Studying a Genetic Mutation in the Cross Sections of the Protein Opa1 by Inserting the

Amino Acid Histidine at Codon 445 using Cloning Techniques

by

Sara Backus

A Research Paper
Submitted in Partial Fulfillment of the
Requirements for the
Bachelor of Science Degree
in
Biochemistry

Under the direction of

Dr Andrew Kehr

Dr. Andrew Kehr Dr. Adam Moser

Loras College
May 2023
 ii

Division of Molecular, Life, and Health Sciences

Chemistry/Biochemistry Program
Loras College
Dubuque, IA

Author: Sara Marie Backus

Title: Studying a Genetic Mutation in the Cross Sections of the
Protein Opa1 by Inserting the Amino Acid Histidine at
Codon 445 using Cloning Techniques
Degree/ Major: BS Biochemistry
Research Adviser: Dr. Andrew Kehr
Month/Year: May 2023
Number of Pages: 36

Abstract

ADOA is the leading cause of blindness with onset occurring within the first ten

years of life. The disease is characterized by the degeneration of the axons made of

ganglion cells that form the optic nerve which is responsible for taking information

retrieved by photoreceptors to the brain. ADOA is a result of mutations occurring in the

large GTPase Opa1. Opa1 is a protein that falls into the category of a Dynamin Superfamily

Protein which have the primary job of performing membrane fission, membrane fusion,

bundling, and helping with innate immune responses within an organism. The definite

structure of Opa1 has not yet been discovered, but it is known that the structure does have

the structural characteristics of a DSP such as a lipid-interacting region, an alpha bundle

region, a stalk region, and a GTPase domain. Therefore, by discovering how mutations of

Opa1 affect the function of the protein, more information can be discovered structurally.

The overarching goal of this research was to successfully clone the disease-causing

mutation R445H into the OPA1 wildtype and to then study the biochemical characteristics

of this mutations.
 iii

In order to achieve this goal, mutated Opa1 DNA was produced using methods

such as site-directed mutagenesis, a restriction enzyme digest, and a bacterial

transformation. A successful method was found using a site directed mutagenesis protocol

with Pfu polymerase, 150 ng of DNA, an annealing temperature of 54ºC, and 4 minutes

per elongation cycle. This protocol resulted in colonies being formed and the DNA being

sent out for sequencing where it was found that the cloning technique was successful, but

a missense mutation (stop codon) was present in the wildtype stock Opa1. After being

provided unmutated wildtype Opa1 DNA, more trials were performed using different site-

directed mutagenesis methods, but none produced the R445H mutation as before. These

trials will be ongoing in order to prepare for future expression, purification, and GTPase

assay of the Opa1 protein in order to gain more knowledge of the structure and function of

the protein and its relationship to ADOA.

 iv

Acknowledgements

There are a plethora of people who have been an influential part of my

educational path and professional growth. First, I would like to extend my gratitude to my

research mentor Dr. Andrew Kehr. Researching in Dr. Kehr’s lab is nothing short of

challenging, but the development of resilience, critical thinking, and overall scientific

knowledge overcomes those challenges or frustrations. I attribute much of my scientific

growth to you and the confidence you have instilled within me as a student. In addition,

the personal support you have provided me throughout these past couple of years has

taught me the importance of building relationships and finding true mentors. In addition,

I would like to draw attention to the other members of the Kehr lab and the small family I

have been a part of because of them. The ongoing support and culture this lab have

provided to me will forever be one of my greatest takeaways from Loras College.

Furthermore, I would like to extend a thank you to the other members of the

science community here at Loras, as well as Loras College itself. The unique opportunity

I have been provided with being responsible for a research project as well as playing a

part in looking to answer greater scientific questions to benefit the greater community has

been life-altering in many ways. But the most important has been in discovering and

developing my own vocation. The intent of growing us, students, beyond science but

better professionals with more fulfilling futures is not something that goes unnoticed.

Finally, I would like to thank my parents. Their ongoing, endless support

throughout not only the past four years, but my entire life has provided me with

opportunities I could never have dreamed of without them. Though they may not
 v

understand the ins and outs of my science aspect of my life, their passion for continuing

to encourage me to keep going is one of the greatest gifts I have been provided with.
 vi

Table of Contents

........................................................................................................................................Page

Abstract .............................................................................................................................. ii

List of Figures ................................................................................................................... vii

List of Tables .................................................................................................................. viii

Introduction ........................................................................................................................ 2

Methods ............................................................................................................................ 10

Site Directed Mutagenesis ......................................................................................... 10

First Mutagenesis Attempt Protocol .......................................................................... 10

Second Mutagenesis Attempt Protocol ...................................................................... 11

Shotgun Mutagenesis ................................................................................................. 12

Dpn1 RE Digest ......................................................................................................... 13

Transformation .......................................................................................................... 14

Heat Shock ................................................................................................................. 14

Plating ........................................................................................................................ 15

PCR Screen ................................................................................................................ 16

Mini Prep ................................................................................................................... 17

Making Plates ............................................................................................................ 18

