Part 12, appearing by page 70-something, seems to be the place to do the reveal. This is a dialogue between Carson and Sean about what Carson hopes to achieve through self-experimentation with a virus that reprograms his nervous system.
I label this post as the progression because I’ve been working and re-working the first 100 pages for several months. To put that into context, the novel has been roughly 320 pages long when the first draft was finished a year ago. Originally, the reveal bubbled to the surface of the story at least 100 pages later. This was not satisfying.
Over the course of those months, the reveal has been moving backwards toward the opening pages making it a better story.
At some point, I will have to pickup at page 120-something and move forward into a new narrative arc. It could easily wipe out half of the original. Future posts will offer their own reveal on that possibility.
These are my notes for the science background for the experiments of Carson Moran and his work at QuantumDotCodes located in Georgetown (south of Seattle).
Our goal was to develop a bifunctional peptide linker that could allow easy attachment of DNA oligonucleotides at one end while the other is modified with a polyhistidine tag to facilitate self-assembly of the full peptide-DNA complex onto QDs via metal-histidine interactions.
We have developed a conjugation strategy based on metal-affinity-driven
interactions between CdSe-ZnS core-shell QDs and proteins or peptides appended with polyhistidine (Hisn) tags. … The His tag drives self assembly by interacting directly with the metallic surface …
- (DNA) DNA conjugates of target DNA are thiolated (sulf-hydride group attached):
- (linker) The synthesis of the peptide module was performed by standard solid-phase peptide synthesis (SPPS) on Rink amide resin to create the desired His6-Cys sequence.
The crude peptide was precipitated, and the final bifunctional reactive linker His6-Cys(Ac)-S-S-Py was obtained through a direct disulfide exchange reaction.
(the initial reactive chemistry for His6 attachment to thiolated-DNA is one of the fastest and most common linkage chemistries used in bioconjugation)
- (tag) Functionalization of free 5‘-thiol DNA with the peptide linker (4) to yield the His6-tailed oligo (5) was rapid and straightforward …
and the mixture was allowed to react anywhere from 1 h to overnight
- (Quantum Dot) The His tag drives self assembly by interacting directly with the metallic surface [of the Quantum Dot]
The QD-peptide-DNA conjugates were further characterized by atomic force microscopy (AFM) imaging, where association of oligos with a central QD was observed only for samples made of QDs mixed with His6-peptide-DNA …
We also demonstrate the potential utility of this His-reactive-peptide modification of DNA by assembling and testing a QD-DNA molecular beacon that specifically detected the presence of its complementary sequence.
The potential of this reactive linker was demonstrated by self-assembling several QD-DNA conjugates as well as a QD-MB construct able to discriminate between different sequences of DNA. A variety of other applications, such as highly luminescent multilabeled hybridization probes, are possible using this construct. Preforming MB sensors with different color QDs and then mixing them may allow “multiplexing”. Beyond nanoparticle-MBs, this selfassembly technique may be applicable to attaching biomolecules to a variety of other similarly prepared surfaces.
And now for the shortened version. Above, you will see a very stylized expression of the process of self-assembly. Again, we will do this by steps:
- Obtain the His6 tags (Polyhistidine-tag)
- Obtain Cy5 dye (a fluorescent/fluorophore nano particle)
- Obtain the Thiolated DNA
- Label the Thiolated DNA with Cy5 dye, the dye becoming a quenched fluorophore;
- Obtain the Quantum Dots
- Combine where:
- The Thiolated DNA, which has an affinity for the one end of the His6 tag, becomes attached;
- The His6 tag, which has an affinity for the DHLA covering of the Quantum Dot, becomes attached;
- (not a production step, but found in the illustration prior to the addition of the complementary DNA) Excite the batch with high energy light as seen in the center of the graphic where:
- the proximity of the Cy5 particle in the His6–Thiolated DNA and the Quantum Dot allows the molecular beacon to absorb energy through FRET (Förster resonance energy transfer) and emit a color shifted light in fluorescence;
- (not a production step, but, rather, the set-up for a test of the marker beacon) Obtain and add Complementary DNA to the process
- the molecular beacon (the His6–Thiolated DNA–Cy5) unzips, and takes on the Complementary DNA;
- (not a production step, but, rather, a test of the marker beacon) The right side of the graphic shows the action the new bioconjugated Quantum Dot undergoes to stimulation by (a now second) high energy light to emit a color shifted light in fluorescence.
