Category Archives: Molecular Biology

Left Helix: Yellow! Rational design of single-chain peptide-based nanostructures

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Nanobiotechnology is a rapidly growing and extremely fascinating field that, in one aspect, aims to rationally and selectively design biological molecules to perform advanced functions (the other aspect is to design non-biological platforms for biological processes). The most common biological molecules used are DNA, RNA and proteins for the reason that they may be conveniently and reliably produced by a variety of processes (PCR, IVT, IVTT, solid-state synthesis, etc). A few days ago I even mentioned one of these methods, DNA nanostructures (sometimes called DNA origami) and the amazing things they can do with biological computing. While there hasn’t been a world-changing breakthrough in the application of these structures yet, there has been some really interesting possibilities demonstrated. One of my favorites comes out of the institute I’m getting my PhD at and describes the application of DNA nanostructures as a platform for the directed assembly of synthetic vaccines.

Fortunately, the field is rich in brilliant scientists thinking of ways to push the boundaries.

In a April 28, 2013 Chemical Biology paper a collaborative group of researchers in Slovenia and UCSF describe the rational design, production and characterization of a three-dimensional nanostructure folded out of a single peptide chain. What is most interesting is that this group strayed from the traditional path of peptide-based structure design. Normally, structural peptide design has been based on motifs and structures which are commonly found in natural proteins. De novo fold design and synthesis has been unsuccessful with the exception of a few cases.

Determination of the sequence-dependent topology of the self-assembled polypeptide.

Only when the structure is properly folded does the YFP assembly and display fluorescence. Image: Gradišar et al, Nature Chemical Biology 2013

The current report describes the application of a platform wherein interacting coiled-coil motifs drove the self-assembly of the protein fold. In this way, the group was able to design a tetrahedral structure which was very unlike protein folds found in nature. They went further and visually characterized their protein using AFM to directly see the small, three-dimensional, pyramid-like shapes. Additionally, they demonstrated the ability to functionalize these structures by attaching split pieces of the fluorescent protein YFP to different vertices of the tetrahedron  (shown above). One during proper folding would the YFP assembly correctly and show fluorescence. When the folding of the structure was disrupted, or if a position was deleted, the structure would align the segments of YFP and no fluorescence was be observed. While, this is a type of experiment which has been performed in other studies (i.e., the assembly of a Malachite Green RNA aptamer in RNA-based nanocube structures), it is an elegant and promising example of rationally designing peptide-based, non-natural structures.

The authors discuss the possibility of using these design principles for making nanostructures with cavities for drug delivery or for orienting synthetic catalytic sites. I’m excited to see what will come from subsequent studies, and also to see which biomolecule will be crowned king of the nanobiotech field – DNA? RNA? Protein?

References

1. Gradišar, H. et al. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments. Nature chemical biology 1–6 (2013).doi:doi:10.1038/nchembio.1248

Happy DNA Day! 5 internet favorites to celebrate 60 years since the discovery of the structure of DNA

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2013_04_25_DNA-Day-HeaderHappy DNA Day! On April 25th every year, we celebrate the 1953 publication of the discovery of the structure DNA in the journal Nature (Click here to read the paper!). 60 years is quite a short amount of time for us to have made such huge strides in understanding this exquisitely complex and exceedingly important biological polymer. All politics aside (we love you, Rosie!), the unbelievable environment of creativity and genius in the early 1950s was most definitely something to celebrate, and I hope you will!

In honor of DNA Day, I wanted to share some of my favorite internet bits about DNA. Did I miss any of your favorites?

1. Visual representation of transcription and translation
PBS’ “DNA. The Secret of Life” is an extremely interesting feature focusing on this wonderful molecule. In the clip below we will see a remarkably accurate representation of the process of DNA transcription (the process of producing mRNA from DNA) and translation (the process of producing protein from mRNA) – in real time!

2. Francis Crick’s letter to his son shortly after discovering the structure of DNA
Reading this letter gives me chills every single time. Once Watson and Crick (with the aid of important discoveries and data collected by Rosalind Franklin and Maurice Wilkins) discovered the structure of DNA, they had a very short amount of time to publish it. There was a literal race to the finish in terms of getting the structure correctly. In fact, chemistry giant Linus Pauling (and twice Nobel laureate) tried to deduce the structure a bit too quickly and incorrectly imagined it as being a triple helix. Shortly after, Crick mailed his son Michael a letter describing in beautiful and elegantly simple terms and figures exactly what the discovery was and how important they felt it might be. Monumental is an understatement. You can read the letter at the New York Times.

