When ignorance is not bliss

Amazingly, it is not only possible but it is well understood; A actual phenomenon that is even expected to naturally exist and play a role in our own cells!

And to be honest, this was quite a surprise to me when I first learned of it.

This post starts with a good example that shows we should not easily succumb to the hubris of dismissing someone’s statements just because these they do not agree with our beliefs.

For example, when I first heard of a triple DNA helix, it was on some obscure website, and because up to that point in all my studies related to DNA I had not yet come across the concept, I just dismissed it as an absurd notion! Not only that, but there was also a feeling of disdain towards the authors of what clearly had to be a nonsense article!

Clearly, I owe them an apology for my premature emotional eruption.

Further, I have to wonder: Does a similar type of hubris-driven scorn happen within the establishment on a regular basis - when in reality even celebrated and proclaimed authorities can clearly be mistaken?

Importantly, how often does this take place in the world of medical experts?

It is a thought I find worth pondering, especially after my own humble, self-reflection.

Considering this, my new working hypothesis is that all communications could carry a message that is true to some degree. I like this concept because it forces one to train open-mindedness even when information is coming in that contradicts one’s own beliefs. After all, that is how a good scientist is supposed to approach any new information: without personal bias.

And hopefully I do not miss any new opportunities to learn about unusual DNA structures because of my biases!

In fact, a triple DNA helix had already been described in 1957, so it is nearly as old as the structure of the canonical double helix DNA (1953). It can be referred to as H-DNA, triple stranded DNA or triplex DNA.

How is triple DNA helix formed?

The canonical DNA double helix (also referred to as B-DNA) is made up of two strands of DNA where each strand is made of four different chemicals that are linked to one another and arranged in a specific order. These chemicals are G, T and C and A. These chemicals can also interact with each other via tiny magnet-like interactions (electromagnetic interactions), so that G especially likes to interact with C while T really likes to interact with A. So much so that the second strand of the DNA is the same sequence arranged to be able to interact with the primary strand of DNA. They are literally mirror copies of each other in reverse. These two strands wound around each other through these complementary interactions of seemingly randomly distributed G, T, C and A chemicals (referred to as bases, or nucleotides) to form the famous double helix. This is also why you can split the double helix DNA into separate strands and use each strand as a template to produce two new identical copies of DNA - a process used every time our cells divide.

The only point of that background though is really just to tell you that G and T (which are bulky in size) are referred to as purines whereas C and A (which are smaller in size) are referred as pyrimidines.

The reason why we needed that information is in order for a triple DNA helix to occur, we need the genetic code to be arranged to have a stretch of purines together (a bunch of Gs and Ts strung together along the strand of DNA which would mean that the complementary second strand of DNA would contain a stretch of pyrimidines, or Cs and As following each other in a stretch).

This is because of the chemical nature of purines: they have the possibility to still make electromagnetic interactions with the chemical bases of an incoming third strand even while they are already engaged in a double helix formation. Thus, the third strand is binding to the purine-rich strand of the DNA duplex.

And what is the incoming third strand made of?

It can be either bunch of purines put together (so G and T bases) or bunch of pyrimidines put together (Cs and As). Either one of these could come in and start binding to the purine rich strand of the double helix. The only aspect that will change is the orientation of the incoming third strand. You can think of it like this: if the incoming third strand is made of Gs and Ts, it will bind the double helix from left to right, and if it’s made of Cs and As, it will bind the double helix from right to left.

Now where that third strand from comes from, is either a new strand of genetic material from elsewhere, or this third strand could be part of the same double helix DNA, just downstream of the DNA sequence that will be targeted for binding with incoming third strand.

This is where sections of genetic code that are repeated come in.

A big chunk of our genomes is made up of repeating units of genetic code.

Now imagine that these repeated units are made of either a purine rich sequence or a pyrimidine rich sequence.

If you have a such a repeat sequence, then you could have a DNA sequence of one such repeat come undone as a DNA double helix, and rather exist as two DNA strands on their own instead of interacting with each other. One such a single DNA strand could then loop back and bind instead to the preceding repeat sequence that is still in the form of a double helix. In this way, you could produce a triple DNA helix. The best image of this is from one of the reviews we used to study the triple stranded DNA.

Adapted from Jain A et al. 2008. Biochimie 90(8):1117-30

Triple helix DNA effects

And voila, apparently this is totally possible in nature and potentially could play some important biological roles. In one obvious way, it clearly changes the organization of the DNA and its three-dimensional topography. This could influence how the DNA is used by proteins that bind to the DNA. This in turn could influence how genes are used (genes being the section of DNA that are used to produce mRNAs which in turn are used as genetic blueprints to produce proteins).

Another aspect of a triple DNA helix is that it would be expected to be highly mutagenic, potentially for a number of reasons. First, because as the triple DNA helix is formed, it means there is still an exposed single stranded DNA that is patiently waiting for its partner strand to come back from the threesome engagement. While it is waiting to reconnect to its partner, it is susceptible to being cut or broken. If such a break in this exposed single strand of DNA were to occur, then in the process of repair it could lead to inadequate repair that introduces a mutation, including, accidentally stitching the strand with a completely different region of DNA genetic material (this can be referred to as recombination or chromosomal translocation). And the single stranded DNA might wait because apparently while triple DNA helix is not as favourably formed as a normal double helix DNA, once it happens, it is very stable and does not come apart as easily.