Designing a NanoVirus has been a work in progress, and if I dare say so (noting a certain irony), an evolution. First, the components:
A quantum dot is a one dimensional construction. It is usually less than 10nm wide (yes, that contradicts the notion of one dimension when it has height, width, and length, but at the quantum scale it is suitable). By selection of material, shape, and size, various optical properties can be set.
|Size (nm)||Emission Peak (nm)||Color|
|7.3 ||655||dark red|
A QDot is often made of toxic elements such as CdSe (Cadmium Selenium). For biomedical applications this needs to be covered without disturbing the optical qualities of the QDot.
To be useful, the quantum dot needs to be functionalized. This means the QDot is attached with strands or layers that can, in turn, selectively bind to larger structures. Another means of attachment is through bio-conjugation which allows for protecting the QDot in a wide variety of harsh media. This leads to the application of DNA or nucleotides.
QDot Coatings for Tagging
The metals used to construct quantum dots are toxic. For biological applications, it is necessary to cover them while still retaining the optical qualities and the possibility of making attachments.
One such coating is Dihydrolipoic acid.
QDot Tags for Functionalization
For a quantum dot to be useful beyond responding to ultraviolet light with fluorescence, a tag is added that allows for attachments. These attachments are simple handles that will attach, in turn, to complex oligonucleotides to become functionalized. These oligonucleotides are chosen to attach, once again, to target RNA/DNA strands. This target will then have a marker beacon to aid viewing the site at which the target resides.
|Probe||Ex (nm)||Em (nm)||MW||Quantum yield|
|Cy3||(512);550||570;(615)||767||QY 0.15 |
Cyanine variants exhibit various optical properties. Within this post, it is limited to being used as a quenched fluorophore that engages through FRET with the quantum dot.
Molecular beacons are hairpin shaped RNA/DNA fragment molecules with an internally quenched fluorophore, the Cy5 above, whose fluorescence is restored when they bind to a target nucleic acid sequence. This is a novel non-radioactive method for detecting specific sequences of nucleic acids. They are useful in situations where it is either not possible or desirable to isolate the probe-target hybrids from an excess of the hybridization probes.
Short DNA or RNA molecules made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, library construction and as molecular probes.
From the work of Paul Rothemund:
DNA Origami is the manipulation of single strands of M13mp18, a folding of a long single strand of this viral DNA aided by multiple smaller “staple” strands.
The simpler description is that DNA can be sliced and diced into short strands that are stiff, and then those strands can be connected/configured into triangles, squares, pentagrams, hexagons…. These planar elements can then be assembled into three dimensional pyramids, cubes, tubes, buckyballs….
From the work of Erik Winfree:
The creation of two-dimensional lattices of DNA tiles using the “double crossover” motif. These tile-based structures provided the capability to implement DNA computing.
DNA computing performs the standard operations of comparing, sorting, counting, and such, but are not intended to replace a word-processor or spreadsheet program.
single guide RNA contains bases in both repeat and spacer regions of a single RNA strand. In a CRISPR application (see below), repeater and spacer has the following meanings:
Repeat–a pattern of bases that are recognized by dCas9 by their being palindromes (the sequence of bases are lined up to read the same both ways). Repeats signal that the pattern of bases that follows the repeat is a spacer.
Spacer–a short pattern of bases that have been stripped out of a virus’ DNA. The length is sufficient to uniquely identify the virus’ past visit, and to engage Cas9 (dCas9 if the spacer is engineered) to act upon the agent that introduced the DNA recently (for a bacteria, it would sense the invasion of a virus based on a past infection).
CRISPR associated protein 9. This protein 9 is specifically associated with the host bacteria Streptococcus pyogenes it inhabits. Protein 9 is a DNA library of virus DNA samples from the bacteria’s past experience of being attacked by viruses. Protein 9 is called a single guide RNA (see above).
Cas9 performs an interrogation on an intruding virus by unwinding that virus’ DNA and checking if it is complementary (matches) to any 20 basepair spacer region of the guide RNA. If the DNA substrate is complementary (matched) to the guide RNA, Cas9 cleaves the invading DNA. This recognition method effectively eliminates the virus.
However, for applications beyond this specific protein 9 and this specific bacteria, Cas9 has the unique ability to bind to essentially any complement (matching) sequence in any genome when provided with a tailored guide RNA.
