Catalyzed induction of targeted proteins by external

lightwaves
Chungpin Liao*
National Formosa University (NFU), Huwei, Taiwan 632
* Correspondent: cpliao@alum.mit.edu

Abstract
German scientist Fritz-Albert Popp's [1] bio-photon experiments had indicated that the
photon absorption/emission process within human body is a communication type of
behavior among DNA's, via which more subtle and effective biological adjustment and
adaptation can be achieved. It is proposed that different nucleobases (A, T, G, C)
within a protein's promoter sector can be differentially excited and thus selectively
activated by external lights of proper wavelengths, inside the body of a living
organism. More precisely, the activated amount of each specific nucleobase is
proportional to the amount of light it absorbs. That is, for a chosen light wavelength,
the distribution of 4 types of nucleobases is largely determined by the 4 absorption
spectra of A, T, G, C, respectively; or more straightforwardly, absorption is activation.
Therefore, by applying one to three lights of different wavelengths, we can accurately
tailor and come up with varying activation distribution desired. A question may arise
as to whether such lightwave sampling of genetic code in the promoter sector can be
correct in sequential order. That is, even though the correct numbers of different
nucleobases can be properly activated by the aforementioned lightwave sampling,
those “woken up” nucleobases most likely will not be in the right order. However,
note that the number of nucleobases on the promoter sector is few, in general, about
6-20 pairs. A few trial-and-errors of light-induced random samplings, in accordance
with the above chosen distribution profile (i.e., promoter population distribution), the
right promoter sequence will be matched every now and then and thus enhanced
transcription of the desired protein be realized at those instants. This photon-caused
protein transcription should be compared with the known routes solely through
activities of RNA or DNA polymerases. In this work, light-enhanced homo-
fermentation of glucose inside the living micro-organism: Lactobacillus delbrueckii
subsp. bulgaricus (also known as Lactobacillus bulgaricus) is used as a system to
verify the proposed protein induction method.

1
Background and motivation

Deoxyribonucleic acid (DNA) is a molecule composed of two chains that coil around each other
to form a double helix carrying genetic instructions for the development, functioning, growth and
reproduction of all known organisms and many viruses. The two DNA strands are also known as
polynucleotides as they are composed of simpler monomeric units called nucleotides. Each
nucleotide is composed of one of four nitrogen-containing nucleobases (cytosine [C], guanine
[G], adenine [A] or thymine [T]), a sugar called deoxyribose, and a phosphate group. The
nucleotides are joined to one another in a chain
by covalent bonds between the sugar of one
nucleotide and the phosphate of the next,
resulting in an alternating sugar-phosphate
backbone. The nitrogenous bases of the two
separate polynucleotide strands are bound
together, according to base pairing rules (A with
T and C with G), with hydrogen bonds to make
double-stranded DNA (see, Fig. 1).

Both strands of double-stranded DNA store the

same biological information. This information is
replicated as the two strands separate. A large
part of DNA (more than 98% for humans) is
non-coding, meaning that these sections do not
serve as patterns for protein sequences. The two Fig. 1. The structure of the DNA double
strands of DNA run in opposite directions to helix. The atoms in the structure are
each other and are thus anti-parallel. Attached to color-coded by element and the detailed
each sugar is one of four types of nucleobases. It structures of two base pairs are shown in
is the sequence of these four nucleobases along the bottom right. [Courtesy of
the backbone that encodes genetic information. Wikipedia]
RNA strands are created using DNA strands as a
template in a process called transcription, where DNA bases are exchanged for their
corresponding bases except in the case of thymine (T), which RNA substitutes for uracil (U).
Under the genetic code, these RNA strands specify the sequence of amino acids within proteins
in a process called translation (see, DNA in Wikipedia [2]).

