Miriam’s comment on: Reversible Changes in Solution pH Resulting from Changes in Thermoresponsive Polymer Solubility

By Miriam I. Otero Rivera
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In this article by Bergbreiter’s group, present the effect of a LCST event on the solution pH of a polymer like poly(N isopropylacrylamide) better known as PNIPAM. They present different experiments were they change pendant groups on PNIPAM and all of these resulting polymer derivatives had lower critical solution temperature (LCST) properties. In the experiments they show how after heating the solution to its LCST the polymer experience a dehydration and the polymer phase separates from solution. In several experiments, the presence of a cationic or anionic pendant groups, that have low or high pH values respectively, its stabilization is dependent of its solvation, and after the LCST event, the species becomes neutral since it is more stable in the dehydrated state resulting from the LCST phenomenon. These experiments show that changes in the environment of the polymers results in great changes in solution acidity and basicity. Also they show that these process of hydration and dehydration is reversible by cooling the solution they obtain the polymer back in solution and the pH was revert to its initial value, this process of heating and cooling was reversible for up to 100 cycles.
Similarly, the article studied the effect of the concentration of the polymer, and the mole % loading of the pendant group (carboxylic acid) in changing the bulk solution pH as a function of temperature. First they saw that if the pH of the solution is significantly different from the pKa of the carboxylic acid (4.76) there is no change in pH. They also saw that at lower mole % loading of carboxylic acid the cloud point curve is most narrow and that at lower concentration of the polymer the clouding curves are broader. Also, studies with solutions containing added salts (LiCl or LiBr) show that the solution’s ionic strength does not significantly affect the ΔpH, although the clouding curve’s onset changes. Finally, they performed experiments with kosmotropic salts, which lower the LCST below room temperature due to the Hofmeister effect, as a result the addition of these salts to solutions lead to the same behavior of changing the pH of the solution, and also the process was reversible. This experiment was visualized using the pH-indicator phenolphtalein. In this experiment they also used visible spectroscopy to show how  reaching  the LCST temperature produce an inflection, before a drastic change in absorbance.  This article show how properties in the pendant group of a polymers change pH of solution as a result of changes in solubility because of changes in temperature, showing the thermoresponsiveness of polymers. This article was one of relevance to our laboratory because we work with LCST events on supramolecular G-quadruplexes. This article can be used as a reference to know the resulting effects on solubility and pH of an LCST event on the systems with which we work in our lab.


Rafa’s comment on: Thermal Switching of Thermoresponsive Polymer Aqueous Solutions

By Rafael A. Brito

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Thermal switches are of great importance to thermal management in a wide variety of applications. A common characteristic associated with thermal switching is thermal conductivity. After noticing the change in thermal conductivity across LCST transitions, Zhiting Tian started researching polymers for this purpose. Poly(N-isopropylacrylamide) (PNIPAM) is the most studied thermoresponsive polymer and has a workable LCST of 32° C. The LCST transition of PNIPAM changes the chain-like formation of the polymer into an aggregation that shows a drastic decrease of thermal conductivity. This sharp change is due to LCST transitions being second-order, which are characterized by being almost instantaneous when the corresponding temperature is reached. Thermal conductivity was measured by applying a powerful approach: the transient thermal grating technique. It is used by heating a solution as a function of position creating a grating of temperature. This grating allows the use of the one-dimensional heat equation, which can be solved to give a relation between the thermal conductivity and temperature. The thermal conductivity can then be calculated using 𝑘 = 𝜌𝑐𝑝α, where 𝜌 is the density,  is the specific heat capacity and α which is a function of temperature. After the setup was completed, solutions of varying concentrations were analyzed. For the solution with the highest concentration, the thermal switching ratio was measured to be 1.15 across the LCST transition. This shows a significant change between the two states of the polymer. The thermal conductivity of the PNIPAM aqueous solutions increases with temperature, the same as with water, until reaching the LCST. Then a drastic change is observed in the solution. The thermal switching ratio of PNIPAM aqueous solutions across the transition keeps increasing with increasing concentration, which is expected from the equation. To explain the thermal conductivity change due to the transition between the two modes of the polymer, the authors used the idea that the homogeneous phase of the solution separates into two phases that increases the thermal interface resistance resulting in a lower effective thermal conductivity.