When Cas9 is used informally as a research or engineered process outside of the specific bacteria, Streptococcus pyogene, it is frequently described as a mutant/mutation of Cas9, or as dCas9 (see following).
dead CRISPR associated protein 9. A Cas9 variant used for both mechanistic studies into Cas9 DNA interrogative binding and as a general programmable DNA binding RNA-Protein complex. Some might prefer to associate the d in dCas9 to the d in endonuclease-deficient.
dCas9 and its guide RNA can be used to interrogate DNA to then
- restore that DNA and release it; or
- remove the matching sequence from that DNA and release it shortened; or
- clip that DNA and disrupt it; or
- remove the matching sequence and insert a new sequence into the DNA and release it; or
- block assembly of transcription factors leading to silencing of specific gene expression; or
- activate genes when fused to transcription activating factors.
A “split-fusion” approach is where the sgRNA (see above) is split into two inactive halves that only regain functionality when the two halves are co-joined at a particular site.
CRISPRs are part of a bacteria’s DNA recognition and adaptive immunity system. This acronym merely names the organization of elements that enable this system, and is not in itself any separable component.
CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of DNA containing short repetitions of base sequences. Each repetition is followed by short segments of “spacer DNA” from previous exposures to a bacterial virus or plasmid. It is pronounced “crisper.”
By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism’s genome can be cut at any desired location.
The CRISPR/Cas9 system is an immune system that confers resistance to bacterial DNA and viruses and provides a form of acquired immunity.
CRISPR activator library for transcriptional activation
CRISPR interfering library for transcriptional repression
Enteric Nervous System
The enteric nervous system (ENS) or intrinsic nervous system is one of the main divisions of the nervous system and consists of a mesh-like system of neurons that governs the function of the gastrointestinal system. It is now usually referred to as separate from the autonomic nervous system since it has its own independent reflex activity.
Some of the microbes in the human body can modify the production of neurotransmitters known to occur in the brain.
From: Microbiota Modulate Behavioral and Physiological Abnormalities Associated with Neurodevelopmental Disorders, Elaine Y. Hsiao, et al.
Bacteroides fragilis corrects gut permeability, alters microbial composition, and ameliorates defects in communicative, stereotypic, anxiety-like and sensorimotor behaviors.
At the structural level, the virus is composed of DNA origami, folded into a shape suited as a delivery mechanism much like the capsid of a virus. QDots functionalized with DNA serve as the vertices and edges of the construction. The QDot heads one or more long tails of DNA.
Within this capsid structure is an DNA tile program, and an sgRNA in a dCas9 (see both above).
I believe in the importance and goodness of Wade’s people.
This band of young men and women take on challenges that hint at success, promise failure, but always reward with rich experience.
Wade’s people are passionate about their work and tightly bonded with their team.
The challenges they choose and the risks that come with them are the essence of today’s start-up culture. Wade’s people bring their edge of creativity to the fringes of town that are more bohemian than corporate campus.
Their challenges occupy a shifting moral landscape that bring them into disturbing contact with complex issues. I believe in how Wade’s people define themselves through their healing recovery from failure.
The following graphic reveals how much data is available to computers on a commercial 747.
This chart reveals nearly two minutes of flight data. It begins at the left, at 20:18:40 (40 seconds after 8:18PM). It continues on to the right, at 20:20:20 (20 seconds past 8:20PM).
As a narrative in the experience of a passenger:
- the 747 is sitting at the end of the runway for the first 10 seconds.
- the 747 has all four throttles full on at 10 seconds in. Maximum thrust is achieved at 10 to 15 seconds later where all four engine lines level off (lines marked EPR for Engine Pressure Ratio).
- Longitudinal Acceleration (the pressure you feel pushing you into the seat back at take-off) also tracks up through this same 10 to 15 seconds.
For the nearly two minutes, it varies between 0.20 and 0.25 G. A 150 pound individual would feel at least 30 pounds of pressure pushing them into the seat back.
- Vertical Acceleration (the pressure you feel pushing you into the seat pan). There is none until lift-off which occurs at 20:19:40. At that time, you feel 0.15 G of force pushing you into your seat with 16 or more pounds of pressure. There is also a corresponding increase of pressure against the seat back.
These are all familiar to the traveler. Other data may be compared to discover all other activities going on during flight, like the pilot pressing the microphone transmit key at the top of the graphic.
One major point of interest. There is a lot of noise in some of the data. Some of it is genuine representations of what is going on. Other data is corrupted.