmaceutical industry. Although it has been known for decades that cationic polymers can transport molecular cargos across the plasma membrane, inefficient cellular delivery continues to impede the development of protein drugs. Our lab recently reported that small, folded proteins containing a minimal cationic motif embedded within a type II polyproline (PPII) helix efficiently cross the plasma membrane of eukaryotic cells. Here we demonstrate that an even smaller cationic motif can be embedded within the α-helix of a small, folded protein to generate molecules that penetrate cells significantly more efficiently than arginine-rich sequences or Tat. Our results suggest that the function of cell permeability can be encoded by judicious placement of as few as 2−3 additional arginine residues on a protein α-helix.

Interaction-Dependent PCR: Identification of Ligand−Target Pairs from Libraries of Ligands and Libraries of Targets in a Single Solution-Phase Experiment

We developed molecular tension probes (TPs) that report traction forces of adherent cells with high spatial resolution, can in principle be linked to virtually any surface, and obviate monitoring deformations of elastic substrates. TPs consist of DNA hairpins conjugated to fluorophore-quencher pairs that unfold and fluoresce when subjected to specific forces. We applied TPs to reveal that cellular traction forces are heterogeneous within focal adhesions and localized at their distal edges.

Photopolymerized poly(ethylene glycol)/poly(L-lysine) hydrogels for the delivery of neural progenitor cells

Interaction-dependent PCR (IDPCR) is a solution-phase method to identify binding partners from combined libraries of small-molecule ligands and targets in a single experiment. Binding between DNA-linked targets and DNA-linked ligands induces formation of an extendable duplex. Extension links codes that identify the ligand and target into one selectively amplifiable DNA molecule. In a model selection, IDPCR resulted in the enrichment of DNA encoding all five known protein−ligand pairs out of 67 599 possible sequences.

An aptamer-functionalized chemomechanically modulated biomolecule catch-and-release system

Neural progenitor cells (NPCs) have shown promise in a number of models of disease and injury, but for these cells to be safe and effective, they must be directed to differentiate appropriately following transplantation. We have developed a photopolymerized hydrogel composed of macromers of poly(ethylene glycol) (PEG) bound to poly(L-lysine) (PLL) that supports NPC survival and directs differentiation. Green fluorescent protein (GFP) positive NPCs were encapsulated in these gels and demonstrated survival up to 17 days. When encapsulated in the gels at a photoinitiator concentration of 5.0 mg/ml, few NPCs (0.5 ± 0.25%) demonstrated apoptosis. Furthermore, 55 ± 6% of the NPCs cultured within the gels in epidermal growth factor (EGF) containing media differentiated into a mature neuronal cell type (neurofilament 200 positive) while the remainder 44 ± 8% were undifferentiated (nestin positive). A small percentage, 1 ± 0.4%, expressed the astrocytic marker glial acidic fibrilary protein. Photopolymerized PEG/PLL gels promote the survival and direct the differentiation of NPCs, making this system a promising delivery vehicle for NPCs in the treatment of injuries and diseases of the central nervous system.

Identification of ligand-target pairs from combined libraries of small molecules and unpurified protein targets in cell lysates.

The efficient extraction of (bio)molecules from fluid mixtures is vital for applications ranging from target characterization in (bio)chemistry to environmental analysis and biomedical diagnostics. Inspired by biological processes that seamlessly synchronize the capture, transport and release of biomolecules, we designed a robust chemomechanical sorting system capable of the concerted catch and release of target biomolecules from a solution mixture. The hybrid system is composed of target-specific, reversible binding sites attached to microscopic fins embedded in a responsive hydrogel that moves the cargo between two chemically distinct environments. To demonstrate the utility of the system, we focus on the effective separation of thrombin by synchronizing the pH-dependent binding strength of a thrombin-specific aptamer with volume changes of the pH-responsive hydrogel in a biphasic microfluidic regime, and show a non-destructive separation that has a quantitative sorting efficiency, as well as the systems stability and amenability to multiple solution recycling.

Novel selection methods for DNA-encoded chemical libraries.

We describe the development and validation of interaction determination using unpurified proteins (IDUP), a method that selectively amplifies DNA sequences identifying ligand+target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. By operating in cell lysates, IDUP preserves native post-translational modifications and interactions with endogenous binding partners, thereby enabling the study of difficult-to-purify targets and increasing the potential biological relevance of detected interactions compared with methods that require purified proteins. In IDUP, target proteins are associated with DNA oligonucleotide tags either non-covalently using a DNA-linked antibody or covalently using a SNAP-tag. Ligand–target binding promotes hybridization of a self-priming hairpin that is extended by a DNA polymerase to create a DNA strand that contains sequences identifying both the target and its ligand. These sequences encoding ligand+target pairs are selectively amplified by PCR and revealed by high-throughput DNA sequencing. IDUP can respond to the effect of affinity-modulating adaptor proteins in cell lysates that would be absent in ligand screening or selection methods using a purified protein target. This capability was exemplified by the 100-fold amplification of DNA sequences encoding FRB+rapamycin or FKBP+rapamycin in samples overexpressing both FRB and FKBP (FRB·rapamycin+FKBP, Kd ≈ 100 fM; FKBP·rapamycin+FRB, Kd = 12 nM). In contrast, these sequences were amplified 10-fold less efficiently in samples overexpressing either FRB or FKBP alone (rapamycin+FKBP, Kd ≈ 0.2 nM; rapamcyin+FRB, Kd = 26 μM). Finally, IDUP was used to process a model library of DNA-linked small molecules and a model library of cell lysates expressing SNAP-target fusions combined in a single sample. In this library×library experiment, IDUP resulted in enrichment of sequences corresponding to five known ligand+target pairs ranging in binding affinity from Kd = 0.2 nM to 3.2 μM out of 67,858 possible combinations, with no false positive signals enriched to the same extent as that of any of the bona fide ligand+target pairs.

Discovery of a Covalent Kinase Inhibitor from a DNA-Encoded Small-Molecule Library × Protein Library Selection

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries.

REACTIVITY-DEPENDENT AND INTERACTION-DEPENDENT PCR

We previously reported interaction determination using unpurified proteins (IDUP), a method to selectively amplify DNA sequences encoding ligand:target pairs from a mixture of DNA-linked small molecules and unpurified protein targets in cell lysates. In this study, we applied IDUP to libraries of DNA-encoded bioactive compounds and DNA-tagged human kinases to identify ligand:protein binding partners out of 32 096 possible combinations in a single solution-phase library × library experiment. The results recapitulated known small molecule:protein interactions and also revealed that ethacrynic acid is a novel ligand and inhibitor of MAP2K6 kinase. Ethacrynic acid inhibits MAP2K6 in part through alkylation of a nonconserved cysteine residue. This work validates the ability of IDUP to discover ligands for proteins of biomedical relevance.