Second, that abandoned single stranded DNA might get tired of waiting for its complementary genetic partner, and start wondering off for some action of its own. It could start invading other sequences of the DNA elsewhere, potentially also contributing to recombinatorial mutations (basically two regions of DNA being spliced together side by side that would not be expected to exist adjacent to each other normally).

And finally, presumably the triple DNA helix structure can be perceived as damage by the repair machinery of the cell and induce attempted repair mechanisms which could increase the likelihood of an accidental misuse of genetic information in the DNA repair process.

Why is this potentially important?

For a couple of reasons.

First, it turns out that naturally occurring sequences capable of forming a triple DNA helix are quite common in human genomes, with a frequency of ~1 in every 50,000 bp, usually in the regions of our genomes used for regulation of how genes are to be used.

The genes that contain long stretches of poly-purine and poly-pyrimidine sequences tend to exhibit an increased likelihood of mutations including those more unusual recombinations or translocations. Some areas of our genomes have “hot spots” for exhibiting mutations, and some of those “hot spot” regions are in or near DNA sequences that have the potential to form triple DNA helix structures. For example, a segment in the regulatory region of the human c-MYC gene that is capable of adopting a triple DNA helix also overlaps with the one of major mutation “hot spots” in that gene which is known to be involved in lymphomas and leukemias.

In addition, genes with a proposed propensity to form a triple DNA helix based on their DNA sequence, also tend to exhibit an increased level of different mRNAs produced (human genes have the capacity to produce multiple but slightly different mRNAs based on how different pieces of the RNA (produced by copying the DNA segments of a single gene) are spliced together). And finally, such genes appear to be used less often compared to other genes.

Second, sequences that are capable of forming a triple DNA helix structure are found in regulatory regions of genes more frequently than would be randomly expected. This would suggest that evolutionarily such a design has been conserved for a purpose. As we mentioned earlier, the altered three-dimensional topography of DNA due to the formation of triple DNA helix would be expected to alter how the DNA is being interacted with, and that could affect how genes are used to produce proteins. The scientific literature is quite abundant with examples of how such a presence of genetic information capable of stimulating a triple DNA helix can influence the use of genes - by either increasing or decreasing their use.

Have triple DNA helix structures been observed in nature?

Yes, they have, mainly inside cells. But whether these are really observed under normal living conditions in humans, that was the part I was not so certain of as there is no compelling evidence that can be easily found. These structures have been observed in cells, for example by using fluorescently labelled single stranded DNA that can bind to that abandoned single stranded DNA that is still waiting for its partner to come back from the triple DNA helix engagement; or by fluorescently labelled antibodies that can recognize the three-dimensional topography of the triple DNA helix. But these experiments did not exactly fit the parameters of physiological conditions. Thus, these might exist naturally, but we might have a very hard time catching their transient existence in action to prove they are really there.

That still does not mean we could not take advantage of it via pharmacological means. And this concept has been toyed with for a long time now! The idea here is seemingly simple: bring a single stranded DNA designed to form a triple DNA helix at a specific site to disrupt a specific gene function, or even to introduce mutations!

Such genetic, single DNA strands are cheekily named triplex-forming oligonucleotides (or TFOs where oligonucleotides are a string of chemical DNA or RNA bases put together). Their first use in the formation of a triple DNA helix was already demonstrated in the 80s, so they have been investigated for quite a long time now. They just have not yet made it into clinical use because of many complications associated with their use, such as stability, delivery to the site, and even the ability to properly target specific site of interest. Another issue is that the introduction of extra genetic material could start binding to other proteins, precluding their intended use inside the cells (btw, this is a concept we still have not investigated with regards to use of mRNA vaccines).

But the advantage of using the TFO approach is that they can be chemically manipulated to our heart’s content, and that is the hope that eventually we will figure out how to use them for clinical purposes, either to regulate gene use, or to introduce mutations (think of it in terms of cancer treatment, where introducing mutations in a caner cell would make such a cell incapable of surviving). In theory, almost any gene could be targeted because apparently almost every gene contains at least one unique TFO binding site (with a total of around 2 million such sites in the human genome altogether, so a lot to pick from).

One neat example of the use of TFOs was to target the c-MYCgene. TFOs were used to introduce DNA breakage and the subsequent DNA repair synthesis in the presence of an antitumor compound, gemcitabine. Gemcitabine, an approved anti-cancer drug, masquerades as a DNA chemical base, and can be incorporated into DNA during its synthesis, but it cannot be recognized by the DNA repair system properly, and it allows extra chemical DNA bases to be introduced, forcing mutations. In this way gemcitabine introduces irreparable errors that prevent proper DNA synthesis and thus leads to cell death. Basically, the compound is used to force cancer cell death. It was shown that introduction of c-MYC-specific TFOs into the mix, significantly increased the effectiveness of gemcitabine in preventing breast tumor cells growth.

This is the hope that scientists cling to when they contrive these wild ideas: that we can help treat diseases.

Whether that works out well or not sometimes may depend on the hubris of human arrogance, but again, here to we have to cling to hope too.

Remember, scientists are supposed to be trained to be very open minded to new and even contradictory concepts in order for new knowledge to take root.

This article has been produced by Merogenomics Inc. and edited by Jason Chouinard, B.Sc. Reproduction and reuse of any portion of this content requires Merogenomics Inc. permission and source acknowledgment. It is your responsibility to obtain additional permissions from the third party owners that might be cited by Merogenomics Inc. Merogenomics Inc. disclaims any responsibility for any use you make of content owned by third parties without their permission.

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