As a summary, they reported the first direct measurement of thermal conductivity change in PNIPAM aqueous solutions across the LCST using a powerful approach, the laser-induced transient thermal grating technique. The results show an abrupt thermal conductivity drop across the transition temperature. The potential of using thermoresponsive polymer aqueous solutions of higher-order phase transitions for thermal switch applications has been demonstrated throughout this paper’s work.


Yanira’s comment on: Intracellular Guest Exchange between Dynamic Supramolecular Hosts

By Yanira Rodríguez Valdéz

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The article by Swaminathan et al. studies the Förster Resonance Energy Transfer (FRET) between two dyes encapsulated in self-assembled nanoparticles made from amphiphilic polymers. The encapsulation of hydrophobic chromophores such as borondipyrromethene (BODIPY) and anthracene inside the hydrophobic interior of these nanoparticles allows their study in aqueous media. In close proximity (inside the same host), BODIPY and anthracene undergo FRET (anthracene = donor, BODIPY = acceptor). The authors observed that mixing nanoparticles loaded with anthracene and nanoparticles loaded with BODIPY has the same effect as loading nanoparticles with both anthracene and BODIPY, which suggests guests being exchanged when nanoparticles are mixed and that they are being captured by the same nanoparticle(s). These studies are performed both in aqueous media and in vitro (HeLa cells), which confirms that the guests can also be exchanged intracellularly.

Two mechanisms for guest transfer between hosts are proposed: 1) guests escape a host and subsequently are captured by another or 2) collision between hosts leads to exchange of guests. They indicate that because of the poor aqueous solubility of the fluorophore guests, mechanism 2 is more likely, but the authors do not perform any experiments to confirm this. They also state that 10-fold dilution with PBS does not affect energy-transfer efficiency. This is not expected if mechanism 2 is at play. Dilution would lead to less frequent collisions between hosts, and therefore, to less guest exchange, and less energy-transfer. The energy-transfer observed could be explained by diffusion, which is more akin to mechanism 1. Poor solubility might not be an issue, because as long as a small amount is soluble, escapes the host and is further entrapped by a host carrying the opposite guest, energy-transfer can still occur.

The authors attempt to determine where the supramolecular assemblies are localized intracellularly by incubating HeLa cells with two sets of nanoparticles, loaded separately with anthracene and BODIPY, and either chlorpromazine or genistein, which inhibit either clathrin- or caveolae-mediated endocytosis, respectively. The results indicated that intracellular fluorescence was reduced to a greater extent by the addition of chlorpromazine, which suggests that the particles are predominantly internalized by clathrin-mediated endocytosis. However, the authors do not perform further experiments that show the particles localized in endosomes and/or lysosomes, which would result from clathrin-mediated endocytosis. They seem to be attempting to rule out any other methods of internalization, but given that chlorpromazine only reduced the intracellular fluorescence to 59%, other methods of internalization could still be taking place.

Swaminathan, 2014. Intracellular Guest Exchange between Dynamic Supramolecular Hosts

Carla’s comment on: Active Targeting of the Nucleus Using Nonpeptidic Boronate Tags

By Carla M. Quiñones

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The main objective of the research described in this article by Rotello and coworkers was to use a synthetic non-peptide targeting motif that accesses the nucleus of the cell through an active transport mechanism. Active and passive transport take place in the cellular membrane as well as in the nuclear membrane. Intracellular targeting is as important as cellular targeting due to their relevance in drug delivery and potential therapies. The two challenges faced by the research group was to deliver the nuclear-targeted protein into the cytosol and to comprehend the mode of nuclear entry. They delivered the protein successfully to the cytosol with the help of a previously synthesised nanoparticle stabilized particle, which encapsulated the proteins of interest and liberated them into the cytosol via membrane fusion. The most significant finding was the efficiency of targeting the nucleus when modifying each protein of interest with benzyl boronate tags (BB tags).
They performed experiments with different proteins modified with BB tags resulting in a successful high-efficiency delivery to the nucleus. To assess the role of boronic acid in the BB tags, they modified GFP with the benzyl tag alone and saw less fluorescence inside the nucleus. This suggests that the boronic acid is necessary in the BB tags for a successful nuclear targeting. Furthermore, they determined if the mechanism of transport into the nucleus was either active or passive. To accomplish this goal, they added Ivermectin, an inhibitor for the α/β importin pathway (active transport), to the cells and also depleted ATP (required for all active transport pathways) in another set of experiments. They delivered GFP with 3 BB tags in both cases and saw a lower nuclear efficiency. They concluded that these modified proteins targeted the nucleus effectively through the importin α/β pathway (active transport) rather than passive transport.
Through the last part of the paper, I was curious of how they acknowledged that this happened the way the proposed since they didn’t explain the mechanism behind the α/β importin pathway, nor the chemistry of how the BB tag contributes to the nuclear targeting. Intrigued by this, I searched more about this phenomenon, but found no relevant studies, probably because this is a novel research field. In general, this was a good article and useful to our lab since we work with supramolecular systems which, in the future, we could adapt this strategy to achieve nuclear targeting.


