A Novel Peptide-Based Encoding System for “One-Bead One-Compound” Peptidomimetic and Small Molecule Combinatorial Libraries

The “one-bead one-compound” (OBOC) combinatorial library method is highly efficient, especially when used with well-established on-bead binding or functional assays. Literally, millions of compounds can be screened concurrently within 1 to 2 days. However, structure determination of peptidomimetic and small molecule compounds on one single bead is not trivial. A novel, highly efficient, and robust peptide-based encoding system has been developed for OBOC peptidomimetic and small molecule combinatorial libraries. In this system, topologically segregated bifunctional beads, which are made by a simple biphasic solvent strategy, are employed for the preparation and screening of an OBOC combinatorial peptidomimetic and small molecule libraries. Testing molecules are on the outer layer, and the coding tags in the interior of the bead do not interfere with screening. The coding tag is a peptide containing a large number of unnatural α-amino acids derived from different building blocks used for generating the peptidomimetic or small molecule. By coupling common building blocks simultaneously to the scaffold of the testing compound and to the side chains of the α-amino acids on the coding peptide, extra synthetic steps are eliminated and the amount of undesirable side products is minimized. Positive bead decoding is easy and straightforward as there is no need for cleavage and retrieval of the coding tag, and positive beads can be sequenced directly with Edman degradation. To demonstrate the efficiency and simplicity of our encoding system, an encoded 158 400-member model peptidomimetic library has been generated and screened for ligands that bind to streptavidin. Potent and novel ligands with clear motifs have been identified

Maximization of Loading and Stability of ssDNA:Iron Oxide Nanoparticle Complexes Formed through Electrostatic Interaction

The use of inorganic nanoparticles (NPs) as vectors for the delivery of oligonucleotides for in vitro and in vivo applications is rapidly gaining momentum. Some of the reasons making them especially good candidates for this purpose are their ease of synthesis in a range of sizes and surface coatings, their propensity to penetrate cell membranes, their stability and biocompatibility, and their unique size-dependent physical properties that impart additional diagnostic and therapeutic tools. Notwithstanding these notable attributes, a major obstacle to their practical use is given by the typically low oligonucleotide loading levels attainable through conventional bioconjugation procedures. This shortcoming is especially worrisome as toxicity concerns have been associated with codelivery of NPs. In this paper we are analytically analyzing the formation of electrostatic complexes between negatively charged ssDNA and positively charged iron oxide nanoparticles (SPIO-NP) with the purpose of identifying the optimal conditions leading to stable formulations at high oligo loading levels. The formation and loading levels of ssDNA:SPIO-NP complexes have been investigated at different oligo:NP ratios and under different ionic strengths through dynamic light scattering, fluorescence quenching experiments, and pull-down assays. Through these studies we have identified optimal conditions for attaining maximal oligo loading levels, and we are proposing a simple model to explain an unusual behavior observed in the formation of the complexes. Finally, we introduce an alternative loading method relying on the electrostatic coloading of an oligo sequence in the presence of a negatively charged PEGylated block copolymer, yielding very stable and high loading PEGylated ssDNA:SPIO-NPs. The findings that we are reporting are of general validity, and similar conditions could be easily translated to the electrostatic formation of ssDNA:NP complexes consisting of different NP materials and sizes

Microwave-Mediated Heterocyclization to Benzimidazo[2,1-<i>b</i>]quinazolin-12(5<i>H</i>)-ones

An effective route to benzimidazo[2,1-b]quinazolin-12(5H)-ones from commercially available o-aryl isothiocyanate esters and o-phenylenediamines is reported. This method accommodates a variety of substituents on either starting material and proceeds under microwave irradiation in the presence of barium hydroxide, conditions that do not hydrolyze methyl ester substituents. The pharmacologically pertinent benzimidazoquinazolinone heterocycle is delivered in excellent yield and purity via both solution- and solid-phase protocols, the latter involving traceless release from the resin

A Novel and Rapid Encoding Method Based on Mass Spectrometry for “One-Bead-One-Compound” Small Molecule Combinatorial Libraries

A novel and efficient encoding method based on mass spectrometry for “one-bead-one-compound” small molecule combinatorial libraries has been developed. The topologically segregated bifunctional resin beads with orthogonal protecting groups in the outer and inner regions are first prepared according to our previously published procedure. Prior to library synthesis, the inner core of each bead is derivatized with 3−4 different coding blocks on a cleavable linker. Each functional group on the scaffold is encoded by an individual coding block containing a functional group with the same chemical reactivity. During the library synthesis, the same chemical reactions take place on the scaffold (outer layer of the bead) and coding blocks (inner core of the bead) concurrently. After screening, the coding tags in the positive beads are released, followed by molecular mass determination using matrix-assisted laser desorption ionization Fourier transform mass spectrometry. The chemical structure of library compounds can be readily identified according to the molecular masses of the coding tags. The feasibility and efficiency of this approach were demonstrated by the synthesis and screening of a model small molecule library containing 84 672 member compounds, with a model receptor, streptavidin. Streptavidin binding ligands with structural similarity (17) were identified. The decoding results were clear and unambiguous

