Bioengineering and characterization of DNA–protein assemblies floating on supported membranes

Boireau, Wilfrid; Bombard, Sophie; Sari, Marie-Agnès; Pompon, Denis

doi:10.1002/bit.10111

Cited by 18 publications

(15 citation statements)

References 25 publications

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“…The self-assembled monolayer (SAM) process, obtained using a previously published procedure (Boireau et al, 2002), leads to the formation of a packed monolayer of OM, which confers a high degree of hydrophobicity to the chip (Fig. 1A).…”

Section: Proof Of Concept Of Entire "On-a-chip" Bia-ms Analysis Of Rsmentioning

confidence: 99%

Revisited BIA-MS combination: Entire “on-a-chip” processing leading to the proteins identification at low femtomole to sub-femtomole levels

Boireau

Rouleau

Lucchi

et al. 2009

Biosensors and Bioelectronics

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We present the results of a study in which biomolecular interaction analysis (BIA, Biacoretrade mark 2000) was combined with mass spectrometry (MS) using entire "on-a-chip" procedure. Most BIA-MS studies included an elution step of the analyte prior MS analysis. Here, we report a low-cost approach combining Biacore analysis with homemade chips and MS in situ identification onto the chips without elution step. First experiments have been made with rat serum albumin to determine the sensitivity and validation of the concept has been obtained with an antibody/antigen couple. Our "on-a-chip" procedure allowed complete analysis by MS/MS(2) of the biochip leading to protein identifications at low femtomole to sub-femtomole levels. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the "on-a-chip" processing to complete the BIA-MS approach in the discovery and analysis of protein complexes.

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Section: Proof Of Concept Of Entire "On-a-chip" Bia-ms Analysis Of Rsmentioning

confidence: 99%

Revisited BIA-MS combination: Entire “on-a-chip” processing leading to the proteins identification at low femtomole to sub-femtomole levels

Boireau

Rouleau

Lucchi

et al. 2009

Biosensors and Bioelectronics

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“…Abbreviations used: AFM, atomic force microscopy; CM, confocal microscopy; DTT, dithiothreitol; SPRI, surface plasmon resonance imagery; PDNA, protein-DNA complex; QD, quantum dot. 1 To whom correspondence should be addressed (email pompon@cgm.cnrs-gif.fr).…”

Section: Building Blocks Structure and Constructionmentioning

confidence: 99%

PDNA as building blocks for membrane-guided self-assemblies

Pompon

Laisné

2007

Biochemical Society Transactions

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Different semi-synthetic PDNAs (protein-DNA complexes), which encompass a protein core engineered from the cytochrome b(5) scaffold, an embedded tuneable redox cofactor, a synthetic linker and a large oligonucleotide, were designed, synthesized and purified to homogeneity. These building blocks can be reversibly attached to Ni-DOGS [1,2-dioleoyl-sn-glycero-3-[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl]-doped supported membranes through a metal chelate bridge with the protein part and be polymerized in a fully controllable manner using a solid-phase synthesis strategy and a stepwise addition of suitable complementary oligonucleotides. The resulting structures could recreate a large range of regular distribution of patterned redox and absorbing centres separated by fully tuneable distances and geometry. Kinetic parameters for the self-assembly of building blocks were determined using SPRI (surface plasmon resonance imagery). Structures of resulting nano-objects were characterized using gel electrophoresis and single molecule approaches following decoration of assemblies with quantum dots.

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“…a unique sulfhydryl residue at the periphery of the 3D structure. Bearing this sulfhydryl moiety, cytochrome b5 is able to (i) react with another cytochrome b5 to form dimeric species through disulfide bridge, (ii) crosslink with specific molecular spacers (linkers) and (iii) perform direct self assembly onto gold surface by chemisorption process leading to a packed and oriented self assembled monolayer (SAM) [4]. This last steep will occur at the surface of the sensor chip and will be analysed in real time by SPR measurements.…”

Section: Introductionmentioning

confidence: 99%

TOF-SIMS structural characterization of self-assembly monolayer of cytochrome b5 onto gold substrate

Aoyagi

Rouleau

Boireau

2008

Applied Surface Science

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Orientation and three-dimensional structure of immobilized proteins on bio-devices are very important to assure their high performance. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is able to analyze upper surface of one layer of molecules. Orientation of immobilized proteins can be evaluated based on determination of a partial structure, representing ensemble of amino acids, on the surface part. In this study, a monolayer of cytochrome b5 was reconstituted onto gold substrate and investigated by surface plasmon resonance (SPR). After freeze-drying, the resulted protein self-assembly was evaluated using TOF-SIMS with the bismuth cluster ion source, and then TOF-SIMS spectra were analyzed to select peaks specific to cytochrome b5 and identify their chemical formula and ensembles of amino acids. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the modified cytochrome b5 and three-dimensional structure of cytochrome b5 registered in the protein data bank. Finally, fragment-ion-generating parts of the immobilized-cytochrome b5 are determined based on the suggested residues and threedimensional structure. These results suggest the actual structure and confirm the expected orientation of immobilized protein.

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Bioengineering and characterization of DNA–protein assemblies floating on supported membranes

Cited by 18 publications

References 25 publications

Revisited BIA-MS combination: Entire “on-a-chip” processing leading to the proteins identification at low femtomole to sub-femtomole levels

Revisited BIA-MS combination: Entire “on-a-chip” processing leading to the proteins identification at low femtomole to sub-femtomole levels

PDNA as building blocks for membrane-guided self-assemblies

TOF-SIMS structural characterization of self-assembly monolayer of cytochrome b5 onto gold substrate

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