Limitations ................................................................................................................. 18

Results and Discussion .................................................................................................... 20

Conclusion ........................................................................................................................ 26

References ......................................................................................................................... 28
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List of Figures

Figure 1. Structure of Eye .................................................................................................. 2

Figure 2. Eye of Patient with ADOA ................................................................................. 2

Figure 3. DSP Structure ..................................................................................................... 3

Figure 4. Opa1 Protein Function ........................................................................................ 4

Figure 5. Pedigree Chart of Patient .................................................................................... 5

Figure 6. Structure of OPA1 .............................................................................................. 6

Figure 7. Arginine vs Histidine .......................................................................................... 7

Figure 8. Where is R445H? ............................................................................................... 8

Figure 9. Colonies of Opa1 DNA .................................................................................... 21

Figure 10. Positive PCR Screen Results .......................................................................... 22

Figure 11. Sequencing with R445H Mutation ................................................................. 23

Figure 12. Sequencing with Missense Mutation .............................................................. 23

 viii

List of Tables

Table 1. First Mutagenesis Attempt Protocol .................................................................. 11

Table 2. Second Mutagenesis Attempt Protocol .............................................................. 12

Table 3. Shotgun Mutagenesis PCR Protocol .................................................................. 13

Table 4. PCR Screen Protocol ......................................................................................... 16

Table 5. SDM Protocols .................................................................................................. 20

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Introduction

Autosomal Dominant Optic Atrophy (ADOA) is the hereditary condition where

the optic nerve in the eye is degenerated. The progressive vision loss normally begins in

the first ten years of life and the severity can range from acute to legal blindness.1,2 It is

known that ADOA causes progressive loss of vision, color vision deficits, a black dot or

blind spot to appear otherwise known as a scotoma, and optic nerve pallor or bright,

white color.1,2 The disease is characterized by the degeneration of the axons made of

ganglion cells that form the back

of the eye, ultimately leading to

the optic nerve which is

responsible for taking information

retrieved by photoreceptors to the

brain (Figure 1).3 In patients with

Figure 1: Structure of the Eye showing where the
ADOA, the signals traveling ganglion cells are related to the rods and cones

through the optic nerve cannot reach the brain which leave the retina very bright in color

showing the trapped signals unable to be recognized (Figure 2).1 The ultimate outcome of

this misconnection

between the retina and

the brain is the

malfunction of the

information exchange

Figure 2 This shows an examination of a control patient on the left and a and the brain receiving
patient with ADOA on the right caused by a mutation in OPA1. Here the
dark spots show the degeneration of the macula where the retina is
unaffected, but brighter as the signals are not able to move out to the
brain.
 3

no signals resulting in blindness.

A multitude of patients with ADOA experience effects just in their vision;

however others may also experience other sensorineural function loss such as hearing

loss, myopathy, peripheral neuropathy, cataracts and, in rare cases, multiple sclerosis-like

illnesses.2 It has been recently discovered that disease-associated OPA1 gene mutations

were identified in families who have a history of autosomal dominant optic atrophy.4

The cause for ADOA has been determined to be caused by dominant mutations in

the OPA1 gene that encodes a large mitochondrial GTPase.5 To better understand the

effect Opa1 has on the eye, the purpose of a dynamin super family protein must be

understood. Dynamin Super Family Proteins, also known as DSPs, are present in all

organisms with the primary job of performing membrane fission, membrane fusion,

bundling, and helping with innate immune responses.6 Likewise, DSPs are specifically

responsible for membrane remodeling and overall membrane dynamics. All DSPs are

characterized with several common structural features that are shown in Figure 3; the

most prominent are a GTPase domain and an adjacent alpha-helical bundle (also referred

to as the stalk region).7

Figure 3 DSP Structure: A general structure of a dynamin super family protein which includes the stalk or
alpha helical region, PH domain, BSE, and G Domain
 4

The GTPase domain and alpha helical bundle allow for membrane fission which is

performed in the crucial process of endocytosis and lipid interactions.6 Similarly, the

lipid-interacting domain is prominent in all DSP structures in some kind of way to

carryout membrane remodeling as well as membrane dynamics. The main purpose of

DSPs are to work cohesively with the membranes to aid in fission and fusion for cell

functions, especially endocytosis.8

Opa1, optic atrophy type 1, is a prominent piece in the inner membrane fusion

within mitochondria and is an example of a DSP.9 Opa1 primarily is responsible for

regulating the shape and conformation of the inner mitochondrial membrane (IMM) by

playing a prominent role in the fusion process and maintaining the mitochondria DNA.10

The interaction of Opa1 with membranes can stimulate higher order assembly,

enhancement of GTP hydrolysis and lead to the deformation of membrane structures in

tubules.11 There is a proteolytic cleavage that happens to the Opa1 protein where a

peptide bond is broken by

enzymes in a hydrolysis

reaction into two forms.12 In

Figure 4, the SOpa1 (soluble

short isoform) and the LOpa1

(membrane-anchored long

isoform) are shown working in

unison to help fusion and Figure 4 OPA1 function: This shows how the S-OPA1
and L-OPA1 work together to perform fusion and
fission in a membrane.9 This fission with other regulators in mammalian cells

shows how the S-Opa1 is cut

 5

from the tether by L-Opa1 by enzymes from the hydrolytic reaction in order to fuse the

membranes.11 The cleavage of proteins leaves the chain of amino acids susceptible to

mistakes or mutations as it is essentially being taken apart and put back together again.

