High-Density Peptide Arrays with Combinatorial Laser Fusing

Combinatorial laser fusing is a new method to produce high-density peptide arrays with feature sizes as small as 10 mu m. It combines the high spot densities achieved by lithographic methods with the cost-efficiency of biofunctional xerography. The method is also adapted for other small molecules compatible with solid phase synthesis

Selective Functionalization of Microstructured Surfaces by Laser-Assisted Particle Transfer

Microcavity arrays represent millions of different reaction compartments to screen for e.g. molecular interactions, exogenous factors for cells or enzymatic activity. We present a novel method to selectively synthesize different compounds in arrays of microcavities with up to 1,000,000 cavities per cm2. In our approach, polymer microparticles with embedded pre-activated monomers are selectively transferred into microcavities with laser radiation. After particle patterning, heating of the particle matrix simultaneously leads to diffusion and coupling of the monomers inside each microcavity separately. This method exhibits flexibility, not only in the choice of compounds, but also in the choice of particle matrix material, which determines the chemical reaction environment. The laser-assisted selective functionalization of microcavities can be easily combined with the intensively growing number of laser applications for patterning of molecules and cells, which is useful for the development of novel biological assays

Method for combinatorial particle manipulation for producing high-density molecular arrays, particularly peptide arrays, and molecular arrays which can be obtained therefrom

application was amended and filed as EP2013/001141 and EP2014/00104

Biomolecule Arrays Using Functional Combinatorial Particle Patterning on Microchips

Biofunctionalization of surfaces in a microarray format has revolutionized biological assay applications. Here, a microarray system based on a microelectronic chip is presented that allows for a versatile combinatorial in situ molecule synthesis with very high density. Successfully demonstrating an application for peptide array synthesis, the method offers a compact approach, high combinatorial freedom, and, due to the intrinsic alignment, high and reproducible precision. Patterning the chip surface with different microparticle types which imbed different monomers, several thousand different molecule types can be simultaneously elongated layer-by-layer by coupling the particle imbedded monomers to the molecules growing on the chip surface. This technique has the potential for a wide application in combinatorial chemistry, as long as the desired monomeric building blocks are compatible with the chemical process

Peptide Arrays with a Chip

Today, lithographic methods enable combinatorial synthesis of >50,000 oligonucleotides per cm(2), an advance that has revolutionized the whole field of genomics. A similar development is expected for the field of proteomics, provided that affordable, very high-density peptide arrays are available. However, peptide arrays lag behind oligonucleotide arrays. This is mainly due to the monomer-by-monomer repeated consecutive coupling of 20 different amino acids associated with lithography, which adds up to an excessive number of coupling cycles. A combinatorial synthesis based on electrically charged solid amino acid particles resolves this problem. A computer chip consecutively addresses the different charged particles to a solid support, where, when completed, the whole layer of solid amino acid particles is melted at once. This frees hitherto immobilized amino acids to couple all 20 different amino acids in one single coupling reaction to the support. The method should allow for the translation of entire genomes into a set of overlapping peptides to be used in proteome research

Identification of RNA 3 ' ends and termination sites in Haloferax volcanii

Archaeal genomes are densely packed; thus, correct transcription termination is an important factor for orchestrated gene expression. A systematic analysis of RNA 3 ' termini, to identify transcription termination sites (TTS) using RNAseq data has hitherto only been performed in two archaea, Methanosarcina mazei and Sulfolobus acidocaldarius. In this study, only regions directly downstream of annotated genes were analysed, and thus, only part of the genome had been investigated. Here, we developed a novel algorithm (Internal Enrichment-Peak Calling) that allows an unbiased, genome-wide identification of RNA 3 ' termini independent of annotation. In an RNA fraction enriched for primary transcripts by terminator exonuclease (TEX) treatment we identified 1,543 RNA 3 ' termini. Approximately half of these were located in intergenic regions, and the remainder were found in coding regions. A strong sequence signature consistent with known termination events at intergenic loci indicates a clear enrichment for native TTS among them. Using these data we determined distinct putative termination motifs for intergenic (a T stretch) and coding regions (AGATC). In vivo reporter gene tests of selected TTS confirmed termination at these sites, which exemplify the different motifs. For several genes, more than one termination site was detected, resulting in transcripts with different lengths of the 3 ' untranslated region (3 ' UTR)

High-Density Peptide Arrays with Combinatorial Laser Fusing

Combinatorial laser fusing is a new method to produce high-density peptide arrays with feature sizes as small as 10 mu m. It combines the high spot densities achieved by lithographic methods with the cost-efficiency of biofunctional xerography. The method is also adapted for other small molecules compatible with solid phase synthesis

Alternative Setups for Automated Peptide Synthesis

Nowadays, lithographic methods facilitate the combinatorial synthesis of >50.000 oligonucleotides per cm(2), an achievement that revolutionized the whole field of genomics. High-density peptide arrays might spark a similar development for the field of proteomics, but all lithographic methods have a peptide specific disadvantage that impairs their use for peptide synthesis: Each monomer must be coupled separately to the solid support. This adds up to an excessive number of coupling cycles, especially when comparing the 4 x 20 coupling cycles that would generate an array of 20meric oligonucleotides, to the 20 x 20 cycles that would yield an array of 20meric peptides. This review mainly discusses one recent development that leads to very high-density peptide arrays: the combinatorial chemical synthesis based on electrically charged solid amino acid particles. Either a colour laser printer or a chip addresses the different charged amino acid particles to a solid support, where the whole layer of solid amino acid particles is melted. Hitherto immobilized amino acids then start to diffuse to the support, where all the 20 different amino acids couple in a spatially defined manner, and in one single coupling reaction to the support. The method should allow for the translation of entire genomes into sets of overlapping peptides to be used in proteome research