Differentiating Parallel and Antiparallel DNA Duplexes in the Gas Phase.

[NNadir]

The paper I'll discuss in this post is this one: Differentiating Parallel and Antiparallel DNA Duplexes in the Gas Phase Using Trapped Ion Mobility Spectrometry (David Butcher, Prem Chapagain, Fenfei Leng, Francisco Fernandez-Lima,* J. Phys. Chem. B 2018 122 27 6855-6861)

When I was a kid, I had the silly notion that biological molecules featuring regular repeating units - peptides, proteins, polysaccharides, nucleic acids and more or less lipids were disinteresting - and to the extent that one could be interested in biological molecules, they had to be natural products with a ton of stereochemistry to confound efforts at total synthesis. In this context, I held the absurd opinion that the main interesting analytical tool was NMR and everything else was ancillary.

I was a stupid kid.

I was forced by circumstances to make myself aware of and knowledgeable about the "regular" biomolecules, which are actually far more exciting and interesting that I could have possibly imagined as a dumb kid.

I almost never think about NMR now; I know it's a powerful tool; and can reveal a lot, but as an old man the analytical tool that is the most amazing to me and the most important is chromatographically coupled mass spectrometry.

The advent of high resolution mass spec has changed the world and gave us one of the best tools to interrogate molecular biology.

Thus I found this paper extremely interesting when I stumbled across it recently in a journal in which I don't spend all that much time.

Some cool stuff about why DNA is decidedly not simple from the intro:

In vivo, DNA strands typically associate in an antiparallel fashion, forming a right-handed duplex with one strand running in the 5′ to 3′ direction, whereas the other strand runs in the 3′ to 5′ direction.1 Adjacent purine and pyrimidine bases on each strand form Watson−Crick base pairs which stabilize the duplex. Genomic DNA that is not undergoing transcription exists largely in this conformation, either in the nucleoid in prokaryotes2 or in the nucleus in eukaryotic cells where duplex DNA is complexed with histones.3 However, other tertiary and quaternary structural motifs can be formed depending on DNA sequence, solvent conditions, molecular crowding, and superhelical torsion. These structures include cruciforms,4 triplexes, G-quadruplexes,5 i-motifs,6 hairpins, and others. Many of these structural motifs have fundamental importance to biological processes in the cell, including transcription, replication, and DNA repair mechanisms,7 and dysfunction of these structural motifs and related protein binding partners is implicated in a wide variety of diseases. For example, the potential formation of the G-quadruplex and imotif by guanine-rich and cytosine-rich sequences in human telomereswhich are often highly extended by overexpression of telomerase in cancerous cells8and a large number of oncogenes9 has led to interest in these structures as drug targets.10

DNA sequences may also associate in a parallel fashion, resulting in a parallel duplex in which both strands run in the same direction. The parallel-stranded duplex is stabilized by the formation of reverse Watson−Crick A-T or G-C base pairs.11 Previous studies have established significant structural and spectroscopic differences between parallel- and antiparallel- stranded complexes.11,12 The formation of parallelstranded duplexes has been observed in vitro in sequences from the genome of Drosophila melanogaster,13 suggesting that parallel-stranded duplexes, like other atypical DNA structural motifs, may be relevant in vivo.

The structure of DNA structural motifs in the gas phase has been characterized using molecular dynamics simulations and various ion mobility spectrometry techniques.14−17 It has been demonstrated that soft ionization techniques such as nanoelectrospray ionization (nESI) can produce desolvated DNA molecular ions which retain a memory of their solution structure...15

...Here, native electrospray ionization combined with trapped ion mobility spectrometry (TIMS) and ultrahigh-resolution time-of-flight mass spectrometry (UHR-TOF-MS) was used for the first time to characterize the conformational space and oligomerization states of two parallel strand-forming oligonucleotides:

psDNA1 (5′-CCATAATTTACC-3′ ) and psDNA2 (5′-CCTATTAAATCC-3′ ). These oligomers have been confirmed to form a parallel-stranded duplex in acidic solution...

The mass spec here has an added dimension in ion mobility, which depends on the cross sectional area of a charged analytic fragment drifting in an electric field in a "drift tube."

(I've heard good things about the Bruker High Resolution Mass Specs used in this paper, even if I have very limited exposure to them, and never actually thought to have a representative in to discuss them.)

Some pictures from the paper:

Figure 1. Mobility profiles for monomers formed by psDNA1 and psDNA2. Negative mode mobility profiles are shown in black and positive mode mobility profiles are shown in red. Mass spectra are shown as insets.

Figure 2. Mobility profiles for dimers formed by psDNA1 (top), psDNA2 (middle), and a mixture of both oligonucleotides (bottom). Gaussian fits to mobility profiles for parallel- and antiparallel-stranded conformations are shown in blue and green, respectively. Mass spectra are shown as insets.

Figure 3. Scheme and candidate structures for psDNA1 and psDNA2 homo and hetero dimers in parallel and antiparallel configuration. Above, the phosphate–sugar backbones of psDNA1 and psDNA2 are shown as black and gray lines and base-pairing mismatches are denoted in red. Below, candidate structures are shown with cytosine, adenine, and thymine residues shown in blue, red and green respectively.

From data collected from the drift tube, one can calculate the cross sectional area of the molecule, that is the "electronic shadow" of the molecule and thus make inferences about its conformation (3D structure):

Reduced mobility values (K0) were correlated with collision cross section (Ω using the Mason−Schamp equation



where z is the charge of the ion, kB is the Boltzmann constant, N* is the number density of the bath gas, and mi and mb refer to the masses of the ion and bath gas, respectively [33]. TIMSMS spectra were analyzed using Compass Data Analysis 5.0 (Bruker Daltonik GmbH) and TIMS Data Viewer 1.4.0.31397 (Bruker Daltonik GmbH).

Some conclusions:

Mobility profiles for the monomers of psDNA1 and psDNA2 display a large number of individual conformations, showing that DNA monomers have great structural heterogeneity in the gas phase. We see two mobility bands for the +4 and −4 dimers formed by psDNA1, psDNA2, and in the mixture, indicating that there are two major conformations of the duplex in the gas phase. Complementary theoretical studies allow us to assign the bands at ∼850 and 900 Å^2 to the paralleland antiparallel-stranded structures, respectively.

We have shown that DNA structures undergo compaction upon transfer to the gas phase, resulting in observed Ω values significantly smaller than theoretical predictions based on solution-phase structures. There is also a significant difference in Ω for parallel- and antiparallel-stranded duplexes measured in positive mode and negative mode. Changes in the pattern of protonation as a result of the ionization process are likely responsible for the significantly smaller Ω values observed in negative mode as compared to positive mode (ΔΩ ≈ 100 Å^2).

Esoteric, but very cool. IMS makes it possible to derive conformational information from mass spectrometry.

Very cool...very cool.

Have a happy Friday tomorrow.