Engineering structure and function using thermoresponsive biopolymers

Abstract

Self-assembly enables exquisite control at the smallest scale and generates order among macromolecular-building blocks that remain too small to be manipulated individually. Environmental cues, such as heating, can trigger the organization of these materials from individual molecules to multipartixcle assemblies with a variety of compositions and functions. Synthetic as well as biological polymers have been engineered for these purposes; however, biological strategies can offer unparalleled control over the composition of these macromolecular-building blocks. Biologic polymers are macromolecules composed of monomeric units that can be precisely tailored at the genetic level; furthermore, they can often utilize endogenous biodegradation pathways, which may enhance their potential clinical applications. DNA (nucleotides), polysaccharides (carbohydrates), and proteins (amino acids) have all been engineered to self-assemble into nanostructures in response to a change in temperature. This focus article reviews the growing body of literature exploring temperature-dependent nano-assembly of these biological macromolecules, summarizes some of their physical properties, and discusses future directions.

Figure 1

Biological polymers represent a rich source for the engineering of nanostructures. Three classes of biopolymers are evaluated within this review: DNA oligonucleotides, polysaccharides, and protein-based sequences. Each has the ability to specifically self-associate, which can modulate the formation of a wide variety of nanostructures. As their association depends on kinetics and/or thermodynamics, these structures are responsive to temperature. All produced from biological sources, they can be engineered by manipulation at the genetic level to varying degrees. The exquisite control exerted by biological synthetic pathways suggests that they are excellent candidates to engineer useful biomaterials. Adapted with permission from the American Chemical Society^{, ,} . Adapted with permission from John Wiley and Sons. Adapted with permission from the Centre National de la Recherche Scientifique and The Royal Society of Chemistry.

Figure 2. Oligonucleotide origami uses single stranded DNA (ssDNA) springs to engineer precision joints

Two arms composed of 3 bundles of double stranded DNA (18 helices total) were linked by a single rigid bundle (6 helices) as well as a ssDNA spring. The length of the spring was modified to produce different angles. Conformational analysis was performed using TEM imaging. Typical particles and histogram distribution are presented with A) 0, B) 11, C) 32, D) 53, and E) 74 bases in the ssDNA springs. The black lines show Gaussian fits to the data. The angles corresponding to the peak values of Gaussian fits were 56.5° (n = 154), 70.2° (n = 213), 97.9° (n = 169), 110.0° (n = 252), 128.2° (n = 204). Bars = 20 nm. Adapted with permission from the American Chemical Society.

Figure 3. Structural versatility of biomaterials generated from gellan gum polysaccharides

A) discs; B) membranes; C) fibers; D) microparticles; E) and F) 3D lyophilized scaffolds. Adapted with permission from John Wiley and Sons.

Figure 4. Peptide-based leucine zippers from GCN4 form nanoropes

Nanorope formation is temperature and cosolute dependent. A) Phase image using tapping mode AFM of nanoropes formed in 1.5 M NaCl. The scale bar represents 500 nm and the z-scale represents 25 nm. B) Phase image of nanoropes formed in 0.75 M (NH₄)₂SO₄ shows even longer structures than those in A). C,D) Circular dichroism was used to study the formation of alpha helices (-Θ_{222 nm} for 144 μM peptide in 10 mM Tris, pH 8.0) as a function of C) cosolute concentration at 25 °C including Na₂SO₄ (●), (NH₄) ₂SO₄ (▲), and NaCl (◆) and glycerol (inset graph) and D) temperature as a function of NaCl concentration, which shows that salt stabilizes the alpha helices until a higher temperature. Adapted with permission from PNAS.

Figure 5. Intracellular assembly and function of elastin-like polypeptide microdomains

A) An amphiphilic ELP (GFP-S48I48) and an ELP monoblock (dsRED-V96) with similar transition temperatures and molecular weight sort into separate microdomains in live cells. B) An ELP fused with the clathrin-light chain (V96-CLC) is soluble at 31 °C but assembles into V96-CLC microdomains above 37 °C. Red = ELP, Green = the angiotensin II receptor (AngIIR) at the cellular membrane. C) V96-CLC microdomains sequester the machinery of clathrin-mediated endocytosis and inhibit the internalization of AngIIR at 37 and 42 °C. The V96-CLC fusion remains soluble at 31 °C and does not affect receptor internalization. (**p<0.0001) Mean ± 95% confidence interval (n=3). Adapted with permission from Wiley–VCH Verlag GmbH & Co ^, .

Figure 6. Resilin: A genetically engineered protein responsive to multiple stimuli

Cryo-TEM micrographs of rec1-resilin for solutions of A) Vitrified rec1-resilin at 20 °C has only dispersed spherical particles approximately 9.5 nm in diameter with poor contrast, which is consistent with soluble proteins. B) Vitrified rec1-resilin just below UCST (4 °C), shows a high-density network of well-dispersed interconnected spherical particles with 5.4 nm diameters and excellent contrast. C) Above LCST, large discrete spherical aggregates with a size range of 100 nm to 130 nm diameters form. D) Increasing solution concentration to 10 mg ml^-1 causes the formation of an interconnected gel particle network at the UCST. Adapted with permission from John Wiley and Sons.