One of the first studies to look at a disease causing Opa1 mutations took place in

2002, when Satoko Shimizu performed a study at Teikyo University Hospital

investigating four Japanese patients from ages six to twenty-one who were diagnosed

with autosomal dominant optic atrophy.4 It was important to note that each patient was

not related to one another, as this mutation contradicts that ADOA is hereditary(Figure

5).5

Figure 5 R445H Pedigree Chart: The pedigree of the family with optic
atrophy studied in Shimizu’s trial. The patient is represented by the shaded
in square, whereas the unaffected family members are an open circle
(female) or square (male). The + represent the wild type OPA1 and - the
R445H mutation. It is important to note that there is no evidence towards
the genotype of this patient’s mom as was deceased when the study began.
 6

Because of the lack of presence of this mutation in the patient’s family members (Figure

5), it is highly probable that this mutation is disease-causing beyond just ADOA as the

patient also suffered from sensorineural hearing loss.4 It is also important to note that

there is no sequencing data on the patient’s biological mother which could insinuate this

mutation is spontaneous and not genetic. In order to look at the patients’ specific gene

sequence, the DNA was extracted from leukocytes of each of the four patients through

their blood. Then, the exons and splicing sites of the OPA1 gene at exons 1-28 of each

patient were amplified in a PCR reaction and sequenced.4 It was found that one patient in

the study with no known family history of dominant optic atrophy had a substitution of G

to A which then results in the amino acid arginine being a histidine at position 455, also

referred to as mutation R445H (Figure 5).

The R445H mutation is located inside the GTPase domain, very close to the stalk

region.4,5 This region can be seen in Figure 6 where the green and blue regions meet

which has been circled.

Figure 6 Structure of Opa1: The structure of OPA1 is characterized by a

GTPase domain (green), a stalk region (blue), a helical bundle domain (yellow), and a
lipid interacting region (pink). These regions are what classify OPA1 as a dynamin super
family protein and its functions.
 7

When any mutation, not only R445H, occurs in Opa1, the function will change

depending on the location of the mutation and how variable the properties of the two

amino acids are. Each different mutation plays a prominent role in aiding researchers in

discovering more about the predicted structure of Opa1, its specific functions, and role

the gene plays in the cause of optic atrophy. But, when specifically looking at the R445H

mutation, the structures of the arginine amino acid to the histidine amino acid (Figure 7)13

must be evaluated in order to draw conclusions and decide as to what tests could be done

to see how the characteristics of these amino acids change Opa1’s structure and function.

Figure 7: The structures of arginine and histidine

Arginine (arg) is more basic in comparison to histidine (his) and also a larger amino

acid.14 One prominent aspect in this conversation is the pKa of each amino acid which is

the property of a compound representing how acidic it is. Arg has a pKa of 12.48 and His

has a pKa in between 6-7, depending on its resonance and state. Both His and Arg can

donate and accept protons under specific conditions, but the major difference comes from

what environment this can occur within. His has a more physiologically relevant pKa as

it can be converted to intermediates in the Citric Acid Cycle. On the other hand, Arg will

be protonated at physiological pH; at this pH, His will mostly be deprotonated. This

difference has a possibility to be relevant in the R445H mutation especially in terms of

ionic interactions. Another conversation could be talked about in terms of flexibility.

 8

Protein folding, ionic interactions, and intermolecular forces are all aspects that could be

altered with the mutated Opa1 instead of the wildtype.

To reinforce the idea that the R445H mutation has the ability to cause more than

ADOA, in 2007, Patrizia Amati-Bonneau and many others reported on eight patients

from six families which were independent of each other that had members showing

mutations in the Opa1 gene can also be responsible for other sensorineural deficits.15 The

syndromic forms of DOA was associated with deafness, ataxia, mitochondrial myopathy,

and chromic progressive ophthalmoplegia.15. The largest result concluding from this

research was that these patients all experienced multiple deletions of mitochondrial DNA

which led the researchers to the question of what part of Opa1 plays a role in the stability

of mitochondrial DNA. Additionally, this research looked specifically at the R445H

mutation and found that the missense mutation here affects the GTPase domain which is

adjacent to the active site of the protein (as seen in Figure 8). This could possibly inhibit

the GTP hydrolysis from occurring by restricting

the protein from utilizing the energy from the

GTPase domain due to reasons that are not

explicitly stated within the research.15 Because

of the lack in the use of energy, this mutation

could possibly impair and interfere with the

binding of nucleotides and can alter the affinity

and hydrolysis in the GTPase domain of the

protein. In Figure 8, the location of the R445H

Figure 8 Where is R445H?: The known structure of
mutation is extremely important in recognizing OPA1 and the location of the mutation of R445H.
(Bonneau)
 9

the variability for effect on the functions related to binding, affinity, and hydrolyzing

when mutated.15

Therefore, though there have been significant developments beyond the Shimizu

trial in 2002, there is a plethora of information still unknown about the role Opa1 plays in

