Nuclear magnetic resonance spectroscopy of nucleic acids

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Nucleic acid NMR is the use of nuclear magnetic resonance spectroscopy to obtain information about the structure and dynamics of nucleic acid molecules, such as DNA or RNA. It is useful for molecules of up to 100 nucleotides, and as of 2003, nearly half of all known RNA structures had been determined by NMR spectroscopy. [1]

Contents

NMR has advantages over X-ray crystallography, which is the other method for high-resolution nucleic acid structure determination, in that the molecules are being observed in their natural solution state rather than in a crystal lattice that may affect the molecule's structural properties. It is also possible to investigate dynamics with NMR. This comes at the cost of slightly less accurate and detailed structures than crystallography. [2]

Nucleic acid NMR uses techniques similar to those of protein NMR, but has several differences. Nucleic acids have a smaller percentage of hydrogen atoms, which are the atoms usually observed in NMR, and because nucleic acid double helices are stiff and roughly linear, they do not fold back on themselves to give "long-range" correlations. Nucleic acids also tend to have resonances distributed over a smaller range than proteins, making the spectra potentially more crowded and difficult to interpret. [3]

Experimental methods

Two-dimensional NMR methods are almost always used with nucleic acids. These include correlation spectroscopy (COSY) and total coherence transfer spectroscopy (TOCSY) to detect through-bond nuclear couplings, and nuclear Overhauser effect spectroscopy (NOESY) to detect couplings between nuclei that are close to each other in space. The types of NMR usually done with nucleic acids are 1H NMR, 13C NMR, 15N NMR, and 31P NMR. 19F NMR is also useful if nonnatural nucleotides such as 2'-fluoro-2'-deoxyadenosine are incorporated into the nucleic acid strand, as natural nucleic acids do not contain any fluorine atoms. [2] [4]

1H and 31P have near 100% natural abundance, while 13C and 15N have low natural abundances. For these latter two nuclei, there is the capability of isotopically enriching desired atoms within the molecules, either uniformly or in a site-specific manner. Nucleotides uniformly enriched in 13C and/or 15N can be obtained through biochemical methods, by performing polymerase chain reaction using dNTPs or NTPs derived from bacteria grown in an isotopically enriched environment. Site-specific isotope enrichment must be done through chemical synthesis of the labeled nucleoside phosphoramidite monomer and of the full strand; however these are difficult and expensive to synthesize. [1] [5]

Because nucleic acids have a relatively large number of protons which are solvent-exchangeable, nucleic acid NMR is generally not done in D2O solvent as is common with other types of NMR. This is because the deuterium in the solvent would replace the exchangeable protons and extinguish their signal. H2O is used as a solvent, and other methods are used to eliminate the strong solvent signal, such as saturating the solvent signal before the normal pulse sequence ("presaturation"), which works best a low temperature to prevent exchange of the saturated solvent protons with the nucleic acid protons; or exciting only resonances of interest ("selective excitation"), which has the additional, potentially undesired effect of distorting the peak amplitudes. [2]

Structure determination

The exchangeable and non-exchangeable protons are usually assigned to their specific peaks as two independent groups. For exchangeable protons, which are for the most part the protons involved in base pairing, NOESY can be used to find through-space correlations between on neighboring bases, allowing an entire duplex molecule to be assigned through sequential walking. For nonexchangable protons, many of which are on the sugar moiety of the nucleic acid, COSY and TOCSY are used to identify systems of coupled nuclei, while NOESY is again used to correlate the sugar to the base and each base to its neighboring base. For duplex DNA nonexchangeable protons the H6/H8 protons on the base correlate to their counterparts on neighboring bases and to the H1' proton on the sugar, allowing sequential walking to be done. For RNA, the differences in chemical structure and helix geometry make this assignment more technically difficult, but still possible. The sequential walking methodology is not possible for non-double helical nucleic acid structures, nor for the Z-DNA form, making assignment of resonances more difficult. [2] [3]