María’s comment on: Artificially Engineered Protein Hydrogels Adapted from the Nucleoporin Nsp1 for Selective Biomolecular Transport

Maria Luisa Adrover


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The main goal of the article by the Olsen group is to present a new protein hydrogel composed of nucleoporin like polypeptides that allow a selective transport of biomolecules into the formed hydrogel. The engineering of this system was inspired by the selectivity of the protein matrix in the nuclear pore complex. The nuclear pore complex is composed of nucleoporins like Nsp1 (16 repeats of a 19 amino acid sequence). These are proteins containing Phe-Gly (FG) repeats that contribute to specific binding of the nuclear transport receptors. The authors took advantage of the finding that individual nucleoporins can form hydrogels in vitro. They designed three different nucleoporin-like polypeptides (NLP) using pentameric (P) coiled-coil domains in the C and N terminus of the polypeptide: P-1NLP-P, P-2NLP-P and P-cNsp1-P (the original nucleoporin).

In their experiments, they conducted rheology studies (using oscillations as a type stress) to evaluate the flow properties of the protein hydrogel. They found that the hydrogel in behaves in some cases as a viscous liquid (when G“ > G`) while in other cases it behaves as an elastic solid (when G` > G“). Also, they noticed by adding hexanediol, the FG repeats were weakened making the hydrogel less elastic. Another study was done using Raman spectroscopy too see any changes in the peaks of the spectra. They saw that by adding hexanediol there was a reduction the Phe vibrational mode of 486 cm-1 meaning that the FG repeats where indeed being disrupted. Lastly, using capillary experiments they studied the selective transport of the protein hydrogels. They studied the selectivity of the biomolecules measuring the fluorescence intensity of: Importin β, IBB-MBP-EGFP (Importin β binding with maltose binding protein and enhanced green fluorescent protein) and MBP-mCherry. Fluorescence studies showed that Importin β was diffused inside P-cNsp1-P hydrogel with the highest intensity, followed by P-2NLP-P and lastly P-1NLP-P. Interestingly, they showed that just by changing one amino acid (Serine to Aspartic Acid) the selectivity for Importin β changed. This suggested that small changes to the nucleoporin like polypeptides affects the selectivity. Adding 10% hexanediol to P-2NLP-P barely showed fluorescence mainly due to the FG disruptions mentioned before. Finally, a successful engineering of hydrogels was achieved showing a high selectivity just like the natural nucleoporin system. A relatively simple polypeptide chain just like the cNsp1 allow the design of nucleoporin-like polypeptides as selective as the protein matrix found inside the nuclear pore complex.

Maxier’s comment on: Macromolecular Crowding Modifies the Impact of Specific Hofmeister Ions on the Coil–Globule Transition of PNIPAM

By Maxier Acosta Santiago

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While the recently discussed Cremer paper described efforts towards understanding the effect on the LCST of PNIPAM by salts of the Hofmeister’s series, Sakota’s article went somewhat deeper into the study of this phenomenon by taking in consideration molecular crowding. Sakota’s group decided to use a PEG polymer as a crowding agent. Crowding may affect the biding of anions from the Hofmeister’s series and the PNIPAM resulting in a change in the LCST. First, they studied the presence of PEG in a PNIPAM solution showing that the crowding agent reduces the LCST. Kosmotropic anions, that decrease the LCST, but chaotropes increase the LCST. For the Hofemeister series effect on LCST we can go back to Luis Prieto’s blog post and Cremer’s paper which explains this effect better.