DNA usually occurs as linear chromosomes. The set of chromosomes in a cell makes up its
genome; the human genome has approximately 3 billion base pairs of DNA arranged into 46
chromosomes. The information carried by DNA is held in the sequence of pieces of DNA called
genes. Transmission of genetic information in genes is achieved via complementary base pairing.
For example, in transcription, when a cell uses the information in a gene, the DNA sequence is
copied into a complementary RNA sequence through the attraction between the DNA and the
correct RNA nucleotides. Usually, this RNA copy is then used to make a matching protein
sequence in a process called translation, which depends on the same interaction between RNA
nucleotides. Only a small fraction of the total sequence of the genome encodes protein, for
example, about 1.5% of the human genome consists of protein-coding exons. Here, we will be
aiming at the creation of targeted proteins. A DNA transcription unit encoding for a protein may
contain both a coding sequence, which will be translated into the protein, and regulatory
sequences, which direct and regulate the synthesis of that protein.

More precisely, the transcription proceeds in the following general steps [3]:
a) RNA polymerase, together with one or more general transcription factors, binds to promoter
DNA (see, Fig. 2).
b) RNA polymerase creates a transcription bubble, which separates the two strands of the DNA
helix. This is done by breaking the hydrogen bonds between complementary DNA
nucleotides.

2
c) RNA polymerase adds RNA nucleotides (which are complementary to the nucleotides of one
DNA strand).
d) RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an
RNA strand.
e) Hydrogen bonds of the RNA–DNA helix break, freeing the newly synthesized RNA strand.
If the cell has a nucleus, the RNA may be further processed. This may include
polyadenylation, capping, and splicing.
f) The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore
complex.

In the above, a promoter is a region of DNA that leads to initiation of transcription of a

particular gene. Promoters are
located near the transcription start
sites of genes, upstream on the
DNA (towards the 5' region of the
sense strand). Promoters are
normally very short, but can also
be about 100–1000 base pairs long.
For transcription to take place, the
enzyme that synthesizes RNA,
known as RNA polymerase, must
attach to the DNA near a gene.
Promoters contain specific DNA
sequences such as response
elements that provide a secure Fig. 2. Illustrative positioning of core promoter for
initial binding site for RNA DNA transcription. A typical example is also shown
polymerase and for proteins called below.
transcription factors that recruit
RNA polymerase. These transcription factors have specific activator or repressor sequences of
corresponding nucleotides that attach to specific promoters and regulate gene expression.

Since it is well-known that human body is microscopically filled with highways of lightwaves
[1], in particular, for the 260 nm wavelength ultra-violet (UV) photons, for possibly the intra-
communication and bio-reaction-regulating purposes, this author was inspired that photons
should play an enhancing role in the DNA transcription processes. In other words, with the light
absorption spectra for all 4 nucleobases known (see, in particular, the 298 K curves in Fig. 3),
under the light irradiation of a particular wavelength, the excited population probability density
distribution among A, T, G, C can be calculated for that frequency.

Further, adjustment of such irradiation frequency can lead to different probability distribution of
excitation (or, absorption). If now, we assume that the excitation (absorption) probability
distribution is essentially the activation probability distribution, i.e., absorption is equal to
activation, then the real code distribution in A, T, G, C in a core promoter can be simulated (or
statistically sampled) via properly choosing the irradiating light frequency. Note that having an
absorption distribution of a shape close to the real nucleobase code distribution (of the promoter)
still does not mean the activation of the relevant genetic code in the correct sequence. However,
since the code length of the promoter sector is usually short, it is expected that after several such
externally-trigged random samplings (or, trial-and-errors), the correct code sequence of the
promoter sector can be hit once in a while and thus the activation button is pushed for targeted
protein generation post these instants.

3
 Fig. 3. Cumulative effect of solvation on the computed UV absorption spectra of
DNA nucleobases. Gas phase spectra at 0K (light blue) and 298 K (green), spectra
upon inclusion of the COSMO environment (purple) and solvent-induced structural
changes (red). [Courtesy of Ref. 4]

Experimental efforts
A glycolysis (or, sugar-breaking) experiment was launched based on the widely-used germ:
Lactobacillus bulgaricus (in full name: Lactobacillus delbrueckii subsp. Bulgaricus), with
measurement effort focused on light-affected feature of the rate-limiting enzyme:
phosphofructokinase (PFK) of the Lactobacillus bulgaricus.

