Who Can Tell Me The Truth?

Who Can Tell Me The Truth?

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BadProtein's avatar
BadProtein
Jan 25

The lie of 'mRNA goes away in day or so' was specifically perpretated in two ways. Moderna made long living mRNA by one technique, and Pfizer used a different technique.

How many people were convinced with Science that had to do with Nature?

Unfortunately, Nature was not used in the mRNA vaccines. Just an inspiration.

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>>>>please analyse the optimisation of the stop codon and its surrounding sequence in the main mrna covid vaccines

That's a fantastic and highly specific question! It hits right at the heart of the sophisticated bioengineering that makes these vaccines so effective. The optimization of the stop codon and its surrounding sequence is part of a larger, meticulous effort to maximize protein production and ensure the mRNA is stable.

Here is an analysis of the key optimization strategies concerning the stop codon and its downstream sequence in the main mRNA COVID-19 vaccines (like Pfizer-BioNTech's BNT162b2 and Moderna's mRNA-1273):

1. Stop Codon Optimization

The primary goal is to ensure efficient and accurate translation termination.

Choice of Stop Codon: The most common and often 'strongest' stop codon in mammalian cells is UAA. While all three stop codons (UAA, UAG, UGA) signal termination, UAA is frequently selected in optimized designs to ensure a clear and definitive stop signal for the ribosome. A weak stop codon could lead to readthrough, resulting in a longer, non-functional protein.

Preventing Readthrough: The choice is critical because the presence of the modified nucleotide N1-methylpseudouridine (Ψ) in the vaccine mRNA can slightly increase the chance of stop codon misreading by a near-cognate transfer RNA (tRNA). To counteract this potential readthrough, a highly efficient stop codon like UAA is crucial.

Slightly cheeky aside: No one wants a vaccine that produces a weird, overly long Spike protein. Accuracy is key!

2. The Surrounding Sequence: The 3' Untranslated Region (3' UTR)

The sequences immediately following the stop codon are the start of the 3' Untranslated Region (3' UTR), a non-coding area that is arguably just as important as the coding sequence itself. Its optimization centers on stability and translation efficiency.

Key Optimization Elements:

Regulatory Elements: The 3' UTR contains binding sites for RNA-binding proteins and microRNAs that heavily influence mRNA stability and half-life in the cell. The vaccine designs incorporate highly stable 3' UTR sequences derived from highly-expressed, long-lived human genes (e.g., β-globin or others).

RNA Secondary Structure: The sequence in the 3' UTR is designed to promote a specific RNA secondary structure (the way the RNA folds back on itself). More stable structures can protect the mRNA from degradation by nucleases, meaning the mRNA lasts longer and produces more Spike protein. Computational algorithms are used to find sequences that have both optimal codon usage and maximal structural stability.

3. The Grand Finale: The Poly(A) Tail

While technically not the sequence surrounding the stop codon, it is the most critical downstream component. It directly follows the 3' UTR.

Function: The Poly(A) tail (a long string of Adenine nucleotides) protects the 3' end of the mRNA from enzymatic degradation and is essential for translation initiation, as it cyclizes the mRNA via interaction with the 5' cap.

Optimization: The synthetic mRNA in the vaccines is engineered with a precisely optimized and often long Poly(A) tail (e.g., around 100-150 nucleotides). A longer poly(A) tail is generally associated with greater mRNA stability and a longer half-life, allowing the production of the antigen to continue for a beneficial period.

Extended Elements: The Moderna vaccine, for instance, includes a specific sequence (like ΨC$\Psi$AG, where Ψ is N1-methylpseudouridine) just before its Poly(A) tail. Recent research suggests this, or the tail itself, can induce a cellular enzyme to re-adenylate (lengthen) the tail inside the cell, further boosting the mRNA's lifespan and efficacy.

In short, the stop codon itself is chosen for termination strength (like UAA), and the downstream region (3' UTR and Poly(A) tail) is meticulously engineered for structural stability and enhanced lifespan to maximize the production of the immunogenic Spike protein. It’s a masterclass in molecular precision!