Autosomal Dominant Optic Atrophy and that mutations in Opa1 can cause a multitude of

other conditions related to the eye and ear. Due to this, the main goal of this research is to

study the biochemical characteristics of the R445H mutation and how the mutation

affects the G-domain dimerization of Opa1. To progress this research, we will try to

insert the mutation R445H into the wildtype Opa1 plasmid through site directed

mutagenesis. Then, a Dpn1 digest would be performed to degrade any leftover wildtype

DNA and then use a heat shock to transform bacteria. From here, a transformation will be

performed where in the end ampicillin resistant colonies should form on the LB +

Ampicillin plates. Using a PCR screen, the colonies would be tested to determine if the

OPA1 gene is present in the bacteria, and a mini prep would then occur to send the

mutated DNA off for sequencing. Any results from this research would add to the current

unknown of the complete structure and function of Opa1. Additionally, it would bring

focus to how Opa1 directly causes optic atrophy to occur in patients with family history

or not. This adds to the overall goal of the scientific community to determine how the

function of OPA1 is affected by the change in structure of the gene due to the switch of

arginine to histidine at codon 445 in the GTPase domain and could shed light upon the

effect different mutations in a similar region of the protein.

 10

Methods

Site-Directed Mutagenesis

Site-Directed Mutagenesis (SDM) is the procedure that uses designed

oligonucleotide primers to insert a desired mutation within a double-stranded DNA

plasmid. This technique allows one to study the effect of switching amino acids of a

protein and how this affects the protein’s structure and function. This basic mechanism

uses two primers: one that contains the desired mutation and the other a complement to

the template DNA strand around the site of mutation. The two primers obtained from

Integrated DNA Technologies in January 2020 are defined by the following sequences: 1)

5’ GCG TTG ATG CAG AAC ATA GCA TTG TTA 2) 3’ CGG TAA CAA TGC TAT

GTT CTG CAA. Once the primers are inserted, the DNA polymerase will extend the

primer and copy the rest of the gene so that the mutated site is present and can be cloned

into the vector plasmid. SDM requires the use of PCR to mix the components required to

insert the mutation R445H into the OPA1 plasmid.

First Mutagenesis Attempt Protocol

The components of this first PCR were 34.4 µL of nuclease free water, 10 µL of

5x pFU buffer, 0.5 µL of 94.4ng/µL of DNA, 200 µM of dNTPs, 25 µM of the 5’ primer,

25 µM of the 3’ primer, 1 µL of the pFU polymerase, and 1 µL of 2% DMSO (dimethyl

sulfoxide). These species were added in this order to a PCR tube which were then placed

into the PCR (BioRad T100 Thermal Cycler) under the following protocol:
 11

Table 1: First Mutagenesis Attempt PCR Protocol

Temp (C°) Time

95° 5 min
95° (35 cycles) 30 s
54° (35 cycles) 30 s
72° (35 cycles) 4 min
72° 5 min
4° ∞ (hold)

In this PCR, the 95°C denaturing cycle denatures the double stranded DNA to allow the

primers to move in. The 54°C annealing cycle brings the primers together to anneal them

into the plasmid vector. This temperature was chosen as the appropriate annealing

temperature because the optimal temperature of the pFU polymerase is 68°C. And,

finally, the 72°C extension time allows for the plasmid to extend off the primers,

essentially inserting R445H into the wild-type Opa1 plasmid.

Second Mutagenesis Attempt Protocol

The second attempt to site-directed mutagenesis involved minor changes in the

PCR mixture components and the annealing temperature in the PCR reaction, as well as

an increased extension time. It was decided that by increasing the amount of DNA in the

sample, there would be a larger target and better opportunity to allow the primers to

attach and extend off the plasmid. Additionally, the annealing temperature was decreased

to avoid complications of approaching the melting point/temperature of the pFU

polymerase during the reaction. Similarly, the extension time of the PCR reaction was

increased at the previous 72°C extension protocol in order to give the polymerase enough

time to extend the primers and clone the R445H mutation inside the vector plasmid

without leaving openings or not fully completing the cloning.

 12

The components of the second PCR reaction were 33.8 µL of nuclease free water,

10 µL of 5x pFU buffer, 1.2 µL of 94.4ng/µL of DNA, 200 µM of dNTPs, 1 µL of the 5’

primer (25 µM), 1 µL of the 3’ primer (25 µM), 1 µL of the pFU polymerase, and 1 µL

of DMSO. The final volume of 50 µL in the PCR reaction was maintained by changing

the DNA volume from 0.5 µL to 1.2 µL and using less nuclease free water. Additionally,

the new PCR reaction protocol is shown below:

Table 2: Second Mutagenesis Attempt Protocol

Temp (C°) Time

95° 5 min
95° (35 cycles) 30 s
53° (35 cycles) 30 s
72° (35 cycles) 8 min
72° 5 min
4° ∞ (hold)

With a longer extension time, one will look to increase the yield of the PCR products

because fewer partial products are synthesized.16 Additionally, by using DMSO within

our PCR reaction mixture, the double strand separation would be increase as well as the

decrease in annealing temperature which reduces the risk of depurination affects due to

high enough temperatures at a long enough period of time.

