1. Nuclear Magnetic Resonance (NMR) Spectroscopy
NMR structural study of a DNA duplex containing two unnatural base pairs
Roisin O’Brien, Weston Preising, Tammy J. Dwyer
Department of Chemistry and Biochemistry, University of San Diego, San Diego, CA 92110
Current Work
Abstract
DNA is the genetic blueprint responsible for the traits of living organisms. The "genetic alphabet" consists of four molecules called nucleobases, represented by the letters A, T, G, and C strung together in a "strand.” A DNA duplex consists of two "strands" held together such that adenine (A) is across from thymine (T), and guanine (G) across from cytosine (C), referred to A-T and G-C "base pairs". Recently, the genetic alphabet or code has been expanded by synthetic biologists in an effort to unveil new insights to the machinery of DNA replication, and potentially develop new medicines to cure diseases like HIV. The new unnatural base pair 5SICS-NaM is the first to be stably replicated by a semi-synthetic E. Coli. organism. This project investigates a novel DNA duplex containing two 5SICS-NaM pairs arranged side-by-side and represents the first example of a stable duplex containing more than one unnatural pair, using a technique called NMR spectroscopy. Thus far, NMR chemical shift assignments for this duplex have been largely completed which provide some information as to the structural configuration of the base pairs. Preliminary results indicate that one of the two adjacent 5SICS-NaM pairs is situated within the duplex, whereas the other has may have the 5SICS base sterically pushed out of the duplex with its paired 5NaM base stacked quite normally. Further analysis of the data should allow us to propose a more precise structure of the DNA duplex as well as provide insight into the versatility of DNA replication machinery to recognize altered DNA structures.
Presently underway is the structure determination of a duplex containing two adjacent 5-SICS:NaM pairs in the self-complimentary sequence:
Why study DNA?
d5SICS
dNaM
2D NOESY spectra provide chemical shift assignments, proton-proton through space interactions including inter-proton distances within 5 Å.
2D COSY spectra provide proton-proton scalar coupling interactions, torsion angle and deoxyribose ring conformational information.
Nuclear Magnetic Resonance (NMR) Spectroscopy
A H8
A H1’
DNA is organized into genes which are organized into chromosomes. Humans have 23 pairs of chromosomes which are compartmentalized into short stretches of DNA called genes. Genes contain three nucleotide segments (codons) of the four natural nucleotides.
The Central Dogma of Molecular Biology. Genetic information flows from DNA to RNA (transcription) and then from RNA to proteins (translation).
Watson and Crick DNA Structure and Base Pairing
Current 2D NMR Results
Double helical structure of DNA. Structure formation formed from nucleotide base pairs on opposite strands that are attached to a deoxyribose sugar-phosphate backbone. The four natural nucleotides of DNA are adenine (A), thymine (T), guanine (G) and cytosine (C). Sequences of these natural bases store the coding information required for creating and maintaining an organism.
Natural Watson-Crick single DNA strand and base pairing. (A) A depiction of a single DNA strand (5’ to 3’) with all four natural bases: adenine, guanine, cytosine and thymine; sugar, is ribose; (B) Watson and Crick base pairing between purines (G & A) and pyrimidines (C & T)
Expanding the Genetic Alphabet
Fingerprint Region. Nucleoside aromatic protons to deoxyribose H1’ of the NOESY spectrum at 25°C. The aromatic proton for G and A is H8, whereas the aromatic proton for C and T is H6. Moving from G1 to G8 and G9 to C12 sequentially via cross peak alignment denotes the “walk” for this region. The gap left by the unnatural d5SICS and dNaM bases suggests a perturbed duplex structure.
d5SICS Walk. Intramolecular aromatic protons of d5SICS in the NOESY spectrum at 25°C (atom labels are given above). The sequential cross peaks from HA to HE denote the ‘walk” for d5SICS. The presence of a methyl group between HB and HC helps orient the assignment of this single, unique d5SICS moiety.
dNaM Walk. Intramolecular aromatic protons of dNaM in the NOESY spectrum at 25°C (atom labels are given above). The sequential cross peaks from HF to HK denote the ‘walk” for dNaM. The presence of a methoxy (OCH3) group adjacent to HK helps orient the assignment of this single, unique dNam moiety.
Future Directions
From NMR spectra, we can tell that the sequences preceding and following the 5SICS-NaM pairs are B-form and symmetric. However, future work involves distinguishing the chemical shift assignments of the undetermined d5SICS and dNaM bases to provide insight on the overall structure. A number of possible confirmations of the two unnatural base pairs could be occurring (as demonstrated below) within the duplex making this particular region unclear. We hope to finalize our investigation of the 2D NMR experimental data in order to begin structural calculations in an effort to eventually produce a final validated structure of the duplex.
Expansion of the Genetic Alphabet. Natural DNA has only four letters (nucleotides) that can make 64 words (codons) but only 20 words (amino acids) with distinct meanings. Adding two unnatural nucleobases (X & Y or d5SICS & dNaM) can make 216 words (codons) with the possibility of 172 distinct meanings (amino acids). The incorporation of novel amino acids can lead to a myriad of new, unique proteins.
The structure of a d5SICS-dNaM pairing. A comparison of d5SICS- dNaM and pyrimidine-pyrimidine bonding via dC-dG. As can be seen, the d5SICS-dNaM has no hydrogen bonding between nucleosides and is completely stabilized by hydrophobic interactions. or or
Our Approach
In 2009, Dr. Floyd Romesberg and his team at Scripps Research Institute designed and synthesized 60 predominantly hydrophobic molecules as potential new DNA bases. The 3600 resulting “base pair” potentials were then screened based on their greatest ability to be incorporated into DNA, replicated and efficiently transcribed with high fidelity. One of the most promising candidates found were the base pairs d5SICS-dNaM, which bond by specific, calibrated hydrophobic interactions. This unnatural base pair demonstrated the accuracy and efficiency of transcription present in natural DNA. In 2014, Romesberg and his team found that a natural DNA duplex, incorporated with a single d5SICS-dNaM base pairing, could be endogenously replicated in E. Coli with an equally high fidelity. This work resulted in the creation of the world’s first semi-synthetic organism. However, the structural determinations of the DNA duplex with d5SICS-dNaM was unknown. In the Dwyer lab at USD, we hope to use 2D NMR techniques to characterize the structure of a natural duplex with two adjacent d5SICS-dNaM pairs incorporated in an attempt to unveil new structure aspects of these unnatural base pairs, d5SICS-dNam, alongside natural nucleotides.
Acknowledgements
USD Faculty Research Grant
NSF MRI grant (NMR)
Alpha DNA