Parameters taken from the spectrum, mainly NOESY cross-peaks and coupling constants, can be used to determine local structural features such as glycosidic bond angles, dihedral angles (using the Karplus equation), and sugar pucker conformations. The presence or absence of imino proton resonances, or of coupling between 15N atoms across a hydrogen bond, indicates the presence or absence of basepairing. For large-scale structure, these local parameters must be supplemented with other structural assumptions or models, because errors add up as the double helix is traversed, and unlike with proteins, the double helix does not have a compact interior and does not fold back upon itself. However, long-range orientation information can be obtained through residual dipolar coupling experiments in a medium which imposes a weak alignment on the nucleic acid molecules. [1] [2]

Recently, solid-state NMR methodology has been introduced for the structure determination of nucleic acids. [6] The protocol implies two approaches: nucleotide-type selective labeling of RNA and usage of heteronuclear correlation experiments.

NMR is also useful for investigating nonstandard geometries such as bent helices, non-Watson–Crick basepairing, and coaxial stacking. It has been especially useful in probing the structure of natural RNA oligonucleotides, which tend to adopt complex conformations such as stem-loops and pseudoknots. Interactions between RNA and metal ions can be probed by a number of methods, including observing changes in chemical shift upon ion binding, observing line broadening for paramagnetic ion species, and observing intermolecular NOE contacts for organometallic mimics of the metal ions. NMR is also useful for probing the binding of nucleic acid molecules to other molecules, such as proteins or drugs. This can be done by chemical-shift mapping, which is seeing which resonances are shifted upon binding of the other molecule, or by cross-saturation experiments where one of the binding molecules is selectively saturated and, if bound, the saturation transfers to the other molecule in the complex. [1] [2]

Dynamic properties such as duplex–single strand equilibria and binding rates of other molecules to duplexes can also be determined by its effect on the spin–lattice relaxation time T1, but these methods are insensitive to intermediate rates of 104–108 s−1, which must be investigated with other methods such as solid-state NMR. Dynamics of mechanical properties of a nucleic acid double helix such as bending and twisting can also be studied using NMR. Pulsed field gradient NMR experiments can be used to measure diffusion constants. [1] [2] [7]

History

Nucleic acid NMR studies were performed as early as 1971, [8] and focused on using the low-field imino proton resonances to probe base pairing interactions. These early studies focussed on tRNA because these nucleic acids were the only samples available at that time with low enough molecular weight that the NMR spectral line-widths were practical. The study focussed on the low-field protons because they were the only protons that could be reliably observed in aqueous solution using the best spectrometers available at that time. It was quickly realized that spectra of the low-field imino protons were providing clues to the tertiary structure of tRNA in solution. The first NMR spectrum of a double-helical DNA was published in 1977 [9] using a synthetic, 30-base-pair double helix. To overcome sever line-broadening in native DNA, sheer-degraded natural DNA was prepared and studied to learn about the persistence length of double-helical DNA. [10] At the same time, nucleosome core particles were studied to gain further insight of the flexibility of the double helix. [11] The first NMR spectra reported for a uniform low molecular weight native-sequence DNA, made with restriction enzymes, was reported 1981. [12] This work was also the first report of nucleic acid NMR spectra obtained at high field. Two dimensional NMR studies began to be reported in 1982 [13] and then, with the advent of oligonucleotide synthesis and more sophisticated instrumentation, many detailed structural studies were reported starting in 1983. [14]

Related Research Articles

The nuclear Overhauser effect (NOE) is the transfer of nuclear spin polarization from one population of spin-active nuclei to another via cross-relaxation. A phenomenological definition of the NOE in nuclear magnetic resonance spectroscopy (NMR) is the change in the integrated intensity of one NMR resonance that occurs when another is saturated by irradiation with an RF field. The change in resonance intensity of a nucleus is a consequence of the nucleus being close in space to those directly affected by the RF perturbation.

In nuclear magnetic resonance (NMR) spectroscopy, the chemical shift is the resonant frequency of an atomic nucleus relative to a standard in a magnetic field. Often the position and number of chemical shifts are diagnostic of the structure of a molecule. Chemical shifts are also used to describe signals in other forms of spectroscopy such as photoemission spectroscopy.