Shotgun Mutagenesis

Later, it was decided that the annealing part of the PCR reaction may not be

working, and it was not ideal to use a trial-and-error method. Therefore, a shotgun

mutagenesis approach was taken. In this kind of PCR reaction, the annealing cycle of the

PCR reaction was split into different zones that have different temperatures. This is ideal

when testing the same samples because many temperatures can be tested at one time to

see which temperature worked most effectively to produce R445H within the plasmid. In
 13

this mixture, the same concentrations were used as outlined in “Second Mutagenesis

Attempt Protocol”, except the volumes were multiplied by a factor of 2.1 in order to

produce a stock solution of 105 µL (enough volume for all five samples). This stock was

then divided into five aliquots of 20 µL and labeled one through five. The PCR protocol

is listed below (Table 3) with the annealing cycle and the corresponding annealing

temperatures for each aliquot of mutagenesis mixture.

Table 3: Shotgun Mutagenesis PCR Protocol and the Corresponding Annealing Temperatures to the right

Temp (C°) Time Temperature zones °C Degrees °C

95° 5 min A #1 56
95° (35 cycles) 30 s
C #2 54.4
56-48.5° (35 cycles) 30 s
D #3 53
72° (35 cycles) 8 min
72° 5 min E #4 51.1
4° ∞ (hold) G #5 48.5

Dpn1 Digest

Each of these types of mutagenesis takes the wildtype OPA1 with the Arginine at

codon 445 and cuts out the Arg to replace it with Histidine. When this occurs, the new

plasmid is left with methylated, original DNA and unmethylated DNA which is the new

plasmid. The goal is now to get rid of the methylated DNA in order to ensure there is no

extra DNA that is not R445H OPA1 gene.

In order to get rid of the methylated DNA (leftover wildtype) from the PCR reaction

from the mutagenesis product, 5µL of Cut Smart Buffer and 1 µL of Dpn1 were added to

the PCR reaction tubes and incubated in a hot water bath at 37°C for one hour. This

digestion used both the cut smart buffer and Dpn1 to select remaining template DNA that
 14

were not mutated during SDM.17 This cleanup process works because Dpn1 is a

restriction enzyme that specifically finds and targets DNA containing methylated adenine

and destroys it.18 From this point all wildtype DNA was eliminated and the unmethylated

DNA could be transformed.

Transformation

Heat Shock

Using DH5α bacteria (a type of competent cell) in this transformation required the

wanted concentrations (150 ng, 225 ng, 300 ng, etc) of the unmethylated DNA to be

added to the tubes of DH5α; using a variety of concentrations allows for more bacteria to

be transformed and colonies to grow in the end. For these wanted concentrations, it has

been found that using 225 ng and 300 ng of unmethylated DNA produces the best results.

Therefore, the unmethylated DNA was added to DH5α bacteria tubes and incubated on

ice. Then, the samples of DH5α and DNA are placed in a hot water bath at 42°C for 90

seconds.

This heat shock is important to transformation as when the cold samples from the

ice are placed into the hot water bath, pores in plasma membrane of the bacteria open up

and take the DNA into the bacterial cell.19 Then, two culture tubes that had been

sterilized in the autoclave were labeled with the corresponding concentration and 1 mL of

LB should be added to each tube aseptically. The aseptic method included using a

Bunsen burner to heat the sides of the LB container and the top of the culture tube to

prevent contamination of the LB. Then, 50 µL of the DNA/DH5α mixture was added to

each corresponding culture tube and put in the shaker for 60 minutes.
 15

Plating

While the culture tubes were in the shaker, two plates were obtained (LB and

Kanamycin) and labeled with all the identifying data. The culture tubes were placed in

the shaker for an hour. Then the full contents of the DNA/DH5α/LB culture tubes were

places into large centrifuge tubes (either 1000µL or 1mL). These tubes were centrifuged

at 16000 xg for twelve to fifteen seconds, allowing for a small pellet to be formed in the

bottom of the tubes. If a pellet had not formed, they were centrifuged again for twelve to

fifteen seconds. The pellet was made up of the DH5α bacteria that has taken up the

mutated plasmid. The supernatant was discarded into the bacterial + bleach waste

container without disturbing the pellet.

In order to plate the pellet, it needs to be resuspended into the remaining

supernatant (normally about 250 µL). A scraping method was used to remove the pellet

and dissolve the bacteria and DNA back into solution. Then, an aseptic method was used

with the “hockey stick” and 95% ethanol to pipet the bacteria and DNA from the tube

onto one of the labeled LB + Kanamycin plates. The bacteria were then spread around the

entire surface of the plate with the hockey stick. These plates were then placed into the

37°C incubator over-night and checked in the morning. If there were colonies, leave in

the incubator for a couple more hours to see if the colonies grow larger. Then, the plates

were taken out of the incubator, wrapped with parafilm, and kept in the refrigerator. If no

colonies were present, mutagenesis was repeated.