Glycolysis, which translates to "splitting sugars", is the process of releasing energy within
sugars. In glycolysis, a six-carbon sugar known as glucose is split into two molecules of a
three-carbon sugar called pyruvate. This multistep process yields two ATP molecules
containing free energy, two pyruvate molecules, two high energy, electron-carrying molecules
of NADH, and two molecules of water.

Glycolysis can occur with or without oxygen. In the presence of oxygen, glycolysis is the first
stage of cellular respiration. In the absence of oxygen, glycolysis allows cells to make small
amounts of ATP through a process of fermentation. In this study, we’ll focus on the light-
influenced PFK reaction rates for the no-oxygen fermentation process.

4
Glycolysis takes place in the cytosol of the cell's cytoplasm. A net of two ATP molecules are
produced through glycolysis (two are used during the process and four are produced). Its 10
key steps are as follows [5] (also,
see Fig. 4):

1) The enzyme hexokinase

phosphorylates or adds a
phosphate group to glucose in a
cell's cytoplasm. In the process,
a phosphate group from ATP is
transferred to glucose
producing glucose 6-phosphate
or G6P. One molecule of ATP
is consumed during this phase.
Fig. 4. Glycolysis process [Courtesy of Wikipedia][5]
2) The enzyme The current experimental interest is the PFK
phosphoglucomutase dynamics in the 3rd step.
isomerizes G6P into its isomer
fructose 6-phosphate or F6P. Isomers have the same molecular formula as each other but
different atomic arrangements.

3) The kinase phosphofructokinase (PFK) uses another ATP molecule to transfer a phosphate
group to F6P in order to form fructose 1,6-bisphosphate or FBP. Two ATP molecules have
been used so far. (This bottle-neck step is our experimental focus, to be elaborated below.)

4) The enzyme aldolase splits fructose 1,6-bisphosphate into a ketone and an aldehyde
molecule. These sugars, dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-
phosphate (GAP), are isomers of each other.

5) The enzyme triose-phosphate isomerase rapidly converts DHAP into GAP (these isomers
can inter-convert). GAP is the substrate needed for the next step of glycolysis.

6) The enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves two functions in

this reaction. First, it dehydrogenates GAP by transferring one of its hydrogen (H⁺)
molecules to the oxidizing agent nicotinamide adenine dinucleotide (NAD⁺) to form NADH
+ H⁺.
Next, GAPDH adds a phosphate from the cytosol to the oxidized GAP to form 1,3-
bisphosphoglycerate (BPG). Both molecules of GAP produced in the previous step undergo
this process of dehydrogenation and phosphorylation.

7) The enzyme phosphoglycerokinase transfers a phosphate from BPG to a molecule of ADP

to form ATP. This happens to each molecule of BPG. This reaction yields two 3-
phosphoglycerate (3 PGA) molecules and two ATP molecules.

8) The enzyme phosphoglyceromutase relocates the P of the two 3 PGA molecules from the
third to the second carbon to form two 2-phosphoglycerate (2 PGA) molecules.

9) The enzyme enolase removes a molecule of water from 2-phosphoglycerate to form

phosphoenolpyruvate (PEP). This happens for each molecule of 2 PGA from Step 8.

5
10) The enzyme pyruvate kinase transfers a P from PEP to ADP to form pyruvate and ATP.
This happens for each molecule of PEP. This reaction yields two molecules of pyruvate and
two ATP molecules.

Comparing the relative reaction rates of the above 3 involved key enzymes, namely, hexokinase
(step 1), phosphofructokinase
(PFK, step 3), and pyruvate
kinase (step 10), of 8, 5, and 12
KCat/KM (or, nM/s),
respectively [6], it is apparent
that PFK is the one resulting in
the bottle-neck step. Thus, PFK
becomes the natural target of our
light-enhancing experimental
investigations. For the latter
purposes, some information of
PFK protein code sequence is
needed, especially, that of the
core promoter sector. Fig. 5 Fig. 5. Beginning protein sequence of PFK of the germ:
shows the beginning part of PFK Lactobacillus bulgaricus
sequence. Then, tracing upstream from the
starting head of such sequence, we located the
core promoter: TTAAATC (underlined, in
Fig. 6).

