Structure probing of nucleic acid

Nucleic acid

Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...

s is the process by which biochemical techniques are used to determine nucleic acid structure

Nucleic acid structure

Nucleic acid structure refers to the structure of nucleic acids such as DNA and RNA It is often divided into four different levels:* Primary structure—the raw sequence of nucleobases of each of the component DNA strands;...

. This analysis can be used to define the patterns which can infer the molecular structure, experimental analysis of molecular structure and function, and further understanding on development of smaller molecules for further biological research. Structure probing analysis can be done through many different methods, which include chemical probing, hydroxyl radical probing, SHAPE, nucleotide analog interference mapping (NAIM), and in-line probing.

Nuclear magnetic resonance spectroscopy

Nucleic acid NMR is the use of NMR spectroscopy to obtain information about the structure and dynamics of nucleic acid

Nucleic acid

Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...

 molecules, such as DNA

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

 or RNA

RNA

Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....

. As of 2003, nearly half of all known RNA structures had been determined by NMR spectroscopy.

Nucleic acid NMR uses similar techniques as 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

Nucleic acid double helix

In molecular biology, the term double helix refers to the structure formed by double-stranded molecules of nucleic acids such as DNA and RNA. The double helical structure of a nucleic acid complex arises as a consequence of its secondary structure, and is a fundamental component in determining its...

 are stiff and roughly linear, they do not fod back on themselves to give "long-range" correlations. The types of NMR usually done with nucleic acids are ¹H or proton NMR

Proton NMR

Proton NMR 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 is used, practically all of the hydrogen consists of the...

, ¹³C NMR

Carbon-13 NMR

Carbon-13 NMR is the application of nuclear magnetic resonance 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...

, ¹⁵N NMR, and ³¹P NMR

Phosphorus-31 NMR

Phosphorus-31 NMR spectroscopy is an analytical technique. Solution 31P-NMR is one of the more routine NMR techniques because 31P has an isotopic abundance of 100% and a relatively high magnetogyric ratio. The 31P nucleus also has a spin of ½, making spectra relatively easy to interpret...

. Two-dimensional NMR methods are almost always used, such as correlation spectroscopy (COSY) and total coherence transfer spectroscopy (TOCSY) to detect through-bond nuclear couplings, and nuclear Overhauser effect

Nuclear Overhauser effect

The Nuclear Overhauser Effect is the transfer of nuclear spin polarization from one nuclear spin population to another via cross-relaxation. It is a common phenomenon observed by nuclear magnetic resonance spectroscopy. The theoretical basis for the NOE was described and experimentally verified...

 spectroscopy (NOESY) to detect couplings between nuclei that are close to each other in space.

Parameters taken from the spectrum, mainly NOESY cross-peaks and coupling constants

J-coupling

J-coupling is the coupling between two nuclear spins due to the influence of bonding electrons on the magnetic field running between the two nuclei. J-coupling contains information about dihedral angles, which can be estimated using the Karplus equation...

, can be used to determine local structural features such as glycosidic bond

Glycosidic bond

In chemistry, a glycosidic bond is a type of covalent bond that joins a carbohydrate molecule to another group, which may or may not be another carbohydrate....

 angles, dihedral angle

Dihedral angle

In geometry, a dihedral or torsion angle is the angle between two planes.The dihedral angle of two planes can be seen by looking at the planes "edge on", i.e., along their line of intersection...

s (using the Karplus equation

Karplus equation

The Karplus equation, named after Martin Karplus, describes the correlation between 3J-coupling constants and dihedral torsion angles in nuclear magnetic resonance spectroscopy:J = A \cos^2 \phi + B \cos\,\phi + C...

), and sugar pucker conformations. 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. 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 oligonuceltides, which tend to adopt complex confromations such as stem-loop

Stem-loop

Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions,...

s and pseudoknot

Pseudoknot

A pseudoknot is a nucleic acid secondary structure containing at least two stem-loop structures in which half of one stem is intercalated between the two halves of another stem. The pseudoknot was first recognized in the turnip yellow mosaic virus in 1982...

s. NMR is also useful for probing the binding of nucleic acid molecules to other molecules, such as proteins or drugs, by seeing which resonances are shifted upon binding of the other molecule.

RNA sequencing

While methods for each type of probing differ in steps, the probing of secondary structure involves certain steps in order to determine the structure. The first step involved is submitting the structural RNA to the probe of interest and incubating over a certain amount of time to allow the reaction to occur. The RNA is then transcribed using reverse transcriptase PCR, where this results in different lengths of bands due to the modification of the RNA at specific sites, which causes the reverse transcriptase to fall off. These bands are then run on a gel with sequencing data determined from RNA sequencing

Sequencing

In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer...