 16

PCR Screen

The purpose of a PCR screen is to determine if the plasmid vector and Opa1 gene

are present in the solution. Though, it cannot tell if it is mutated, it shows that the gene

has been transformed. To perform this screen, master mix obtained from

ThermoScientific which includes 2x concentrated solution of polymerase, dNTPs, and

other components required for PCR were combined with 3’ primer, 5’ primer, nuclease

free water, and ½ DNA from the colony. Taq polymerase and buffer was used within the

master mix as well as the reverse primer T7 terminator and forward OPA1 5’ primer, for

rationale see below Table 4. To retrieve the colony, an inoculation loop/water was used

aseptically to place half the colony into the master mix inside the PCR tube. The other

half of the colony was vigorously swished in a culture tube that had 5 mL of LB and 5 µL

of kanamycin. Once the colony was put into both places, the culture tubes were placed in

the shaking incubator at 37°C and the PCR tubes undergo another PCR reaction (Table

4).

Table 4: PCR Screen Protocol

Temp (°C) Time

95° 5 min
95° (35 cycles) 30 sec
52° (35 cycles) 30 sec
68° (35 cycles) 1 min
68° 5 min
40° ∞ (hold)

An agarose gel was used in order to run the colony PCR product. On this gel,

bands should be seen at 800-830 base pairs. If so, this shows that the plasmid is present

and can be mini-prepped and sent out for sequencing. Then, about 24 hours later, the
 17

culture tubes were checked for cloudiness which means that the bacteria were still

present, and the plasmid was within it.

Mini Prep

The goal of a mini prep is to purify the hopefully mutated plasmid out of the

bacteria and away from any extrachromosomal DNA. To do this, the contents of the

cloudy, bacteria present culture tube were transferred to a conical tube. This conical tube

was centrifuged for five minutes; a pellet then formed, the supernatant was bleached and

discarded in the bacterial waste jug, then 1 mL of cell wash (50 mM Tris and 100 mM

NaCl) was used and then vortexed until the pellet was resuspended back into solution.

The full volume of the conical tube was placed into 1.5 mL centrifuge tubes and

centrifuged at 16,100 g for thirty seconds; again, a pellet was formed, and the supernatant

was discarded. Then, 200 µL of Buffer 1 was added and the pellet was resuspended using

a scraping method, 200 µL of Buffer 2 was added and the mixture was gently inverted to

mix the solutions. Following this, 400 µL of Buffer 3 was added and mixed gently. Now,

the tube was centrifuged at 16,100 g for thirty minutes. The zymo-spin column was

inserted into the collection tube and the supernatant from the centrifugation was poured

into the top of the column and centrifuged for one minute. The flow through was

discarded into a waste beaker and 400 µL of plasmid wash buffer was added, centrifuged

for one minute, and discarded again. Then, 30 µL of DNA elution buffer was added and

sat for about sixty seconds, centrifuged for a minute afterwards, and 20 µL of DNA

elution buffer was then added and centrifuged once again. Following this, a nanodrop

reading was performed to ensure the concentration is great enough to perform sequencing

(260/280 of 1.80 is ideal). If so, it will be sent out for sequencing.

 18

Making Plates
Though not directly related to Opa1 and the mutation R445H, making plates was

essential to carrying out the transformation process and for having good lab etiquette for

the others in lab. To make the plates, a mix of 15 µL of Kanamycin and 85 µL of LB

(lysogeny broth) was made and the mix was then spread onto the empty plates. It is

important to make sure the plates are on a straight or even table, so the resin does not

form unevenly; then let the plates sit for a day or two. Additionally, the antibiotic

Kanamycin was used because DH5α bacteria are resistant to this antibiotic, therefore if

the unmethylated DNA is taken up by bacteria (during transformation) colonies should

form on the plates.

Limitations
As with most research and scientific experiments, there are factors that play into

research that impeded the research from being successful. The first limitation brought

about was the presence of a missense mutation which produced a stop codon within the

stock wildtype DNA. Due to the stop codon produced, this mutation inhibits the ability to

use the cloned OPA1 gene with the R445H mutation within the plasmid. Therefore,

though the “original mutagenesis” protocol deemed successful, the outcome was not

favorable due to the missense mutation. Second, many of the culture plates (Kanamycin

and LB) were becoming moldy after being in the incubator for about twenty-four hours

after transformation and trying to grow colonies. New LB was made to try and inhibit this

issue, but that did not aid in the cause. It was suspected that the airflow within the

laboratory was not ideal for pouring plates, therefore the plates needed to be poured in a

sterile laminar flow hood. Though this limitation was fixed, there were many colonies

that formed on the plates that were also accompanied by mold which made it exceedingly
 19

difficult to try and get those colonies off the plate to PCR screen. Finally, it was brought

to the lab’s attention that the DMSO that had been used in the entirety of the laboratory

was stored improperly at room temperature and the stock was from over fifty years ago.