Would you like a deeper dive into how the codon optimization in the main coding sequence (before the stop codon) further contributes to this enhanced protein expression?

moderna's sequence just before the Poly(A) tail is interesting... tell me about that

That is the juicy detail we're looking for. The sequence right before the Poly(A) tail in the Moderna mRNA-1273 vaccine, often cited as a small, non-Adenosine sequence (specifically, a segment like m$\PsiCm\Psi$AG where Ψ is the modified uridine), is fascinating because of its unexpected, dynamic role in the cell.

Here is the breakdown of why this little linker sequence is so clever and effective:

1. It's a Remnant, and a Trigger

The sequence is essentially a vestige—a few non-adenosine nucleotides that are often a scar from the DNA template used in the manufacturing process (in vitro transcription). However, scientists have realized it has a profound biological function.

2. The Great Decoy and the TENT5A Twist

Recent research (using sophisticated techniques like nanopore sequencing) revealed a two-step process in the host cells:

Step 1: The Initial Attack (Decoy) When the mRNA is first translated, the cell's natural degradation machinery (specifically, the CCR4-NOT deadenylation complex) targets the 3' end. This degradation process often begins by rapidly removing that terminal m$\PsiCm\Psi$AG sequence. It's almost like a disposable cap.

Step 2: The Re-Adenylation Boost Once this short sequence is removed, something remarkable happens, particularly in key immune cells like macrophages: the vaccine mRNA induces the expression of a cytoplasmic enzyme called TENT5A poly(A) polymerase.

TENT5A recognizes the exposed end of the Poly(A) tail and starts re-extending it, sometimes boosting the length from ∼100 nucleotides up to ∼200 nucleotides. This process is called re-adenylation.

3. The Functional Outcome: Turbo-Charging Stability

Why does this matter?

Feature Impact on Vaccine Performance

Increased Stability A longer poly(A) tail means the mRNA molecule is significantly more stable and has a longer half-life inside the cell.

Enhanced Antigen Production Longer stability translates directly to a longer period of Spike protein production, leading to a much higher overall dose of antigen and, consequently, a stronger immune response (higher antibody production).

Cell-Specific Enhancement The TENT5A-mediated re-adenylation is observed most strongly in macrophages, which are critical antigen-presenting cells (APCs) at the injection site. This mechanism seems to be key to Moderna's high efficacy.

Essentially, the Moderna design includes a sequence that initiates degradation, but the resulting change tricks the cell into actively repairing and super-charging the mRNA molecule in the exact cells needed for robust immunity.

The Pfizer-BioNTech vaccine (BNT162b2) also has a composite Poly(A) tail but appears to rely less on this specific TENT5A re-adenylation mechanism, highlighting a fascinating difference in the "black belt" optimization strategies of the two platforms.

Would you be interested in an analysis of the specific N1-methylpseudouridine (Ψ) modification that is foundational to both the Moderna and Pfizer-BioNTech vaccines?

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BadProtein's avatar
BadProtein
Jan 25

SARS-CoV-2 spike protein interactions with amyloidogenic proteins:

Potential clues to neurodegeneration

Danish Idrees, Vijay Kumar

Available online 24 March 2021

a b s t r a c t

The post-infection of COVID-19 includes a myriad of neurologic symptoms including neurodegeneration.

Protein aggregation in brain can be considered as one of the important reasons behind the neuro-degeneration. SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) binds toheparin and heparin binding proteins. Moreover, heparin binding accelerates the aggregation of thepathological amyloid proteins present in the brain. In this paper, we have shown that the SARS-CoV-2 S1RBD binds to a number of aggregation-prone, heparin binding proteins including Ab, a-synuclein, tau,prion, and TDP-43 RRM. These interactions suggests that the heparin-binding site on the S1 proteinmight assist the binding of amyloid proteins to the viral surface and thus could initiate aggregation of these proteins and finally leads to neurodegeneration in brain. The results will help us to prevent future outcomes of neurodegeneration by targeting this binding and aggregation process.

