Sequence-Specific Detection of Unlabeled Nucleic Acid Biomarkers Using a "One-Pot" 3D Molecular Sensor.

DNA and RNA biomarkers have not progressed beyond the automated specialized clinic due to failure in the reproducibility necessary to standardize robust and rapid nucleic acid detection at the point of care, where health outcomes can be most improved by early-stage diagnosis and precise monitoring of therapy and disease prognosis. We demonstrate here a new analytical platform to meet this challenge using functional 3D hydrogels engineered from peptide and oligonucleotide building blocks to provide sequence-specific, PCR-free fluorescent detection of unlabeled nucleic acid sequences. We discriminated at picomolar detection limits (<7 pM) "perfect-match" from mismatched sequences, down to a single nucleotide mutation, buried within longer lengths of the target. Detailed characterization by NMR, TEM, mass spectrometry, and rheology provided the structural understanding to design these hybrid peptide-oligonucleotide biomaterials with the desired sequence sensitivity and detection limit. We discuss the generic design, which is based on a highly predictable secondary structure of the oligonucleotide components, as a platform to detect genetic abnormalities and to screen for pathogenic conditions at the level of both DNA (e.g., SNPs) and RNA (messenger, micro, and viral genomic RNA).

A modular self-assembly approach to functionalised ß-sheet peptide hydrogel biomaterials.

Two complementary ß-sheet-forming decapeptides have been created that form binary self-repairing hydrogels upon combination of the respective free-flowing peptide solutions at pH 7 and >0.28 wt%. The component peptides showed little structure separately but formed extended ß-sheet fibres upon mixing, which became entangled to produce stiff hydrogels. Microscopy revealed two major structures; thin fibrils with a twisted or helical appearance and with widths comparable to the predicted lengths of the peptides within a ß-sheet, and thicker, longer, interwoven fibres that appear to comprise laterally-packed fibrils. A range of gel stiffnesses (G' from 0.05 to 100 kPa) could be attained in this system by altering the assembly conditions, stiffnesses that cover the rheological properties desirable for cell culture scaffolds. Doping in a RGD-tagged component peptide at 5 mol% improved 3T3 fibroblast attachment and viability compared to hydrogel fibres without RGD functionalisation.

Assembly pathway of a designed alpha-helical protein fiber.

Interest in the design of peptide-based fibrous materials is growing because it opens possibilities to explore fundamental aspects of peptide self-assembly and to exploit the resulting structures--for example, as scaffolds for tissue engineering. Here we investigate the assembly pathway of self-assembling fibers, a rationally designed alpha-helical coiled-coil system comprising two peptides that assemble on mixing. The dimensions spanned by the peptides and final structures (nanometers to micrometers), and the timescale over which folding and assembly occur (seconds to hours), necessitate a multi-technique approach employing spectroscopy, analytical ultracentrifugation, electron and light microscopy, and protein design to produce a physical model. We show that fibers form via a nucleation and growth mechanism. The two peptides combine rapidly (in less than seconds) to form sticky ended, partly helical heterodimers. A lag phase follows, on the order of tens of minutes, and is concentration-dependent. The critical nucleus comprises six to eight partially folded dimers. Growth is then linear in dimers, and subsequent fiber growth occurs in hours through both elongation and thickening. At later times (several hours), fibers grow predominantly through elongation. This kinetic, biomolecular description of the folding-and-assembly process allows the self-assembling fiber system to be manipulated and controlled, which we demonstrate through seeding experiments to obtain different distributions of fiber lengths. This study and the resulting mechanism we propose provide a potential route to achieving temporal control of functional fibers with future applications in biotechnology and nanoscale science and technology.

Flow linear dichroism of some prototypical proteins.

Flow linear dichroism (LD) spectroscopy provides information on the orientation of molecules in solution and hence on the relative orientation of parts of molecules. Long molecules such as fibrous proteins can be aligned in Couette flow cells and characterized using LD. We have measured using Couette flow and calculated from first principles the LD of proteins representing prototypical secondary structure classes: a self-assembling fiber and tropomyosin (all-alpha-helical), FtsZ (an alphabeta protein), an amyloid fibril (beta-sheet), and collagen [poly(proline)II helices]. The combination of calculation and experiment allows elucidation of the protein orientation in the Couette flow and the orientation of chromophores within the protein fibers.

A de novo self-assembling peptide hydrogel biosensor with covalently immobilised DNA-recognising motifs.

We report here the first experimental evidence of a self-assembling three-dimensional (3D) peptide hydrogel, with recognition motifs immobilized on the surface of fibres capable of sequence-specific oligonucleotide detection. These systems have the potential to be further developed into diagnostic and prognostic tools in human pathophysiology.

Templating silica nanostructures on rationally designed self-assembled peptide fibers.

Nature presents exquisite examples of templating hard, functional inorganic materials on soft, self-assembled organic substrates. An ability to mimic and control similar processes in the laboratory would increase our understanding of fundamental science, and may lead to potential applications in the broad arena of bionanotechnology. Here we describe how self-assembled, alpha-helix-based peptide fibers of de novo design can promote and direct the deposition of silica from silicic acid solutions. The peptide substrate can be removed readily through proteolysis, or other facile means to render silica nanotubes. Furthermore, the resulting silica structures, which span the nanometer to micrometer range, can themselves be used to template the deposition of the cationic polyelectrolyte, poly-(diallyldimethylammonium chloride). Finally, the peptide-based substrates can be engineered prior to silicification to alter the morphology and mechanical properties of the resulting hybrid and tubular materials.