Supporting Information

BasePairPuzzle: Molecular models for manipulating the concept of

hydrogen bond and base pairs in nucleic acids
Jiro Kondo *, † and Shota Nakamura ‡

†
Department of Materials and Life Sciences, Faculty of Science and
Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, 102-8554 Tokyo,
Japan.
‡
StudioMIDAS Inc., 3-2-11 Honcho, Ageo, 362-0014 Saitama, Japan.

*
Corresponding Author: j.kondo@sophia.ac.jp

Pages 2-3: Reaction paper

Pages 4-9: Representative responses from students
Pages 10-12: Survey data
Pages 13-14: DIY instruction

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 Reaction paper
Full Name:
Student ID:

Work #1. Find the rule that bases attract each other.
Find the rules under what conditions the parts of BasePairPuzzle attract or repel, and
describe them.

Work #2. Find complementary base pairs.

Find the A-T and G-C base pairs that Watson and Crick discovered in 1953. Once you
find them, check their stability. For DNA to form an undistorted double helix, the A-T
and G-C base pairs must be the same width. Also, to form an antiparallel right-handed
helix, the A and T or G and C must be in cis configuration to each other, and the resulting
A-T and G-C (or T-A and C-G) base pairs must be geometrically identical.
Describe anything you notice or wonder about.

Work #3. Try to create base pairs with A and C or G and T.

Can you create base pairs with A and C or G and T that are not complementary?
Describe anything you notice or wonder about.

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Work #4. Explore the mechanism of the DNA mutation that causes sickle cell disease.
Sickle cell disease is caused when a specific position in the gene that codes for
hemoglobin is replaced from an A-T base pair to a T-A base pair. You can find out for
yourself how the mutation occurs. There are many ways to answer.

Work #5. Create base triplets or quartets using three or more parts.
Bases form not only pairs but also triplets and quartets, which allow DNA and RNA to
have specific functions.
Describe anything you notice or wonder about.

(Questionnaire) Please feel free to describe your impressions of the BasePairPuzzle.

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 Representative responses from students

Work #1. Find the rule that bases attract each other.
- Bonding occurs when the combination is blue-white-blue or blue-white-
red, i.e., when white is sandwiched between blue and red.
- Blue and white, or red and white attract each other, blue and red, or red
and red repel each other.
- Balls of the same color repelled, and blue and red balls repelled. White
and red and white and blue balls attracted each other.
- Based on the structure of the molecule, I assumed that red was oxygen,
blue was nitrogen, and white was hydrogen. Since bonding occurred
between red and white, or blue and white, hydrogen bonding might occur
between oxygen and hydrogen, or nitrogen and hydrogen.
- The parts of the molecular model had to be placed parallel to each other
to attract each other.
- Balls of the same size repelled each other, and balls of different sizes
attracted each other.
- I thought that the electrons and polarity of the atoms might be responsible
for the attraction and retraction of the balls to each other.
- Whenever parts are joined together, a white ball is involved.

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Work #2. Find complementary base pairs.
- When A-T and G-C were placed in a cis shape and superimposed, they
were very similar in size and shape.
- The G-C base pair is formed by three hydrogen bonds, so the two model
parts were strongly attracted to each other. On the other hand, the A-T
base pair was relatively fragile.
- When A and T or C and G were placed in the cis configuration with
respect to each other, once one hydrogen bond was formed, the remaining
hydrogen bonds formed spontaneously.
- The A-T base pair had the same shape, whether it was cis or trans. But
the G-C base pair did not.
- When I tried to make the G-C base pair formed through three hydrogen
bonds, I could only make a cis-type base pair. On the other hand, when I
tried to make the A-T base pair consisting of two hydrogen bonds, I could
make three different base pairs.
- The cis G-C base pair was more stable than the cis A-T base pair due to
the greater number of hydrogen bonds.
- I was able to create various base pairs by combining G and C or A and T,
and I thought that the most stable one might be the Watson-Crick base
pair.
- Since A-T base pairs are less stable than G-C base pairs, I felt that DNA
double helix with more A-T base pairs is more likely to unravel.
- The G-C base pair is formed through three hydrogen bonds, so it took
much force to break the base pair.