3. The Nobel prize lectures of the co-discoverers of the DNA helix
While many believe that not all true contributors to the discovery of the structure of DNA were rightly credited, we shouldn’t discount the efforts of those who were. The awarding of the Nobel Prize in Physiology or Medicine in 1962 (less than a decade after the discovery) to Francis Crick, James Watson and Maurice Wilkins “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material” was a tremendous boon to biological research and the lectures given during the ceremony are worth a careful read. Click here to read Crick’s, Watson’s or Wilkins’ lectures.

4. Cold Spring Harbor Laboratory’s “DNA From the Beginning”
This is a great resource for those unfamiliar with the fundamental experiments leading to our understanding of the structure, function and use of DNA as well as those who just need a refresher. With useful animations, videos and clear text, this website delivers complex information in a nice, digestible package. Visit CSHL’s DNAftb now here.

5. Paul Rothemund: The astonishing promise of DNA folding (TED)
One of my favorite topics, bar none. DNA origami is a fascinating field in nanobiotechnology which attempts to use the base-pairing ability of DNA to form complex 3-dimensional structures with the hopes that they can be used as materials for nano-scale devices for use in applications such as computing and drug delivery. In this talk Paul Rothemund, one of the founding scientists of the field, discusses the promise of DNA origami, what has been achieved, and what he hopes for the future. (Though RNA is looking like an equally, if not more promising, addition to the playing field)

 

Minecraft as a tool for biological models? Maybe! – A neuron built out of Minecraft bricks

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minecraft_neuronThe meshing of the world of computer gaming and biological research has steadily grown in the past couple of years – occasionally with really fantastic results. The best example that comes to mind is the solving of the previously elusive crystal structure of a retroviral protease from the Mason–Pfizer monkey virus aided by users playing FoldIt – a game wherein players explore different conformations of protein folding in order to achieve the theoretically lowest energy level. Essentially, it’s a (fun and emotionally rewarding) method to crowd-source research. The solution was published in the Impact Factor 12.71 (2011) journal Nature Structural and Molecular Biology.

While not quite as scientifically impacting (yet), Reddit user AllUpInHyuh posted images of a neuron model produced in the online game Minecraft - a game in which users interact to build, modify and interact in a world composed of Lego-like building blocks. The images show a structure composed of dynamic-type bricks which, in total, represent action potentials, ion fluxes and membrane boundaries of a neuron. As of this posting, a video of the model in action is unavailable.

This represents a very interesting perspective of biological modeling wherein users/researchers may actually “exist within” the systems they are modeling. Imaging following, real-time, an mRNA strand being produced, spliced, transported, translated and then degraded not by observing a rendering from a pre-made program, but by following a model produced by known physiological constraints of proteins, ions and other biomolecules involved. The possibilities are extremely exciting!

Click here to view the images of the Minecraft neuron model.

Seeing… quadruple? Quantitative visualization of G-quadruplex DNA expands our knowledge of genome structure and organization

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Researchers over the past 10 years have suggested the existence of 4-strand DNA structures in cells. This goes contrary to the now-60-year-old understanding of DNA has a strict double-helix-type molecule.

It turns out that the hypotheses, molecular modeling and in vitro biochemical tests were right – quadruplex DNA does exist! These motifs, termed G-quadruplex structures, are composed of guanine-rich sequences and exhibit a high thermodynamic stability and parallel-strand organization. Specifically, they “comprise two or more G-tetrads that form when four guanines  are held in a planar arrangement through Hoogsteen hydrogen  bonding, with additional stabilization provided by a monovalent cation coordinated to the O6 lone pairs of each guanine” (Source).

Presence of G-quadruplex DNA at various stages of the cell cycle.

Presence of G-quadruplex DNA at various stages of the cell cycle. Biffi et al, 2013.

By using specifically engineered antibody approach to investigating these G-quadruplex structures in DNA, researchers at the University of Cambridge have provided the first quantitative evidence of their existence in vivo and further explored their role in genomic structure and organization.

It turns out that these structures are highly stable, and can be further stabilized by small molecule ligands. The additional observation that these structures are more numerous during cell division provides an interesting therapeutic opportunity; because pro-cancerous genes, or oncogenes, usually act to speed a cell through cell division, specifically targeting these G-quadruplex structures with a small molecule ligand might halt rapidly dividing cells and poise them for destruction.