CIDNP, often pronounced like "kidnip", is a nuclear magnetic resonance (NMR) technique that is used to study chemical reactions that involve radicals. It detects the non-Boltzmann (non-thermal) nuclear spin state distribution produced in these reactions as enhanced absorption or emission signals.

<span class="mw-page-title-main">Nuclear magnetic resonance spectroscopy</span> Laboratory technique

Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy or magnetic resonance spectroscopy (MRS), is a spectroscopic technique to observe local magnetic fields around atomic nuclei. This spectroscopy is based on the measurement of absorption of electromagnetic radiations in the radio frequency region from roughly 4 to 900 MHz. Absorption of radio waves in the presence of magnetic field is accompanied by a special type of nuclear transition, and for this reason, such type of spectroscopy is known as Nuclear Magnetic Resonance Spectroscopy. The sample is placed in a magnetic field and the NMR signal is produced by excitation of the nuclei sample with radio waves into nuclear magnetic resonance, which is detected with sensitive radio receivers. The intramolecular magnetic field around an atom in a molecule changes the resonance frequency, thus giving access to details of the electronic structure of a molecule and its individual functional groups. As the fields are unique or highly characteristic to individual compounds, in modern organic chemistry practice, NMR spectroscopy is the definitive method to identify monomolecular organic compounds.

Carbon-13 (C13) nuclear magnetic resonance is the application of nuclear magnetic resonance (NMR) spectroscopy to carbon. It is analogous to proton NMR and allows the identification of carbon atoms in an organic molecule just as proton NMR identifies hydrogen atoms. 13C NMR detects only the 13
C
 isotope. The main carbon isotope, 12
C
 is not detected. Although much less sensitive than 1H NMR spectroscopy, 13C NMR spectroscopy is widely used for characterizing organic and organometallic compounds.

<span class="mw-page-title-main">Proton nuclear magnetic resonance</span> NMR via protons, hydrogen-1 nuclei

Proton nuclear magnetic resonance is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the structure of its molecules. In samples where natural hydrogen (H) is used, practically all the hydrogen consists of the isotope 1H.

Nuclear magnetic resonance spectroscopy of proteins is a field of structural biology in which NMR spectroscopy is used to obtain information about the structure and dynamics of proteins, and also nucleic acids, and their complexes. The field was pioneered by Richard R. Ernst and Kurt Wüthrich at the ETH, and by Ad Bax, Marius Clore, Angela Gronenborn at the NIH, and Gerhard Wagner at Harvard University, among others. Structure determination by NMR spectroscopy usually consists of several phases, each using a separate set of highly specialized techniques. The sample is prepared, measurements are made, interpretive approaches are applied, and a structure is calculated and validated.

The heteronuclear single quantum coherence or heteronuclear single quantum correlation experiment, normally abbreviated as HSQC, is used frequently in NMR spectroscopy of organic molecules and is of particular significance in the field of protein NMR. The experiment was first described by Geoffrey Bodenhausen and D. J. Ruben in 1980. The resulting spectrum is two-dimensional (2D) with one axis for proton (1H) and the other for a heteronucleus, which is usually 13C or 15N. The spectrum contains a peak for each unique proton attached to the heteronucleus being considered. The 2D HSQC can also be combined with other experiments in higher-dimensional NMR experiments, such as NOESY-HSQC or TOCSY-HSQC.

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<span class="mw-page-title-main">Residual dipolar coupling</span>

The residual dipolar coupling between two spins in a molecule occurs if the molecules in solution exhibit a partial alignment leading to an incomplete averaging of spatially anisotropic dipolar couplings.

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Experimental approaches of determining the structure of nucleic acids, such as RNA and DNA, can be largely classified into biophysical and biochemical methods. Biophysical methods use the fundamental physical properties of molecules for structure determination, including X-ray crystallography, NMR and cryo-EM. Biochemical methods exploit the chemical properties of nucleic acids using specific reagents and conditions to assay the structure of nucleic acids. Such methods may involve chemical probing with specific reagents, or rely on native or analogue chemistry. Different experimental approaches have unique merits and are suitable for different experimental purposes.