Using the population distribution of the

obtained above Lactobacillus bulgaricus
promoter, i.e., A: 42.8%, T: 42.8%, G: 7.1%,
C: 7.1% (taking into account the double helix
sequences made up of A-T and C-G pairs),
the proposed light-enhancing activation of the
promoter is arranged, using the known
absorption data of A, T, C, G, respectively
(see, Fig. 3). The calculated proper light
wavelength is found to be 254 nm (ultra violet
light, UV-C) to simulate the promoter
sequence population distribution. With this,
Fig. 7 conveys the idea properly, i.e., having
an absorption distribution of a shape close to Fig. 6. Located PFK core promoter at the
the real population distribution. After that, a upstream (underlined) of the marked
series of random swapping of the codes will beginner of the protein sequence.
take place while the shining light is on, and
every now and then whenever the correct promoter sequence is hit, the PFK protein is
activated and further produced by the living germ.

To test the proposed concept, an experimental setup was arranged as follows. All equipment
and apparatus were subjected to sterilization before use, by going through water boiling and
UV irradiation. Then, two vessels were prepared with each containing 200 cc whey (i.e., liquid
remaining after the milk has been curdled and strained), 10 g glucose, and 5 g Lactobacillus
bulgaricus powder. The two samples were then separately put in 40C-preheated constant
temperature chambers for 6 hours. One of them acted as the reference group, and stayed in dark
during the whole experiment. The other was the experiment group and was subjected to the

6
aforementioned 254
nm UV light
irradiation
continuously during
the whole span of
experiment. After this
cultivation period,
solutions from both
(a) Population distribution (b) Absorption distribution
groups (28 cc each)
were taken through
Fig. 7. (a) Base pair probability distribution within the core
12-hour ultrasonic
promoter sector (TTAAATC) of the rate-limiting
agitation (for cracking
phosphofructokinase (PFK) enzyme; (b) Absorption-simulated
the germ cell
distribution among base pairs A, T, G, C, under the tenuous
membranes and
irradiation of 254 nm UV light
releasing of proteins),
6-hour spinning (to secure the upper level fluid) in the first place.

Subsequently, enzyme PFK of Lactobacillus bulgaricus in both groups were extracted through
precipitation via salting-out (using ammonium sulphate) and spinning, adding of de-ionized
water, and precipitation via isoelectric focusing (through adjusting solution pH value to reach
PFK’s isoelectric point). Then, in each group de-ionized water of the amount 20cc was added
to the above precipitates again to first secure the PFK characteristic spectrum (i.e., a valley at
248 nm, a peak at 278 nm) for its identification proof. Then, using the same solutions from both
groups, laser beam penetration measurements of the resultant PFK concentration were carried
out.

Through the well-known photonic absorption formula of Beer-Lambert law, i.e.,

𝐴 = − log10 𝑇𝑟𝑎𝑛𝑠𝑚𝑖𝑠𝑠𝑖𝑜𝑛 = log10 (1⁄𝑇𝑡𝑟𝑎𝑛𝑠𝑚𝑖𝑠𝑠𝑖𝑜𝑛) , where A, Transmission are
attenuation and transmission fraction, respectively, the measurement results are tabulated in the
following (under the 240 nm probing light of varying intensities):

1) Intensity: 14000 count (~1.4x10-14W/cm2)

Reference Experiment
Initial A 0.6 0.6
Transmission 25% 25%
Absorption 75% 75%
Experiment Reference
Final A 0.9 1.2
Transmission 13% 6%
Absorption 87% 94%
Respective gains in PFK 12% 19%
Enhancement % (19-12)/12 = 58%

2) Intensity: 14000 count (~1.4x10-14W/cm2)

Reference Experiment
Initial A 0.25 0.25
Transmission 56% 56%
Absorption 44% 44%
Experiment Reference
Final A 0.45 0.6

7
 Transmission 35% 25%
Absorption 65% 75%
Respective gains in PFK 21% 31%
Enhancement % (31-21)/21 = 48%

3) Intensity: 14000 count (~1.4x10-14W/cm2)

