Week 2 HW: DNA Read, Write, & Edit

Synucleinopathies are a group of chronic and progressive neurodegenerative diseases characterized by the abnormal accumulation of misfolded alpha-synuclein protein. This misfolded protein aggregates into insoluble inclusions known as Lewy bodies, which are found in neurons and glial cells. The major synucleinopathies include Parkinson’s disease, Dementia with Lewy bodies, and Multiple system atrophy. These disorders typically appear in late adulthood and progressively worsen over time.

Alpha-synuclein is a neuronal protein encoded by the SNCA gene in humans. Under normal physiological conditions, it plays a role in synaptic vesicle regulation and neurotransmitter release. However, in neurodegenerative conditions, the protein undergoes misfolding and aggregation, forming toxic oligomers and fibrils that disrupt cellular homeostasis and ultimately lead to neuronal death.

Because protein misfolding and aggregation are central mechanisms in many neurodegenerative diseases, studying alpha-synuclein at the sequence level provides important insight into disease pathology and potential therapeutic strategies.

`>sp|P37840|SYUA_HUMAN Alpha-synuclein OS=Homo sapiens OX=9606 GN=SNCA PE=1 SV=1MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA`<br>

Sequence taken from UNIPROT

The resulting optimized nucleotide sequence is:
atggatgtgtttatgaaaggcctgagcaaagcgaaagaaggcgtggtggcggcggcggaa aaaaccaaacagggcgtggcggaagcggcgggcaaaaccaaagaaggcgtgctgtatgtg ggcagcaaaaccaaagaaggcgtggtgcatggcgtggcgaccgtggcggaaaaaaccaaa gaacaggtgaccaacgtgggcggcgcggtggtgaccggcgtgaccgcggtggcgcagaaa accgtggaaggcgcgggcagcattgcggcggcgaccggctttgtgaaaaaagatcagctg ggcaaaaacgaagaaggcgcgccgcaggaaggcattctggaagatatgccggtggatccg gataacgaagcgtatgaaatgccgagcgaagaaggctatcaggattatgaaccggaagcg

To improve expression efficiency, I optimized the alpha-synuclein coding sequence for Escherichia coli, a commonly used bacterial expression system in molecular biology. Using the Reverse Translate tool with an E. coli codon usage table, the most frequently used codons were selected for each amino acid. This increases the likelihood of efficient transcription and translation when the gene is expressed in bacterial cells.

Step 1: Gene Synthesis and Cloning
The codon-optimized alpha-synuclein DNA sequence is first chemically synthesized and inserted into a plasmid vector. The plasmid contains a strong promoter (such as T7), a ribosome binding site, an antibiotic resistance gene for selection, and a transcription terminator. This plasmid serves as a vehicle to introduce and express the gene inside bacterial cells.

Step 2: Transformation into Bacterial Cells
The recombinant plasmid is introduced into competent Escherichia coli cells through heat shock or electroporation. The bacteria are then plated on antibiotic-containing media so that only cells that successfully incorporate the plasmid survive.

Step 3: Transcription (DNA → mRNA)
Inside the bacterial cell, RNA polymerase binds to the promoter and transcribes the alpha-synuclein DNA sequence into messenger RNA (mRNA). During transcription, thymine (T) in DNA is replaced by uracil (U) in RNA.

Step 4: Translation (mRNA → Protein)
The mRNA binds to ribosomes, which read the sequence in sets of three nucleotides (codons). Each codon specifies one amino acid. Transfer RNAs (tRNAs) deliver the corresponding amino acids, which are linked together to form the alpha-synuclein protein. Because the gene was codon-optimized for E. coli, translation efficiency is increased.

Step 5: Protein Folding and Purification
After translation, the protein folds into its functional structure. The bacterial cells can then be lysed, and the protein purified using methods such as affinity chromatography, ion exchange chromatography, or size exclusion chromatography.

In summary, the optimized DNA sequence is transcribed into RNA and translated into alpha-synuclein protein, enabling large-scale production for research on neurodegenerative diseases such as Parkinson’s disease.

The human SNCA gene primarily encodes the 140-amino-acid alpha-synuclein protein. However, alternative splicing can generate shorter isoforms (e.g., 126 or 112 amino acids), demonstrating how one gene can produce multiple protein variants.

Below is the alignment of the designed DNA sequence with its corresponding RNA and translated protein:

(i) What DNA would you want to sequence and why?
For this project, I would focus on genes commonly mutated in adenocarcinoma endometrial, such as TP53, PTEN, PIK3CA. Sequencing these genes allows identification of mutations that drive tumor progression and informs targeted therapy strategies.

Example source: NCBI Gene Database for TP53: https://www.ncbi.nlm.nih.gov/gene/7157

(ii) Sequencing technologies to use and why:
- I would use next-generation sequencing (NGS, second-generation) due to its high throughput and accuracy for detecting somatic mutations.
- Input preparation: DNA extraction from tissue, fragmentation, adapter ligation, PCR amplification.
- Sequencing steps: Cluster generation on flow cell, cyclic reversible termination (base incorporation), imaging, base calling.
- Output: FASTQ files with read sequences, aligned to human reference genome to identify mutations.

(i) What DNA would you want to synthesize and why?
To simulate or study therapeutic interventions, I could synthesize DNA fragments of TP53, PTEN, or PIK3CA with specific mutations. These could be used in cell line models to test gene expression, protein production, or biosensor detection in a controlled environment.

Example applications:
- Genetic circuits for biosensors embedded in the DIU to detect tumor markers.
- DNA origami structures for controlled drug release or as scaffolds for sensor integration.

(ii) Technology to perform DNA synthesis and why:
- I would use oligonucleotide synthesis and assembly methods (Twist Bioscience, IDT), which are precise and scalable.
- Essential steps: Phosphoramidite synthesis, purification, assembly into larger constructs.
- Limitations: Sequence length constraints (usually <1–2 kb per construct), cost for large-scale synthesis, and potential errors in repetitive sequences.

(i) What DNA would you want to edit and why?
I would target mutated TP53 or PTEN alleles in endometrial cells. Editing these genes could theoretically restore normal tumor suppressor function, providing a model for localized gene therapy integrated with DIU-mediated monitoring.

For Example: The Company Colossal Biosciences demonstrates genome engineering in animals; similarly, precise editing tools can model human therapeutic strategies.

(ii) Technology to perform DNA edits and why:
- I would use CRISPR/Cas9 or base editors for targeted nucleotide correction.
- Essential steps: Design guide RNA complementary to the mutation, deliver Cas9 and guide RNA into cells, allow DNA repair mechanisms to introduce the correct sequence.
- Input preparation: DNA template or plasmid with Cas9 and guide RNA, target cells (e.g., endometrial cell line), transfection reagents.
- Limitations: Off-target effects, variable editing efficiency, delivery challenges in vivo.