November
6, 2015 -- University
of Utah
“We
are one step closer to understanding the underlying chemistry that leads to
genetic diseases,” says Cynthia Burrows, distinguished professor and chair of
chemistry at the university and senior author of a new study published Nov. 6
in the online journal Nature Communications. “We have a way of marking
and copying DNA damage sites so that we can preserve the information of where
and what the damage was.”
Jan
Riedl, a University of Utah postdoctoral fellow and the study’s first author,
says 99 percent of DNA lesions – damage to the chemical bases known as A, C, G
and T that help form the DNA double helix – are repaired naturally. The rest
can lead to genetic mutations, which are errors in the sequence of bases and
can cause disease. The new method can “identify and detect the position of
lesions that lead to diseases,” he says.
Burrows
says: “We are trying to look for the chemical changes in the base that can lead
the cell to make a mistake, a mutation. One of the powerful things about our
method is we can read more than a single damaged site [and up to dozens] on the
same strand of DNA.”
The
chemists say their new method will let researchers study chemical details of
DNA lesions or damage. Such lesions, if not repaired naturally, accumulate over
time and can lead to mutations responsible for many age-related diseases,
including colon, breast, liver, lung and melanoma skin cancers; clogged
arteries; and neurological ailments such as Huntington’s disease and Lou
Gehrig’s disease.
“A
method capable of identifying the chemical identity and location in which
lesions appear is crucial for determining the molecular etiology [cause] of
these diseases,” Burrows and colleague write in their study.
The
research was funded by the National Institutes of Health. Burrows and Riedl
conducted the study with University
of Utah
Method detects DNA damage that can lead to disease
DNA is
a molecule, shaped like a double-stranded helix that contains the genetic
instructions that living organisms use to develop, function and reproduce. Each
strand is made of numerous copies of four chemical bases – A, C, G and T –
linked by a backbone of sugar and phosphate. When two strands are connected in
a double helix, only two kinds of base pairs can form C-G (or G-C) and A-T (or
T-A).
The
new method for finding DNA lesions combines other, existing techniques.
First,
the researchers find the damage and cut it out of the DNA the same way a cell
does naturally, using what is called “base excision repair,” the discovery of
which won a Nobel Prize in Chemistry this year for Tomas Lindahl, a scientist
in England.
Second,
an “unnatural base pair” is inserted at the snipped-out DNA damage site to
label it. Instead of natural base pairs C-G and A-T, the Utah
chemists used a so-called third or unnatural base pair invented by chemists at
the Scripps Research Institute in California
This graphic
shows a new method, developed at the University
of Utah
Third,
the DNA with the damage site labeled by an unnatural third base pair is then
amplified or copied millions of times using a well-known existing method called
PCR, or polymerase chain reaction. Burrows says the new study’s key innovation
was to use base excision repair to snip out the damage and then to insert the
unnatural base pair at the damage site, making it possible to make millions of
copies of the DNA – a process that normally would be prevented by the damage.
Fourth,
another chemical label, named 18-crown-6 ether, is affixed to the unnatural
base pair on all the DNA strands, which are then read or sequenced using a kind
of nanopore sequencing developed a few years ago by Burrows and Utah
People
are born with their genome or genetic blueprint of 3 billion base pairs, “and
then stuff happens,” Burrows says. “There’s damage from oxidative stress due to
inflammation and infection, too much metabolism, or environmental chemicals.”
The
new method seeks “molecular details that define how our genome responds to what
we eat and the air we breathe, and ends up being healthy or not,” she says.
DNA
lesions happen more than 10,000 times a day in a single human cell. A lesion
can be a missing base, a base that has changed chemically or a break in the DNA
backbone. That many lesions may seem like a lot, but with 3 billion base pairs
in the genome of a single cell, the damage only affects about one of every
300,000 base pairs.
Details
of the study
The
chemists tested their method on a gene named KRAS that, when mutated,
can cause lung or breast cancer.
Burrows
says most DNA sequencing methods reveal mutations because the methods read the
bases A, C, G and T. When sequencing reveals one of those bases out of place,
that is a mutation. “However, what you don’t know is what chemistry – what
modification – caused that mutation,” she says,
But
scientists can’t find a single strand of DNA in a solution in a reasonable
time. They need to make millions to billions of copies of the DNA so they can
sequence it and locate the gap where the damage was. But the damage itself is
“a train wreck” that either prevents making copies of the DNA or makes copies
with errors, Burrows says.
So
once the damaged base has been excised to create a gap, the chemists insert the
third or unnatural base pair at then damage site as a way to label it while at
the same time allowing millions of copies of the labeled DNA to be made.
Burrows
says the key innovation in the new method is using base excision repair and
unnatural base pairs “to copy the DNA and retain the information about damage
that was in the original molecule.”
The
chemists next use PCR amplification to make millions of copies of the DNA by
heating it until strands in the double helix separate. The strands are put in a
solution with lots of A, C, G and T nucleotides – the bases attached to pieces
of DNA backbone. A polymerase enzyme is attached to the end of each strand of
DNA, and then moves along the strand grabbing T, G, C and A nucleotides to make
a second strand. Each DNA strand quickly becomes two. The number of strands
reaches the millions in hours.
“We
need that many because, if we just have one molecule, we can’t find it,”
Burrows says. “It’s like saying, ‘There’s one fish in that pond; try to catch it.’
But if there are a bunch of identical fish, you can catch any one of them and
still have dinner.”
Once
the chemists have millions of DNA strands with the damage labeled by an
unnatural base pair, they then use nanopore sequencing to locate the damage. Burrows
says to imagine the DNA strand is a long piece of yarn with a number of knots
tied in it to represent labeled DNA lesions. Grab one end of the yarn with one
hand, and pull it through a couple of fingers on the other hand. The fingers
represent the nanopore that detects the knots or damage as they pass between
the fingers.
To
do nanopore sequencing, the DNA’s damage sites, which are labeled with
unnatural bases, are labeled again with a chemical named 18-crown-6 ether that
basically makes the “knots” on the DNA “yarn” bigger and easier to find as the
DNA strand moves through a pore-shaped protein bathed in electrically charged
fluid. Changes in electric current allow chemists detect the bases A, C, G and
T and the labeled unnatural bases.
Finding
the labeled unnatural bases identifies the DNA damage site to within 10 base
pairs. Burrows says the nanopore sequencing method needs improvement or
replacement by next-generation sequencing to actually pinpoint the damage
sites.
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