.

Chemical methods

RNA chemical probing can involve many different chemicals which serve to modify specific bases at certain sites to show certain locations available for specific modification type.

Hydroxyl radical probing

Probing with hydroxyl radical

Hydroxyl radical

The hydroxyl radical, •OH, is the neutral form of the hydroxide ion . Hydroxyl radicals are highly reactive and consequently short-lived; however, they form an important part of radical chemistry. Most notably hydroxyl radicals are produced from the decomposition of hydroperoxides or, in...

s involves an additional step, as hydroxyl radicals are short lived in solution they need to be generated. This can be done using H₂O₂, ascorbic acid, and Fe(II)-EDTA complex which is attached to the backbone through EDTA. The Fe(II) along with ascorbic acid generates hydroxyl radicals which then can react with the nucleic acid molecules. Hydroxyl radical probing is often used in conjunction with chemical probing of nucleic acid molecules that are thought to associate with proteins, this is due to the modification done by hydroxyl radicals. Hydroxyl radicals attack the ribose/deoxyribose ring and this results in breaking of the phosphate backbone, which is independent of secondary structure, as all backbone is accessible, but is instead resultant of protein or tertiary structure protection. Probing with hydroxyl radicals shows the protection of structured nucleic acids by the proteins thought to be associated or folding on itself, where cleavage again results in a band formed through gel electrophoresis (after RT-PCR in the case of RNA) that is shorter than the full nucleic acid depending upon where it is cleaved. In this case since the last nucleotide is not modified the band length is indicative of the base that was cleaved. When examining the gel produced by running the gels on a band the areas of various strength of protection where areas of stronger protection for hydroxyl radicals can be said to have tighter association with a protein, or if no protein associates with the nucleic acid it can be caused by the tertiary fold.

DMS

Dimethyl sulfate

Dimethyl sulfate

Dimethyl sulfate is a chemical compound with formula 2SO2. As the diester of methanol and sulfuric acid, its formula is often written as 2SO4 or even Me2SO4, where CH3 or Me is methyl...

, known as DMS, is a chemical that can be used to modify nucleic acids in order to determine secondary structure. DMS modifies certain bases through methylation

Methylation

In the chemical sciences, methylation denotes the addition of a methyl group to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation with, to be specific, a methyl group, rather than a larger carbon chain, replacing a hydrogen atom...

. One set of methylation products that is used for RNA is the methylation of N1 adenosine and N3 of cytosine which prevents the natural hydrogen bonds to form between bases modified by DMS. This enables modification sites to be detected by RT-PCR, as modified sites cannot be basepaired, which results in the reverse transcriptase to fall off and different band sizes. DMS may modify adenines and cytosines that are single stranded, base paired at the end of a helix, or in a base pair next to a GU pair. Detection of these modifications is done by examination of a gel and looking at bands present. One thing of note is that since modification prevents the addition of nucleotides at the modification site each of the bands generated is shifted by one base down on the gel. Nucleotides may be protected from DMS modification by base pairing, tertiary contacts, or protein-RNA interactions. This can be used to begin to develop a model of secondary structure for the RNA molecule. Structure probing by DMS allows for detection of secondary structure changes due to binding of RNA molecules along with detecting changes in tertiary structure.

DMS modification can also be used for DNA, for example in footprinting DNA-protein interactions.

CMCT

1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate known as CMCT is another chemical that is used in structure probing. CMCT like DMS serves to modify the exposed bases of specific nucleotides, which are uridine

Uridine

Uridine is a molecule that is formed when uracil is attached to a ribose ring via a β-N1-glycosidic bond.If uracil is attached to a deoxyribose ring, it is known as a deoxyuridine....

, and to a smaller extent guanine

Guanine

Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...

. CMCT reacts primarily with N3 of uridine and N1 of guanine modifying two sites responsible for hydrogen bonding on the bases. Modification using CMCT is analogous in detection to DMS as modification prevents basepairing at specific nucleotides which are then detected using rt-PCR and running of the PCR products on an agarose gel as shown in Figure 1. Structure probing of RNA by CMCT indicates the presence of uridine and guanine in single stranded regions by accessibility to modification or the presence of uridine and guanine in double stranded regions by protection from CMCT, which is the absence of a band. CMCT, like DMS can detect secondary and tertiary structure changes, but still has the same weaknesses as the method of modification is the same as DMS.