Carbohydrate NMR spectroscopy is the application of nuclear magnetic resonance (NMR) spectroscopy to structural and conformational analysis of carbohydrates. This method allows the scientists to elucidate structure of monosaccharides, oligosaccharides, polysaccharides, glycoconjugates and other carbohydrate derivatives from synthetic and natural sources. Among structural properties that could be determined by NMR are primary structure, saccharide conformation, stoichiometry of substituents, and ratio of individual saccharides in a mixture. Modern high field NMR instruments used for carbohydrate samples, typically 500 MHz or higher, are able to run a suite of 1D, 2D, and 3D experiments to determine a structure of carbohydrate compounds.

Nuclear magnetic resonance decoupling is a special method used in nuclear magnetic resonance (NMR) spectroscopy where a sample to be analyzed is irradiated at a certain frequency or frequency range to eliminate fully or partially the effect of coupling between certain nuclei. NMR coupling refers to the effect of nuclei on each other in atoms within a couple of bonds distance of each other in molecules. This effect causes NMR signals in a spectrum to be split into multiple peaks. Decoupling fully or partially eliminates splitting of the signal between the nuclei irradiated and other nuclei such as the nuclei being analyzed in a certain spectrum. NMR spectroscopy and sometimes decoupling can help determine structures of chemical compounds.

<span class="mw-page-title-main">Nuclear magnetic resonance</span> Spectroscopic technique based on change of nuclear spin state

Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field and respond by producing an electromagnetic signal with a frequency characteristic of the magnetic field at the nucleus. This process occurs near resonance, when the oscillation frequency matches the intrinsic frequency of the nuclei, which depends on the strength of the static magnetic field, the chemical environment, and the magnetic properties of the isotope involved; in practical applications with static magnetic fields up to ca. 20 tesla, the frequency is similar to VHF and UHF television broadcasts (60–1000 MHz). NMR results from specific magnetic properties of certain atomic nuclei. Nuclear magnetic resonance spectroscopy is widely used to determine the structure of organic molecules in solution and study molecular physics and crystals as well as non-crystalline materials. NMR is also routinely used in advanced medical imaging techniques, such as in magnetic resonance imaging (MRI).

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<span class="mw-page-title-main">Chemical shift index</span> Laboratory technique

The chemical shift index or CSI is a widely employed technique in protein nuclear magnetic resonance spectroscopy that can be used to display and identify the location as well as the type of protein secondary structure found in proteins using only backbone chemical shift data The technique was invented by David S. Wishart in 1992 for analyzing 1Hα chemical shifts and then later extended by him in 1994 to incorporate 13C backbone shifts. The original CSI method makes use of the fact that 1Hα chemical shifts of amino acid residues in helices tends to be shifted upfield relative to their random coil values and downfield in beta strands. Similar kinds of upfield and downfield trends are also detectable in backbone 13C chemical shifts.

Nitrogen-15 nuclear magnetic resonance spectroscopy is a version of nuclear magnetic resonance spectroscopy that examines samples containing the 15N nucleus. 15N NMR differs in several ways from the more common 13C and 1H NMR. To circumvent the difficulties associated with measurement of the quadrupolar, spin-1 14N nuclide, 15N NMR is employed in samples for detection since it has a ground-state spin of ½. Since14N is 99.64% abundant, incorporation of 15N into samples often requires novel synthetic techniques.