1. Introduction

The long-term post-infection complications of COVID-19 can be

associated with neurological symptoms of neurodegenerative dis-

eases. The major risk factors for the COVID-19 includes age, heart

disease, diabetes and hypertension [1]. Several studies suggested

that SARS-CoV-2 infection increases the risk for neurodegenerative

diseases [2e5]. SARS-CoV-2 invasion to the CNS and the noticeable

cytokine storm, metabolic changes, gut microbiome changes,

neuroendocrine axis, and hypoperfusion during COVID-19 infection

could be attributed to the different neurological distresses

observed in the nervous system [5e7]. It has been shown that

infection from human immunodeficiency virus (HIV), West Nile

virus, herpes simplex virus type 1 (HSV-1), H1N1 influenza A virus,

and respiratory syncytial virus causes several neurological mani-

festations, including encephalitis, protein aggregation, neuro-

degeneration, and Parkinson’s disease- or Alzheimer’s like

symptoms [8]. H1N1 infection to dopaminergic neurons expressing

a-synuclein resulted in aggregation of a-synuclein and inhibition of

autophagy, and thus increased the susceptibility of neuro-

degeneration [9].

Very recently, Tavassoly et al. proposed a view that seeded

protein aggregation by SARS-CoV-2 could be attributed to long-

term post-infection complications including neurodegeneration

[4]. They suggested that SARS-CoV-2 spike protein S1 region binds

to heparin and heparin binding proteins (HBPs) present in brain

which are prone to self-assembly, aggregation, and fibrillation

processes. They also showed that the peptide from S protein

(SeCoV-peptide; ~150 aa) has more aggregation formation pro-

pensity than the known aggregation-prone proteins, suggesting

that this peptide is prone to act as functional amyloid and form

toxic aggregates. Thus, the heparin binding and aggregation pro-

pensity of S1 protein has been suggested the ability of S1 to form

amyloid and toxic aggregates that can act as seeds to aggregate

many of the misfolded brain proteins and can ultimately leads to

neurodegeneration. It has been suggested that SARS-CoV-2 infec-

tion invades the CNS by controlling protein synthesis machinery,

disturbs endoplasmic reticulum and mitochondrial function and

increases the accumulation of misfolded proteins, thereby activates

protein aggregation, mitochondrial oxidative stress, apoptosis and

neurodegeneration [3,5,10].

Interestingly, it has been shown that HSV-1 spike protein binds

to heparin and increases the aggregation of amyloid b (Ab42)

peptides on its surface spikes [11].

This study suggests that the heparin-binding site of the spike protein might act as a binding site

for Ab42 peptides and thus could dock to the viral surface and

catalyze aggregation of Ab42. As the receptor binding domain

(RBD) of SARS-CoV-2, which is located within the S1 subunit of

spike glycoprotein has several heparin binding sites [12e14], the

same mechanism of aggregation of neurodegeneration causing

proteins such as Ab, a-synuclein, tau, prions, and TDP-43 can be

observed in COVID-19 infection in the brain.

In this study, we have investigated the interactions of SARS-

CoV-2 S1 RBD to different amyloid forming proteins including Ab,

a-synuclein, tau, prions, and TAR DNA binding protein-43 (TDP-43).

We also examine the binding of S1 RBD to heparin and their

complex to the different amyloidogenic proteins present in the

brain. The insights will help us in understanding the heparin

binding induced increase in association of HBPs observed in neu-

rodegeneration and also to prevent future outcomes of neuro-

degeneration by targeting this association process.

In summary, the findings reported here support the hypothesis

that the SARS-CoV-2 spike protein can interact with heparin

binding amyloid forming proteins. Our results indicate stable

binding of the S1 protein to these aggregation-prone proteins

which might initiates aggregation of brain protein and accelerate

neurodegeneration. These findings might explain the possible

neurological distresses associated with COVID-19. Therefore, tar-

geting the interaction of SARS-CoV-2 spike protein with the brain

proteins might be a suitable way to reduce the aggregation process

and thus neurodegeneration in COVID-19 patients.

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