Kethoxal

1,1-Dihydroxy-3-ethoxy-2-butanone, known as kethoxal, is used like DMS and CMCT, where treatment with kethoxal causes the modification of guanine, specifically altering the N1 and N2 by covalent interaction. The modification by kethoxal prevents single stranded guanine nucleotides to become modified, so that when reverse transcriptase reaches the modified guanine it falls off resulting in a band. After rt-PCR the products will be of different lengths where a band present indicates modification of a guanine base, and the absence of a band indicates that the base was not available for modification. Using this gel in combination with DMS and CMCT a model for structural RNAs can be formed through comparison between the gels which indicate protected and accessible positions in all the bases, which can be used to form a preliminary model.

SHAPE

Selective 2′-hydroxyl acylation analyzed by primer extension, or SHAPE, takes advantage of reagents that preferentially modify the backbone of RNA in structurally flexible regions. Reagents such as N-methylisotoic anhydride (NMIA) and 1-methyl-7-nitroisatoic anhydride (1M7) undergo hydrolysis to form adducts on the 2'-hydroxyl of the RNA backbone. Compared to the chemicals used in other RNA probing techniques, these reagents have the advantage of being largely unbiased to base identity, while remaining very sensitive to conformational dynamics. Nucleotides which are constrained (usually by base-pairing) show less adduct formation than nucleotides which are unpaired. Adduct formation is quantified for each nucleotide in a given RNA by extension of a complementary DNA primer with reverse transcriptase and comparison of the resulting fragments with those from an unmodified control. SHAPE therefore reports on RNA structure at the individual nucleotide level. This data can be used as input to generate highly accurate secondary structure models. SHAPE has been used to analyze diverse RNA structures, including that of an entire HIV-1 genome. The best approach is to use a combination of chemical probing reagents and experimental data.

In-line probing

In-line probing does not involve treatment with any type of chemicals or reagents to modify exposed RNA structures. This type of probing assay uses the structure dependent cleavage of RNA, as areas that are single stranded are more flexible and over time will degrade, as RNA structure is not always stable. The process of in-line probing is often used to determine changes in structure due to ligand binding as this can result in different cleavage patterns. The process of in-line probing involves incubation of structural or functional RNAs over a long period of time, can be several days, but varies in each experiment, and then running the incubated products on a gel to visualize the bands. This experiment is often done using two different conditions: 1) with ligand and 2) in the absence of ligand. Cleavage results in shorter band lengths and is indicative of areas that are not basepaired, as those that are tend to be less sensitive to spontaneous cleavage. In line probing is a functional assay in ligand binding changes of RNA because it can show directly the change in flexibility and binding of regions of DNA in response to a ligand, as well as compare that response to analogous ligands. An in-line probing assay is then commonly used in dynamic studies, specifically when examining riboswitches

Nucleotide analog interference mapping

Nucleotide analog interference mapping (NAIM) is the process of using nucleotide analogs, molecules that are similar in some ways to nucleotides but lack function, to determine the importance of a functional group at each location of an RNA molecule. The process of NAIM is to insert a single nucleotide analog into a unique site. This can be done by transcribing a short RNA using T7 RNA polymerase

T7 RNA polymerase

T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA in the 5'→ 3' direction.- Activity :T7 polymerase is extremely promoter-specific and only transcribes DNA downstream of a T7 promoter. The T7 polymerase also requires a DNA template and Mg2+ ion as...

, then synthesizing a short oligonucleotide containing the analog in a specific position, then ligating them together on the DNA template using a ligase. The nucleotide analogs are tagged with a phosphorothioate, the active members of the RNA population are then distinguished from the inactive members, the inactive members then have the phophorothioate tag removed and the analog sites are identified using gel electrophoresis and autoradiography. This indicates a functionally important nucleotides, as cleavage of the phosphorothioate by iodine results in an RNA that is cleaved at the site of the nucleotide analog insert. By running these truncated RNA molecules on a gel, the nucleotide of interest can be identified against a sequencing experiment Site directed incorporation results indicate positions of importance where when running on a gel, functional RNAs that have the analog incorporated at that position will have a band present, but if the analog results in non-functionality, when the functional RNA molecules are run on a gel there will be no band corresponding to that position on the gel. This process can be used to evaluate an entire area, where analogs are placed in site specific locations, differing by a single nucleotide, then when functional RNAs are isolated and run on a gel, all areas where bands are produced indicate non-essential nucleotides, but areas where bands are absent from the functional RNA indicate that inserting a nucleotide analog in that position caused the RNA molecule to become non-functional