Cancer biologists like to say that cancer cells “go or grow,” and that by halting their migration we can target specifically dividing cells. Discoveries such as those described in the Nature Chemistry paper published earlier today might provide some practical opportunities for taking advantage of this deadly behavior.

The researchers hope to further their investigation by structurally mapping the locations which are prone to developing these structures in order to gain insight into their physiological functionality at the genome-wide level.

This landmark discovery shifts our thinking by shattering the iconic image of DNA as only a double-helical structure and excites me about future discoveries fueled by this breakthrough.

Read more at PhysOrg and Nature.

References

 

Creative gene synthesis during the holiday season

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While constructing a Gateway cassette I BLASTed one of my sequences and noticed an interesting gene sequence similarity: apparently the company DNA 2.0 sends out a newsletter during the holiday season that contains the Tomten gene. The sequence for this gene is:

ORIGIN      
        1 ggggacaagt ttgtacaaaa aagcaggcta ggaggataac atatgacgca gatgaccgaa
       61 aacgatgaag aaccgattaa cactcacgaa ggtcgtatcc cgcaattcac gcacgagatg
      121 atcgactgga tcaacacgga gcgttgccaa ctggactcta ctgcgcgctc tagcgaaaac
      181 gacgcatctc cggcgcgtaa actgatcaac ggcctgatcg gtcatacggc cctgctggca
      241 cgcgaagcct ccctggaaga accgcagaat acgcacatct ctctgcaaaa cgagctgtat
      301 ttcgctcgca tgctggctac cgaaattaac acccatatct cttggatcaa taccgagcgt
      361 aacatcggcc atactaccca cgaaccggcc ctggagatgc agcagaacat ctctgcatgg
      421 gcgaacgatg agcgcgaacg ctctaaccag tggctgatcg aaagctggca tatcaccgaa
      481 cagaacccta ttaacgaggc aaatgacttc atccgttcta atcagtgggg tctgcaatgg
      541 agccagaacg cccgtcagca gtttacccag ccttctcatg ctaaggaaac tcacgaaacc
      601 cagatgactg aagccctgca aaatgaaatc tccgcgtggg ctaaagagtc cgaagcatcc
      661 cagaactctg gtcgtgaaga aaccattaac ggcagcttcc gtcagatgga caacgctacc
      721 tggcagcctc agattaatac ccagtgaacc cagctttctt gtacaaagtg gtcccc
//

Not so weird, right? Now, carefully look at the translation:

MTQMTENDEEPINTHEGRIPQFTHEMIDWINTERCQLDSTARSSENDASPARKLINGLIGHTALLAR
EASLEEPQNTHISLQNELYFARMLATEINTHISWINTERNIGHTTHEPALEMQQNISAWANDERERS
NQWLIESWHITEQNPINEANDFIRSNQWGLQWSQNARQQFTQPSHAKETHETQMTEALQNEISAWAK
ESEASQNSGREETINGSFRQMDNATWQPQINTQ

Are you seeing it yet? Let me help – trade out all of the letter Q appearances (Q = glutamine) for the letter O:

MTOMTENDEEPINTHEGRIPOFTHEMIDWINTERCOLDSTARSSENDASPARKLINGLIGHTALLAR
EASLEEPONTHISLONELYFARMLATEINTHISWINTERNIGHTTHEPALEMOONISAWANDERERS
NOWLIESWHITEONPINEANDFIRSNOWGLOWSONAROOFTOPSHAKETHETOMTEALONEISAWAK
ESEASONSGREETINGSFROMDNATWOPOINTO

And finally, add a few spaces and line breaks:

M TOMTEN
DEEP IN THE GRIP OF THE MIDWINTER COLD 
STARS SEND A SPARKLING LIGHT
ALL ARE ASLEEP ON THIS LONELY FARM
LATE IN THIS WINTER NIGHT
THE PALE MOON IS A WANDERER
SNOW LIES WHITE ON PINE AND FIR
SNOW GLOWS ON A ROOFTOP SHAKE
THE TOMTE ALONE IS AWAKE
SEASONS GREETINGS FROM DNA TWO POINT O
A-ha! Those sneaky scientists at DNA 2.0 synthetically constructed (and submitted to GenBank!) a gene, which they named Tomten, that contains the first paragraph of the 1881 poem “Tomten” by Viktor Rydberg. The poem describes a small mythical creature from Scandinavian folklore named a tomte, who is alone in the cold on Christmas night, pondering the mysteries of life and death. Read the full poem here. Note the start codon, haha!
I love finding little science-y surprises!