Reference Experiment
Initial A 0.6 0.6
Transmission 25% 25%
Absorption 75% 75%
Experiment Reference
Final A 0.8 1.0
Transmission 16% 10%
Absorption 84% 90%
Respective gains in PFK 9% 15%
Enhancement % (15-9)/9 = 67%

4) Intensity: 7000 count (~7x10-15W/cm2)

Reference Experiment
Initial A 0.6 0.6
Transmission 25% 25%
Absorption 75% 75%
Experiment Reference
Final A 1.15 1.2
Transmission 7% 6%
Absorption 93% 94%
Respective gains in PFK 18% 19%
Enhancement % (19-18)/18 = 6%

5) Intensity: 7500 count (~7.5x10-15W/cm2)

Reference Experiment
Initial A 0.6 0.6
Transmission 25% 25%
Absorption 75% 75%
Experiment Reference
Final A 1.1 1.2
Transmission 8% 6%
Absorption 92% 94%
Respective gains in PFK 17% 19%
Enhancement % (19-17)/17 = 12%

Summary and conclusions

It was shown experimentally that tenuous UV lights alone, at the proper frequency in the
intensity range of 10-15-10-14 W/cm2 (for 6 hours), can considerably enhance the bottle-neck
fermentation reaction (by 10% ~ 60%) involving the enzyme PFK in the glycolysis occurring
inside the cells of Lactobacillus bulgaricus. In short, adjusting the light frequency so that its
activated (by being absorbed by the PFK protein) nucleobases in the PFK promoter sector
render a similar distribution profile to that of the nucleobase population probability distribution

8
(for the PFK promoter sector) would be key to this proposed methodology, which increased the
production of PFK and thus enhancement of the above bottle-neck fermentation reaction.

It was also reminded that having a simulated absorption distribution of the shape
close to the real genetic code distribution (of the promoter) still does not guarantee
the activation of nucleobases in the correct promoter sequence order. Thus, it is only
more feasible when the code length of the promoter is short enough (which is
usually so) such that after several random samplings (or, trial-and-errors) owing to
the persistent external photon irradiation, the correct nucleobase sequence of the
promoter can be matched in a relatively short interval and thus the activation button
is readily pushed for targeted protein generation. This kind of matching situations
happen every now and then, in a random fashion. Approaches with more than one
single light wavelength can also be adopted, as long as the nucleobase population
density distribution profile can be simulated by all involved external lights altogether.

The application and ramification of such light-caused manipulation of targeted

proteins within living organisms can be wide-ranging, far-reaching, and cross-
disciplinary. Nonetheless, the detailed biochemical mechanisms and dynamics
following the above light-caused promoter activation still remain somewhat unclear
at this point.

References:

[1] Prof. Fritz-Albert Popp, see, e.g., in https://www.biontologyarizona.com/dr-fritz-albert-popp/.

[2] DNA, in https://en.wikipedia.org/wiki/DNA.

[3] Transcription, in https://en.wikipedia.org/wiki/Transcription_(biology).

[4] Marin Sapunar, Wolfgang Domcke, Nada Doslic, “Supporting information for UV absorption
spectra of DNA bases in the 350-190 nm range: Assignment and state specific analysis of
solvation effects,” in Electronic Supplementary Material (ESI) for Physical Chemistry Chemical
Physics. This journal is © the Owner Societies 2019.

[5] Glycolysis, in https://en.wikipedia.org/wiki/Glycolysis.

[6] https://www.brenda-enzymes.org/all_enzymes.php?ecno=2.7.1.1&table=KCat_K
M_Value#TAB, and
https://www.brenda-enzymes.org/all_enzymes.php?ecno=2.7.1.11&table=Turno
ver_Number, and
https://www.brenda-enzymes.org/all_enzymes.php?ecno=2.7.1.40&table=Engin
eering

Acknowledgement:

This work is a derivative from my undergraduate student Mr. Jian, Ting-Yu’s Special
Topic research result. He was rather talented and knowledgeable in biochemistry in
general.