References

  1. 1 2 3 4 5 Fürtig, Boris; Richter, Christian; Wöhnert, Jens; Schwalbe, Harald (2003). "NMR Spectroscopy of RNA". ChemBioChem. 4 (10): 936–962. doi:10.1002/cbic.200300700. PMID   14523911. S2CID   33523981.
  2. 1 2 3 4 5 6 7 Wemmer, David (2000). "Chapter 5: Structure and Dynamics by NMR". In Bloomfield, Victor A.; Crothers, Donald M.; Tinoco, Ignacio (eds.). Nucleic acids: Structures, Properties, and Functions. Sausalito, California: University Science Books. ISBN   0-935702-49-0.
  3. 1 2 Addess, Kenneth J.; Feigon, Juli (1996). "Introduction to 1H NMR Spectroscopy of DNA". In Hecht, Sidney M. (ed.). Bioorganic Chemistry: Nucleic Acids. New York: Oxford University Press. ISBN   0-19-508467-5.
  4. ↑ Kan, Lou-sing; Ts'o, Paul O. P. (1986). "Nuclear Magnetic Resonance Studies of Nucleic Acids". In Chien, Shu; Ho, Chien (eds.). NMR In Biology and Medicine . New York: Raven Press. ISBN   0-88167-231-9.
  5. ↑ Kojima, C; Ono, A; Ono, A; Kainosho, M (2002). Solid-phase synthesis of selectively labeled DNA: Applications for multidimensional nuclear magnetic resonance spectroscopy. Methods in Enzymology. Vol. 338. pp. 261–283. doi:10.1016/S0076-6879(02)38224-7. ISBN   9780121822392. PMID   11460552.
  6. ↑ Marchanka, Alexander; Simon, Bernd; Althoff-Ospelt, Gerhard; Carlomagno, Teresa (2015). "RNA structure determination by solid-state NMR spectroscopy". Nature Communications. 6: 7024. Bibcode:2015NatCo...6.7024M. doi:10.1038/ncomms8024. PMC   4432599 . PMID   25960310.
  7. ↑ Robinson, B.H.; Drobny, G.P. (1995). "[19] Site-specific dynamics in DNA: Theory and experiment". Nuclear Magnetic Resonance and Nucleic Acids. Methods in Enzymology. Vol. 261. pp. 451–509. doi:10.1016/S0076-6879(95)61021-9. ISBN   978-0-12-182162-3. ISSN   0076-6879. PMID   8569508.
  8. ↑ Kearns, David; Patel, Dinshaw; Shulman, Robert (1971). "High Resolution Nuclear Magnetic Resonance Studies of Hydrogen Bonded Protons of tRNA in Water". Nature. 229 (5283): 338–339. Bibcode:1971Natur.229..338K. doi:10.1038/229338a0. PMID   4927207. S2CID   4290896.
  9. ↑ Early, Thomas; Kearns, David; Burd, John; Larson, Jacquelynn; Wells, Robert (1977). "High resolution proton nuclear magnetic resonance investigation of the structural and dynamic properties of d(C15A15)·d(T15G15)". Biochemistry. 16 (3): 541–551. doi:10.1021/bi00622a031. PMID   836800.
  10. ↑ Early, Thomas; Kearns, David (1979). "1H nuclear magnetic resonance investigation of flexibility in DNA". PNAS. 76 (9): 4165–4169. Bibcode:1979PNAS...76.4165E. doi: 10.1073/pnas.76.9.4165 . PMC   411531 . PMID   291958.
  11. ↑ Feigon, Juli; Kearns, David (1979). "1H NMR investigation of the conformational states of DNA in nucleosome core particles". Nucleic Acids Research. 6 (6): 2327–2337. doi:10.1093/nar/6.6.2327. PMC   327853 . PMID   461191.
  12. ↑ Early, Thomas; Kearns, David; Hillen, Wolfgang; Wells, Robert (1980). "A 300 MHz and 600 MHz proton NMR study of a 12 base pair restriction fragment: investigation of structure by relaxation measurements". Nucleic Acids Research. 8 (23): 5795–5812. doi:10.1093/nar/8.23.5795. PMC   324342 . PMID   6258152.
  13. ↑ Feigon, Juli; Wright, John; Leupin, Werner; Denny, WA; Kearns, David (1982). "Use of two-dimensional NMR in the study of a double-stranded DNA". J. Am. Chem. Soc. 104 (20): 5540–5541. doi:10.1021/ja00384a069.
  14. ↑ Address, Kenneth; Feigon, Juli (1996). "Introduction to 1H NMR Spectroscopy of DNA". In Hecht, Sidney (ed.). Bioorganic Chemistry: Nucleic Acids. New York: Oxford University Press. ISBN   0-19-508467-5.
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