When the article begins to look at the presence of PEG at different salt concentrations, they see a close correlation between the LCST and the Hofmeister series. Yet, for the chaotropes ClO4⁻ and SCN⁻, the presence of PEG lead to a larger increase in the LCST. From here they decide to apply different theories to explain the results. Within the examined theories they discuss around thermodynamics of the system. Their explanation evolves as follows, even if the organization via LCST of PNIPAM is not thermodynamically favorable, the overlapping excluded volumes of PEG and the PNIPAM particles increase the translational entropy of water molecules in solution, which makes the formation of the system possible.

Although this paper brings something new to the table to discuss (molecular crowding and LCST), I do have some concerns. When taking into consideration so many different factors like molecular crowding, salt, and the responsive system itself, we should look deeper into the behavior. To limit certain factors, they maintained certain constant concentrations throughout the paper. Yet, in the discussion, I feel they lacked additional experimental results or computational studies (using molecular and/or coarse-grained simulations) to support their theoretical thermodynamics analysis.

Minelise’s comment on: Synthesis and Direct Observation of Thermoresponsive DNA Copolymers

By Minelise E. Rivera

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In this paper, Li and Schroeder use single molecule techniques to have a direct observation on DNA-PNIPAM copolymers. First, they synthetized DNA-PNIPAM copolymers using a two-step strategy based on polymerase chain reaction (PCR) for generating linear DNA backbones containing dibenzocyclooctyne-dUTP, then grafted thermoresponsive side branches (PNIPAM) onto DNA backbones using copper-free click chemistry. Subsequent single molecule fluorescence spectroscopy studies unveiled more clearly the molecular heterogeneity association with the stretching and relaxing of the polymer above and below their LCST. Their results showed that intramolecular conformational dynamics of DNA-PNIPAM copolymers are affected by properties of the branches like molecular weight, density, leading to a change in transition temperatures. In other words, the single molecule technique provided a better understanding in a molecular perspective of chemically heterogeneous and stimuli-response polymers.

As I read this paper and looked for information to better understand it, I was amazed by the details with which they worked with throughout their study. I would have thought of working better with a bunch of them instead of just single molecules. It didn’t cross my mind that someone was going to, not only synthesized the molecule, but also study its characteristics. It was very interesting to learn about the methods that they used for characterization and synthesis. It got me wondering if those methods were the only ones that would work in this case and why. But, what I think that was very useful for me is that I got to understand better the importance of the LCST and the role that it played in their system. It reminded me of our project in which the SGQ self-assembles into the SHS and how it is to study it and understand its influence in the SHS as it was important for the copolymers with which it was worked with in the paper.

Kiara’s comment on: A 2,7-diamino-1,4,8-triazanaphthalene derivative selectively binds to cytosine bulge DNA only at a weakly acidic pH

By Kiara N. Villa Del Valle

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This paper focuses reports how through a change in the pH, their compound gets protonated and through hydrogen bonds, the compound and the base binds. Lastly, the better-known DNA structure the double helix, but there are other varieties such as hairpin loops, interior loops, multi-branched loops and bulge loops. Bulge loops, are when an extra base winds up on one side of the DNA strand. This can happen when the helix missing a base or an extra one was inserted during the DNA copying process. This anomaly can cause cancer or triplet diseases. They first performed experiments to determine the pH dependence if the azaDANP. They changed the pH from 1.0 to 9.0 and it shows a hypochromic and hypsochromic shifts as the pH was changing. They also plotted the absorption in terms of the pH and determined a pKaH (pKa of the protonated form) of 4.3, a lower pKaH of 6.8 for a previously described compound. This decrease was due to the decreased basicity of the ring nitrogen by the substitution at C4-N. To get information about the azaDANP binding to the C-bulge, they simulated possible complexes between azaDANP and cytosine, it showed that the protonation likely occurs at N1 for the complex formation. They performed more experiments to measure the absorption spectra of azaDANP in presence of T, G, A and C bulged DNA, at pH 7.0 and 5.5 at room temperature. We want to focus in this, at 5.5 pH there is a new absorption band at 407 nm that in neutral pH is not observed and therefore suggesting that the formation is pH sensitive. The thermal stability of the azaDANP-C-bulge was investigated with absorption with variable temperature. We can see that the peak observed at 407 nm at 2 °C decreases as the temperature is increased to 80 °C. This shows the equilibrium of azaDANP and GCG/CC DNA to form a binary complex and, therefore, appearing to be temperature dependent.