To safely store DMSO, it should be kept frozen and in the freezer at all times. This

mishap interferes with yield of the PCR reaction and specificity because DMSO is

responsible for binding to the DNA and preventing the reannealing of single-stranded

DNA.20 All these limitations have been accounted for but took time and trial and error to

figure out.
 20

Results and Discussion

Looking deeper into the findings of the Shimizu in his 2002 paper, the mutation

R445H of OPA1 became the focus in the cloning process. Throughout the duration of this

research, a multitude of techniques were utilized in order to clone Opa1 with the arginine

to histidine mutation into codon number 445. As stated previously, the site-directed

mutagenesis (SDM) method was used in a few forms: with one annealing temperature

(many trials at different temperatures) and in a shot-gun method of the annealing

temperatures. In Table 5 below a summary of the methods used and their different

characteristics are described. Success, in this case, means that the method produced

colonies that were sent out for sequencing that was confirmed to switch arginine for

histidine at codon 445.

Table 5: Different Methods used for Site Directed Mutagenesis. Successful SDM is highlighted in green.

Summary and Success of Methods Utilized

Enzymes Q5 Pfu

55.5, 54.8,

Site Directed Annealing Temperatures 54, 53.7,

Mutagenesis (°C) 52.3, 51.1

5’ and 3’

Primers primers

(1065,1066)

Interesting enough, Trial 1, which was characterized by using Pfu polymerase and buffer,

an annealing temperature of 54.0 °C and the original 5’ and 3’ primers (sequences

referenced in the methods section) was the best cloning technique chosen through the
 21

various trials. The first step in determining that this site-directed mutagenesis method was

successful was to determine if any colonies were produced from the PCR reaction when

the product is plated with the DH5α bacteria. The growth of a bacterial colony would allow

us to assume that the Opa1 DNA was obtained by the DH5α bacteria as this bacteria is

resistant to the kanamycin antibiotic which is present on the plate. Figure 9 depicts the 2

colonies that were grown in trial 1 and used in a PCR screen. Both colonies were 1 𝜇𝐿 of

DNA.

Figure 9: Colonies with Opa1 in the DH5-alpha bacteria that were

sent out for sequencing

After determining the colonies were from bacterial growth and not contamination

due to the size and placement, a PCR screen was done on the colony samples in order to

determine if the Opa1 DNA was actually taken up by the DH5α bacteria. This ensures that

the sample sent out for sequencing is valid and worth-while. Figure 10 shows the PCR gel

with the positive control in lane 1, colony 1 in lane 2, and colony 2 in lane 3. When looking

at the gel, 800 base pairs is a match in DNA, and 200 base pairs is referred to as primer

dimers.
 22

Figure 10: PCR Screen Results: The first lane shows the ladder of the base pairs, the second lane is colony 1 and

the third lane is colony 2

Due to the predicted success of the SDM by the PCR screen, the NanoDrop was used to

determine the quality of the DNA sample. Colony 1 resulted in a concentration of 121.4

ng/ 𝜇𝐿 and a 260/280 of 1.89. On the other hand, colony 2 had a concentration of 110.4

ng/ 𝜇𝐿 and a 260/280 of 1.80. The NanoDrop has an allowed deviation of 2% which was

found on the manufacturer’s website. The concentration for these samples shows much

DNA is present in a microliter of sample. The ideal value of the 260/280 is 1.8 to ensure

purity and not an excessive amount of RNA contamination (260/280 ratio of 2.0). These

measurements were deemed successful; thus, the samples were sent out for sequencing.

Referring to the second attempt of mutagenesis and shot gun approach for the

SDM methods in the methods, section, each trial failed to provide consistent colonial

growth and, therefore success. In few cases, bacterial growth was present on the LB +

Kanamycin plates, but when tested with the PCR screen, no DNA was present, hence

leading to the belief that the colonies were grown due to contamination.
 23

The goal of this research was to clone the R445H mutation in the wildtype Opa1.

In Figure 11, it was confirmed that the plasmid was successfully mutated at codon 445

where the arginine amino acid was replaced with a histidine amino acid. The first line

below shows the sequence alignment of the expected product, and the second and third

lines show the sequences of the two predicted mutated products sent out; this successful

mutation is characterized with the CAT three letter code.

Figure 11: Sequencing for Trial 1 of SDM showing successful mutation (CAT) at codon 445

Sequencing is not only important for showing if the SDM process was successful in making

the desired mutation, but also if other mutations occurred within the plasmid during this

process. This purpose was specifically useful in this research project as shown in Figure

12 where a nonsense mutation producing a stop codon was found. In this sequencing, there

is a “T” for thymine instead of the predicted or expected “G” for guanine. At first it was

assumed something within the used protocol allowed for this second mutation to be made.