Supplementary Methods from <i>In vivo</i> optical imaging of human lymphoma xenograft using a library-derived peptidomimetic against α4β1 integrin

Supplementary Methods from In vivo optical imaging of human lymphoma xenograft using a library-derived peptidomimetic against α4β1 integri

Single Cell MicroRNA Analysis Using Microfluidic Flow Cytometry

<div><p>MicroRNAs (miRNAs) are non-coding small RNAs that have cell type and cell context-dependent expression and function. To study miRNAs at single-cell resolution, we have developed a novel microfluidic approach, where flow fluorescent <em>in situ</em> hybridization (flow-FISH) using locked-nucleic acid probes is combined with rolling circle amplification to detect the presence and localization of miRNA. Furthermore, our flow cytometry approach allows analysis of gene-products potentially targeted by miRNA together with the miRNA in the same cells. We demonstrate simultaneous measurement of miR155 and CD69 in 12-O-tetradecanoylphorbol 13-acetate (PMA) and Ionomycin stimulated Jurkat cells. The flow-FISH method can be completed in ∼10 h, utilizes only 170 nL of reagent per experimental condition, and is the first to directly detect miRNA in single cells using flow cytometry.</p> </div

Multiplexed detection of CD69 and miR155 using imaging and flow-FISH.

<p>A. Images of Jurkat cells showing RCA amplified miR155 signal as green dots in the cytosol, CD69 proteins stained with Qdot705 as red, and the nucleus is in blue. B. Median values of multiplexed miR155 fluorescence and CD69-Qdot705 fluorescence collected via flow cytometry.</p

Data_Sheet_1_Alternative Splicing of MXD3 and Its Regulation of MXD3 Levels in Glioblastoma.pdf

The transcription factor MXD3 is an atypical member of the MYC/MAX/MXD transcriptional network and has been previously shown to be an important regulator of cell proliferation. MXD3 has been shown to be overexpressed and to be required for medulloblastoma and acute lymphoblastic leukemia cell proliferation. In this study we leveraged datasets from The Cancer Genome Atlas to examine MXD3 across several cancers. We find that MXD3 transcripts are significantly overexpressed in ~72% of the available datasets. The gene itself is not frequently altered, while the promoter appears to be hypomethylated. We examine the possibility that aberrant regulation of the MXD3 message is the cause of abnormal MXD3 expression across cancers. Specifically, we looked at MXD3 alternative splicing in glioblastoma multiforme (GBM) and find notable functional differences between the splice variants. The 3′UTR confers differential message stability. Furthermore, the different coding sequences lead to different protein stabilities and localizations. Altogether, these data extend our knowledge of MXD3 in the context of human cancers while characterizing a previously unstudied splice variant of MXD3.</p

Structure −Activity Relationship Studies of Targeting Ligands against Breast Cancer Cells

A series of LXY3 (1) analogues were designed and synthesized. Their binding affinity was demonstrated using MDA-MB-231 breast cancer cells adherence inhibition assay. Further structure−activity relationship was obtained. Analogue 29 was discovered to have 3.5-fold increase of the binding affinity. Fluorescent microscopy and in vivo and ex vivo imaging studies demonstrated that 29 is an efficient in vivo targeting agent against α3 integrin of MDA-MB-231 breast tumor xenograft implant

LNA Flow-FISH detection of miR155 in Jurkat cells activated by PMA and Ionomycin.

<p>Jurkat cells stimulated with PMA and Ionomycin for 0, 8, 16, 20, and 24 h were loaded in duplicate into the 10-chamber chip as shown in A. The top 5 chambers were hybridized with miR155 probe, whereas the bottom 5 chambers were hybridized to scrambled probe for negative control. B. overlay of miR155 fluorescence histograms measured by LNA flow-FISH. C. miR155 median fluorescence normalized as a percentage of the maximum fluorescence, showing significant increase from 0 h at 16, 20, and 24 h stimulation. (* p<0.01) The increase at 8 h was not statistically significant (** p>0.01). D. RT-qPCR of miR155 fold changes in Jurkat cells treated with PMA and Ionomycin, also showing significant miR155 increase starting at 16 h stimulation, but not at 8 h. (** p>0.01, * p<0.01).</p