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- A-T had the same stability, whether cis or trans, but G-C was more stable
in cis than trans.
- When A and G were in the anti conformation, base pairs with large widths
were formed, whereas when A and G were in the syn conformation, base
pairs with small widths were formed.
- The G-C base pair was stable and unbreakable but broke easily when
twisted, so I felt that bases tend to align parallel to each other.
- I was surprised because I could make many other forms of G-C and A-T
base pairs besides those discovered by Watson and Crick.
- I could make various forms of G-C and A-T base pairs, but I felt that the
base pair discovered by Watson and Crick was relatively stable among
them.

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Work #3. Try to create base pairs with A and C or G and T.
- When I tried to create a cis base pair with A and C, I could only make
unstable base pairs formed through a single hydrogen bond. However,
when I tried using G and T, I was able to create a cis base pair that was as
stable as the cis A-T base pair.
- I could create A-C and G-T base pairs. I also made A-G and C-T base
pairs. It could mean that base pairs other than the complementary A-T and
G-C base pairs might also be formed in our cell.
- The cis C-A base pair was unstable but could be created. On the other
hand, the cis G-T and A-G base pairs were quite stable even though they
are not complementary base pairs.
- I think DNA mutations are more likely to occur because I could create
many types of base pairs.
- The G-T base pair is as stable as the A-T base pair but slightly distorted.
- In the case of the G-U base pair, the shape was the same whether it was
cis or trans configuration. In the case of the A-C base pair, the cis
configuration was unstable, but the trans configuration was stable.
- If I didn't care about the combination and arrangement of the bases, I
could make any number of base pairs.
- I thought that only the combination of A and T or G and C could form a
stable base pair, so I was surprised that many other base pairs could be
formed. Could mistakes be easily made in DNA replication and
transcription?

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Work #4. Explore the mechanism of the DNA mutation that causes sickle
cell disease.
- A-T → G-T → G-A → T-A
- A-T → A-G → T-G → T-A
- A-T → A-C → T-C → T-A
- A-T → C-T → C-A → T-A
- A-T → A-U → T-U → T-A

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Work #5. Create base triplets or quartets using three or more parts.
- I thought the triplet would be a three-dimensional structure, but what I
created was all flat.
- I created a G quartet, which was square and very stable.
- I made a quartet with four G bases, which was very stable. I also created
a quartet using two T bases and two C bases, but it was not stable.
- Since the triplets and quartets were more stable than I expected, it would
not be surprising to form such structures in vivo.
- I tried to see if I could attach another base to an A-T base pair or a G-C
base pair; I found that G and A have more atoms that participate in
hydrogen bonding, so they can easily make triplets.
- After making various triplets, I noticed that G is more likely to form
triplets than other bases.
- I was able to create triplets and quartets that are three-dimensional rather
than planar, but can such structures be formed in vivo?
- Various combinations of bases could be used to make triplets, but stable
triplets were limited.
- It was easier to make a triplet if the purine base was placed in the center,
like the C-A-T triplet.
- I could construct some stable triplets, but I could only make unstable
quartets.

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 Survey data

We conducted active learning using the BasePairPuzzle in Fundamental

Biochemistry at Sophia University, a course for first and second-year
university students. Before and after using the BasePairPuzzle, students were
asked the question, "What is a hydrogen bond?".

Student #1
Before:
A hydrogen bond is an intermolecular force that forms a special dipole-
dipole when a hydrogen atom bonds to a strongly electronegative atom. It
contributes to the secondary structure of polymers, such as amino acids.
After:
Hydrogen bonds are responsible for holding DNA, protein and other
macromolecules. Because of the difference in electronegativity, the H atom
bears a large partial positive charge, and the atom bonded to H bears a large
partial negative charge. Hydrogen bonds have limited distance and angle.

Student #1 seems to have realized that hydrogen atoms and their bonding
partners are positively and negatively polarized, respectively, and that
hydrogen bonding distances and angles are restricted, by feeling the
attraction and repulsion of the neodymium magnets embedded in the
molecular models.

Student #2
Before:
I remember learning that hydrogen bonds are quite strong. One important
role they play is to give water the strength of adhesion and cohesion, to
stick tightly to each other and others.
After:
The lecture and the toys helped me understand how hydrogen bonds can
facilitate bonding and folding to create various structures. It was
interesting to imagine how only certain arrangements of the bases will
allow for the most bonds can be made.

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Student #2 seems to understand by creating several base pairs that hydrogen
bonds are involved in intra- and intermolecular interactions and the
formation of molecular structures. Like Student #1, Student #2 also seems to
have realized that the distances and angles of hydrogen bonds are limited.

Student #3
Before:
What I know about hydrogen bonds is that it is an intermolecular force that
occurs due to the difference in charge depending on the location of the
molecule. Due to lone pairs, the molecule does not have an equal charge
throughout, so the more negative parts of the molecule are attracted toward
the positive side of another molecule. It is also why water boils at a much
higher temperature than what the mass suggests. I think it is also what
causes water tension.
After:
After looking at the model in class, it became clear that hydrogen bonding
can only occur in certain parts of the molecule, where the lone pairs are.

Student #3 seemed to already have a good understanding of hydrogen

bonding before taking this lecture. By touching the BasePairPuzzle,
identifying the position where the neodymium magnet is embedded (i.e., the
position of the lone pair), and experiencing the attraction between it and the
hydrogen atom, the student seems to deeply understand the importance of
the lone pair in the hydrogen bond.

Student #4
Before:
H bonds are a type of intermolecular force, and it is one of the more
substantial types, requiring more energy to break apart. The polarity of the
molecule also exists.
After:
As understood earlier, H bonds create polarity, which is an important
concept for us biologically. It tells us the reason for the unique properties
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 of water, which is explained by polarity, including cohesion and surface
tension. I also learned that there is limited distance and angle due to where
H bonds can occur - lone pairs are negative, while H atoms are positive,
resulting in electrostatic attraction. This property is important when
considering the binding positions of drugs in biomolecules.

Student #4 mentions the polarity that molecules have. The student seems to
have understood by using the BasePairPuzzle that hydrogen bonding is a
kind of electrostatic interaction and is key to molecular recognition of drugs.

Student #5
Before:
Hydrogen bonds are chemical bonds formed between a hydrogen atom and
another molecule/atom. Usually, they are weaker than ionic bonds and
covalent bonds.
After:
Hydrogen bonds are the electrostatic attraction between lone pairs of a
highly electronegative atom and a hydrogen atom bonded to a highly
electronegative atom.

Before taking this lecture, student #5 seemed to have a vague understanding

of hydrogen bonds as chemical bonds formed between hydrogen atoms and
other atoms. After this lecture, like the other students, he mentioned that
hydrogen bonds are the electrostatic attraction. This must be due to the
experience of intermolecular interactions by feeling attraction and repulsion
of neodymium magnets.

All the students’ responses confirmed that they would have gained a sensory
understanding of hydrogen bonding by using this molecular model. In
particular, many students mentioned that they noticed that the distances and
angles of hydrogen bonds are limited and that hydrogen bond is a kind of
electrostatic attraction. It is not easy to understand these things from
chemical structural formulas written on paper or whiteboard. Undoubtedly,
being able to experience the hydrogen bond and the base pairing on one’s
hand is the primary advantage of learning with the BasePairPuzzle.
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 DIY instruction

(i) Download the STL files (Supporting Information). You can download
the STL files also at our BasePairPuzzle website.
https://jkondo.wixsite.com/basepairpuzzle/diy-english
(ii) Use the downloaded STL file to output the base parts on your 3D
printer. Using resin with different colors (orange for A, green for C,
cyan for G, and purple for U) is recommended. Fluorescent polymers
can also be used, as shown in Figure 9 in the main text.
* For making fluorescent models, we used the Lumisis filament from
Central Techno Corporation.

(iii) Use commercially available ink to color hydrogen white, nitrogen blue,
and oxygen red. After coloring, dry well using a hair dryer.
* We used acryl gouache inks from Turner Colour Works Ltd.

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(iv) Use glue to embed commercially available cylindrical neodymium
magnets (4 mm in diameter and 4 mm in height) into the holes. At this
point, be careful that the S-pole faces outward at the hydrogen
positions, and the N-pole faces outward at the nitrogen and oxygen
positions.

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