Figure 12: Sequencing on Mutated Opa1 Showing the Point Mutation

This sequencing showed that the wildtype Opa1 was successfully cloned with the R445H

mutation, but the switch from guanine to the thymine resulted in a nonsense mutation

which is also known as a stop codon. This means that the mutated version of the Opa1

produced an unviable plasmid and can not be used to research on. It was later found after
 24

other successes in lab that there was this mutation in the provided stock wildtype of

Opa1, not an issue within our protocol. Therefore, a new stock of Opa1 had to be

obtained.

As stated in the methods, more trials of SDM were performed with the different

characteristics as stated in Table 5. Though with the different trials, there was no success

in terms of growing colonies or sending out a colony for sequencing. Common results

were no colonies growing, or the colony sent out for sequencing resulting in non-mutated

wildtype Opa1. There were a multitude of factors that can have attributed to these results.

The first is the notion that the 5’ and 3’ primers for mutation R445H were not meeting all

the necessary requirements to be a primer. These requirements are a G-C clamp on each

side of the mutation at codon 445 and to be around 60 base pairs long. With addressing

these requirements as well as the melting temperatures of the primers, it could be a

concern that the melting temperature of the primers is too high which could lead to the

inability of the primers to pull apart from the DNA they attached to in the beginning

cycles of the SDM PCR protocol. This would be due to the bonds created and being too

strong. Therefore, in the continuation of this research as I have moved into the written

portion of my thesis, a new student has taken on the R445H Opa1 project by starting with

addressing these primer concerns and redesigning these primers to be more effective and

appropriate in terms of annealing temperature and length.

In addition to addressing the primer design, a method called touch-down PCR

would be the next step in the SDM protocol trials. As stated in the methods section, a

plethora of annealing temperatures were used in order to create the mutation within the

wildtype Opa1, but success could not be replication with the new DNA from the NIH.
 25

This touchdown method would take one sample of the primers and DNA and allow the

first annealing cycle to be at the initial desired temperature. The following cycles would

then decrease each time in order to cover a plethora of temperatures, instead of trying to

pinpoint the exact annealing temperature needed.

Following the successes of this cloning process in the future, more insight and

experiments like GTPase assays could be performed in order to look how the R445H

mutation not only impacts the structure of Opa1, but the function as well.
 26

Conclusion

In summary, the overarching goals amongst this research were to (1) use a variety

of protocols relating to site-directed mutagenesis in order to determine the most effective

protocol in order to clone the mutation R445H into the wildtype Opa1 DNA and (2) study

the biochemical characteristics of the R445H mutation and how it affects the G-Domain

dimerization of Opa1. All-in-all these findings would be able to help answer structural and

functional questions that are still unknown about the Opa1 protein.

The most efficient method found to clone the R445H mutation into the wildtype

DNA was characterized by the use of a Pfu polymerase, an elongation time of 4 minutes a

cycle, 150 ng DNA, and an annealing temperature of 54ºC. The use of a restriction enzyme

digest with Dpn1 was determined to be appropriate, followed by a bacterial transformation

using the bacteria DH5α in order to uptake the Opa1. Additionally, using the antibiotic

Kanamycin in order to grow colonies also was a success as the bacteria is resistant to this

particular antibiotic.

To reiterate, this protocol did deem successful with a positive PCR screen with a

band at the correct bp representing Opa1 plasmid DNA; additionally, the sequencing

confirmed that the Arg amino acid was replaced with a His at codon 445, but a missense

mutation (producing a stop codon) was found in the product as well. This missense

mutation was not a result of the SDM and transformation, but instead found in the stock

wildtype DNA. The presence of the “UAA” stop codon prohibited the research from

moving forward with protein expression, purification, and assays. After receiving

unmutated wildtype Opa1 from the NIH, this successful protocol could not be replicated

over various trials. Additionally, new protocols such as shot-gun mutagenesis with a
 27

variety of annealing temperatures were utilized, but none deemed successful with fully

incorporating the R445H mutation into the plasmid.

With attempts still ongoing in the Kehr lab to clone the R445H mutation into the

Opa1 plasmid, the scope of the research will expand and cover the production of this

mutation, protein expression of the mutated Opa1, protein purification, and a GTPase

assay. The first steps into revamping these protocols are to redesign the 5’ and 3’ primers

used in the SDM process. Additionally, a new protocol called touchdown PCR could be

used as a site-directed mutagenesis attempt allowing one product to undergo various

annealing temperatures in one cycle. Furthermore, the success of cloning Opa1 DNA into

the mutated R445H version will not allow for further research to be done, but the eventual

ability to compare the structure and function of the wildtype Opa1 protein to the mutated

R445H Opa1.

Such discovery would aid in the overall determination of the differences in the

function of OPA1 with the presence of His instead of Arg. Furthermore, this discovery

could fill in a small piece of new information about the structure of Opa1 and could provide

insight into the larger picture of how and why these mutations relate to the pathology of

ADOA and how other diseases such as deafness could be related to Opa1 mutations too.

With looking deeper into the pathology of ADOA and its activity/interactions, possible

treatment options and investigation of cures for ADOA could come to the table.
 28

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