Aikawa, 2017. A 2,7-diamino-1,4,8-triazanaphthalene derivative selectively binds to cytosine bulge DNA only at a weakly acidic pH

José’s comment on: Intelligent, Biodegradable, and Self-Healing Hydrogels Utilizing DNA Quadruplexes

By José M. Martinez

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This paper is about making of a water based gel made from the supramolecular interactions of guanosine rich oligonucleotides. They attached these oligonucleotides symmetrically into the ends of PEG chains of variable lengths and conformations. This was made through High Efficiency Liquid Phase (HELP) synthesis. This synthetic strategy, as its name says, occurs in a liquid phase instead of the usual solid phase synthesis. The purpose of this is to allow them to obtain greater quantities of product more easily without risking the effectiveness. These DNA-PEG-DNA monomers have the ability, when diluted and presented with a cation (K+ or Na+), to self-assemble its DNA moieties through non-covalent interactions to for G-quadruplexes. Those strong interactions change the physical properties of the solution by forming a hydrogel. In the study, it was proven that the concentration of the cation required to trigger the self-assembly is relatively small. It is even compared to the concentration of salts found in body-related fluids like sweat, saliva, and tears. The hydrogel also has the capability to heal itself and interact by diffusion with another of the same kind. Finally, it was found that by adding a few extra nucleotides to the DNA moiety of the monomer and a matching strand for that part, the G-quadruplexes could be disassembled, returning the gel back to its liquid phase. All the previously stated properties indicate potential uses of this hydrogel for biomedical purposes.
Regarding myself, I learned mostly about the molecular aspect of gels and how they are formed. It was very interesting seeing the role that the supramolecular interactions played in the formation of this particular gel and the experiments made to investigate further into its properties.

JMM: Tanaka, 2017. Intelligent, Biodegradable, and Self-Healing Hydrogels Utilizing DNA Quadruplexes

Diana’s comment on: Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

By Diana Silva Brenes

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Relating molecular structure to function is the first step and one of the greatest challenge to understand nature’s designs or to make novel “functional designs” of our own. This paper by the Chilkoti group begins with statistical analysis of some of the most relevant proteins displaying LCST and UCST behavior. By analyzing the peptide sequences, the authors identify as common motif for both behaviors a high glycine & proline content. Furthermore, for LCST abundance of aromatic residues seems to be a requirement whereas UCST peptides seem to be encoded by a pair of zwitterionic residues.
To test if these observations lead to LCST/UCST phenomena, over 80 model peptides were recombinantly synthesized and their thermoresponsive behavior was measured by UV absorbance while changing the temperature. Each peptide presented the predicted behavior, giving support to their observations. Furthermore, by comparing a few selected examples, they show how an increase in hydrophobicity leads to an increased UCST cloud point and how eliminating one of the residues from azwitterionic pair turns a UCST peptide to an LCST peptide.
The LCST and UCST behavior is, however, a complex phenomenon dependent on protein-protein versus protein-water interactions, which in turn are modulated by more factors aside from the sequence of the protein. The possible scenarios are limitless, and the authors give insight on the most significant: peptide length, concentration, and pH (charge state of protonable atoms).
The robustness of the behavior encoded in the rules they found can be seen by a hybrid peptide containing both an LCST portion and a UCST one. The resulting peptide displays both behaviors, albeit at different temperatures from the “parent” sequences.
Finally, the authors show that searching for the characteristics they determined as important for LCST/UCST behavior throughout the human proteome produces examples of proteins whose function could very well be related to a thermoresponsive behavior, highlighting the applicability of their observations to understand the phenomena that make life as we know it possible.

Quiroz, 2015. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers