Raman and infrared studies of nucleosides at high pressures: II. Cytidine.

Raman and mid-infrared (MIR) spectra have been recorded for crystalline cytidine at pressures up to 10 GPa at room temperature. Broadening and positive wavenumber shifts are observed for most of the Raman and MIR peaks with increasing pressure. However, some of the MIR peaks associated with hydrogen-stretching modes display a negative wavenumber shift as a result of charge transfer effects. Evidence of a phase transition near 4 GPa is presented and attributed to a change in the conformation of the five membered sugar ring.

An efficient synthesis of mimetics of neamine for RNA recognition.

As mimetics of neamine, several 4-heterocyclic 2-deoxystreptamine derivatives were chemically synthesized for RNA recognition. Conversion of 4-methylthiomethyl-5,6-di-O-acetyl-diazido-2-deoxystreptamine to the 4-chloromethyl derivative followed by reactions with different nuclophilic reagents gave the 4-heterocyclic 2-deoxystreptamine derivatives in satisfactory yields.

Spontaneous base flipping in DNA and its possible role in methyltransferase binding.

Recent crystallographic studies showed that HhaI and other methyltransferases flip their target DNA base completely out of a DNA helix. This base flipping is also a key feature in a number of other enzyme-catalyzed processes involving DNA. The mechanism of base flipping by these enzymes remains elusive. Based on a full atomic level description of bond rotational motions we have studied the energetics of flipping a base in a B-DNA duplex in the absence of the enzyme. We have also investigated the effect of the restraints from enzyme-distorted DNA backbone on the movement of a flipped base in several methytransferase bound DNA crystal structures. Our study on crystal B-DNA helices showed that a base could be flipped at an energy cost close to the enthalpy observed for base pair opening in premelting thermal fluctuations. This suggests that spontaneous base flipping in DNA due to thermal fluctuation may be achieved. Analysis of several crystal HhaI and HaeIII methyltransferase DNA duplex structures showed that the enzyme induced DNA backbone distortion severely restricts the movement of the flipped base, which indicates that during base flipping the backbone needs to adopt a substantially different conformation than that observed in the x-ray (enzyme-bound) structures. Our results suggest the possible role of thermally induced transient base opening in facilitating recognition and binding of methyltransferases and other enzymes.

Base opening in RNA and DNA duplexes: implication for RNA stability.

The energetics of a low-energy single base opening in several RNA duplex crystal structures has been calculated and compared to DNA duplexes. Base opening in RNA appears to have an overall preference towards the major groove, similar to results previously reported for B-DNA. Movement of each of the adenine, uracil, and cytosine bases into the minor groove is blocked by a high-energy barrier due to severe close contact with neighboring bases. Guanine bases are able to open towards both grooves because of the unique orientation of the base that avoids steric clash along the opening pathway. RNA bases are found to have a substantially smaller major groove opening extent than that of their B-DNA counterparts. A comparison with base opening behavior of A-DNA duplexes suggests that this difference results from helix constraint associated with A-form backbone conformation. The reduced opening extent correlates with the RNA duplex stability and is consistent with observed slower imino proton exchange rates in RNA duplexes.

Identification of ligands for RNA targets via structure-based virtual screening: HIV-1 TAR.

Binding of the Tat protein to TAR RNA is necessary for viral replication of HIV-1. We screened the Available Chemicals Directory (ACD) to identify ligands to bind to a TAR RNA structure using a four-step docking procedure: rigid docking first, followed by three steps of flexible docking using a pseudobrownian Monte Carlo minimization in torsion angle space with progressively more detailed conformational sampling on a progressively smaller list of top-ranking compounds. To validate the procedure, we successfully docked ligands for five RNA complexes of known structure. For ranking ligands according to binding avidity, an empirical binding free energy function was developed which accounts, in particular, for solvation, isomerization free energy, and changes in conformational entropy. System-specific parameters for the function were derived on a training set of RNA/ligand complexes with known structure and affinity. To validate the free energy function, we screened the entire ACD for ligands for an RNA aptamer which binds L-arginine tightly. The native ligand ranked 17 out of ca. 153,000 compounds screened, i.e., the procedure is able to filter out >99.98% of the database and still retain the native ligand. Screening of the ACD for TAR ligands yielded a high rank for all known TAR ligands contained in the ACD and suggested several other potential TAR ligands. Eight of the highest ranking compounds not previously known to be ligands were assayed for inhibition of the Tat-TAR interaction, and two exhibited a CD50 of ca. 1 microM.

Measuring dissociation constants of RNA and aminoglycoside antibiotics by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has been used to determine the dissociation constants (K(D)s) and binding stoichiometry for tobramycin and paromomycin with a 27-nucleotide RNA construct representing the A-site of the 16S ribosomal RNA. K(D) values determined by holding the ligand concentration fixed are compared with K(D) values derived by holding the RNA target concentration fixed. Additionally, the effect of solution conditions such as the amount of organic solvent present and the amount of salt present in the solution on the K(D) measurement is investigated. It is shown that the preferred method for determining dissociation constants using ESI-MS is holding the RNA target concentration fixed below the expected K(D) and titrating the ligand. K(D) measurements should also be carried out at as high as possible salt concentration to minimize nonspecific binding due primarily to electrostatic interactions. For tobramycin, two nonequivalent binding sites were found with K(D1) = 352 nM and K(D2) = 9 microM. For paromomycin, there is only one binding site with K(D) = 52 nM.

The RNA-binding domain of ribosomal protein L11 recognizes an rRNA tertiary structure stabilized by both thiostrepton and magnesium ion.

Antibiotics that inhibit ribosomal function may do so by one of several mechanisms, including the induction of incorrect RNA folding or prevention of protein and/or RNA conformational transitions. Thiostrepton, which binds to the 'GTPase center' of the large subunit, has been postulated to prevent conformational changes in either the L11 protein or rRNA to which it binds. Scintillation proximity assays designed to look at the binding of the L11 C-terminal RNA-binding domain to a 23S ribosomal RNA (rRNA) fragment, as well as the ability of thiostrepton to induce that binding, were used to demonstrate the role of Mg(2+), L11 and thio-strepton in the formation and maintenance of the rRNA fragment tertiary structure. Experiments using these assays with both an Escherichia coli rRNA fragment and a thermostable variant of that RNA show that Mg(2+), L11 and thiostrepton all induce the RNA to fold to an essentially identical tertiary structure.

Enhanced gas-phase hydrogen-deuterium exchange of oligonucleotide and protein ions stored in an external multipole ion reservoir.

Rapid gas-phase hydrogen-deuterium (H-D) exchange from D(2)O and ND(3) into oligonucleotide and protein ions was achieved during storage in a hexapole ion reservoir. Deuterated gas is introduced through a capillary line that discharges directly into the low-pressure region of the reservoir. Following exchange, the degree of H-D exchange is determined using Fourier transform ion cyclotron resonance mass spectrometry. Gas-phase H-D exchange experiments can be conducted more than 100 times faster than observed using conventional in-cell exchange protocols that require lower gas pressures and additional pump-down periods. The short experimental times facilitate the quantitation of the number of labile hydrogens for less reactive proteins and structured oligonucleotides. For ubiquitin, we observe approximately 65 H-D exchanges after 20 s. Exchange rates of > 250 hydrogens s(-1) are observed for oligonucleotide ions when D(2)O or ND(3) is admitted directly into the external ion reservoir owing to the high local pressure in the hexapole. Partially deuterated oligonucleotide ions have been fragmented in the reservoir using infrared multiphoton dissociation (IRMPD). The resulting fragment ions show that exchange predominates at charged sites on the 5'- and 3'-ends of the oligonucleotide, whereas exchange is slower in the core. This hardware configuration is independent of the mass detector and should be compatible with other mass spectrometric platforms including quadrupole ion trap and time-of-flight mass spectrometers.

Mass spectrometry as a drug discovery platform against RNA targets.

Mass spectrometry continues to play an increasingly important role in the drug discovery arena. In many drug discovery and biotechnology environments, mass spectrometry constitutes an integral component of the entire drug discovery and development process, from lead identification to quality control of bulk drug substance. Prominent among emerging drug discovery strategies are those which target cellular RNAs. Antisense drugs elicit a therapeutic response by hybridizing a synthetic oligonucleotide to a single stranded region of a target RNA; alternatively, highly structured regions of RNA represent an interesting class of drug target as the complex structural elements often comprise regulatory or functional domains where small molecules can bind with high affinity and specificity. In this paper, we review work performed within the last few years where mass spectrometry has been used to identify and study molecules that bind to RNA drug targets.

A gated-beam electrospray ionization source with an external ion reservoir. A new tool for the characterization of biomolecules using electrospray ionization mass spectrometry.

In this work we describe a micro-electrospray ionization source equipped with an atmospheric pressure external ion shutter. The solenoid-activated shutter prevents the electrospray plume from entering the inlet capillary unless triggered to the 'open' position. When in the 'closed' position, a stable electrospray plume is maintained between the electrospray ionization (ESI) emitter and the electrically isolated face of the shutter. When the shutter is triggered, a 'slice' of ions is allowed to enter the inlet capillary and is subsequently accumulated in an external ion reservoir comprised of a radio frequency only (rf-only) hexapole and a pair of electrostatic elements. Following ion accumulation in the external ion reservoir, intact molecular ions of proteins, oligonucleotides, and noncovalent complexes can be stored for extended intervals (>30 minutes) prior to being transferred to the Fourier transform ion cyclotron resonance (FTICR) trapped ion cell for mass analysis. By introducing reactive gases directly into the external ion reservoir during the storage interval, ion-molecule reactions, such as H/D exchange, can be performed at high effective pressures. This scheme obviates the need for the long reaction times and delays associated with restoring base pressure in the trapped ion cell and allows H/D exchange reactions to be conducted in a fraction of the time required using conventional in-cell exchange approaches. The back face of the shutter arm contains an elastomeric material which can be positioned to seal the inlet to the mass spectrometer resulting in lower base pressure in the ion reservoir and the FTICR cell. Additionally, it is noted that blocking the ESI plume during non-accumulation events results in reduced fouling of the source electrodes and longer times between required source cleaning.

Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry.

We have developed methods for studying the interactions between small molecules and RNA and have applied them to characterize the binding of three classes of aminoglycoside antibiotics to ribosomal RNA subdomains. High-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of aminoglycosides and multiple RNA targets simultaneously. Signal overlap among RNA targets was avoided by the addition of neutral mass tags that direct each RNA target to a unique region of the spectrum. In addition to determining binding affinities, the locations of the binding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycoside/RNA complex. Specific complexes were observed for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomycin, and lividomycin, whereas apramycin preferentially formed a complex with the eukaryotic subdomain. We show that differences in binding between paromomycin and ribostamycin can be probed by using an MS-MS protection assay. We have introduced specific base substitutions in the RNA models and have measured their impact on binding affinity and selectivity. The binding of apramycin to the prokaryotic subdomain strongly depends on the identity of position 1408, as evidenced by the selective increase in affinity for an A1408G mutant. An A1409-G1491 mismatch pair in the prokaryotic subdomain enhanced the binding of tobramycin and bekanamycin. These observations demonstrate the power of MS-based methods to provide molecular insights into small molecule/RNA interactions useful in the design of selective new antimicrobial drugs.

Multiplexed screening of neutral mass-tagged RNA targets against ligand libraries with electrospray ionization FTICR MS: a paradigm for high-throughput affinity screening.

We demonstrate that binding of mixtures of aminoglycosides can be measured simultaneously against multiple RNA targets of identical length and similar (or identical) molecular weight. Addition of a neutral mass tag to one of the RNA targets shifts the detected peaks to a higher mass/charge ratio, where complexes with small molecules can be identified unambiguously. An appropriately placed neutral mass tag does not alter RNA--ligand binding. The utility of this strategy is demonstrated with model RNAs corresponding to the decoding region of the prokaryotic and eukaryotic rRNAs and a mixture of five aminoglycosides. Complexes are observed between the aminoglycoside library and the prokaryotic rRNA model, while no aminoglycoside was observed to bind to the mass-tagged eukaryotic rRNA model. The differential binding data is consistent with the eukaryotic A-site rRNA having a different conformation compared with the prokaryotic A-site that prevents entry and binding of neomycin-class aminoglycosides. Mass spectrometric analysis of neutral mass-tagged macromolecular targets represents a new high-throughput screening paradigm in which the interaction of multiple targets against a collection of small molecules can be evaluated in parallel.

Raman and infrared studies of nucleosides at high pressures: I. Adenosine.

Room temperature Raman and infrared (IR) spectra of crystalline adenosine at pressures between 1 atm and 10 GPa are reported. Vibrational modes were identified through assignments in the literature. Many modes were found to increase in frequency with pressure; however, some irregularities were observed. Discontinuities were observed in numerous Raman and IR modes near 2.5 GPa, indicating a phase transition. The modes associated with the glycosidic bond shift significantly down in frequency near this pressure, suggesting a weakening of the associated bond. The IR modes associated with hydrogen-stretching motions were found to decrease in frequency with pressure.

Infrared multiphoton dissociation in an external ion reservoir.

In this work we present a novel scheme for performing infrared multiphoton dissociation (IRMPD) external to the mass analyzer in an external ion reservoir consisting of an rf-only multipole and a pair of electrostatic lens elements. Ions generated by electrospray ionization (ESI) are accumulated in an rf-only hexapole and dissociated by irradiation at 10.6 microns from a CW CO2 laser in the source region of the mass spectrometer. This scheme is unique from other IRMPD schemes as dissociation occurs in a spatially distinct region of the spectrometer and is independent of the mass spectrometry platform used to analyze the fragment ions. The effectiveness of the technique is demonstrated with ESI IRMPD FTICR mass spectrometry of a 20-mer phosphorothioate oligonucleotide. A comparison of the external IRMPD scheme with nozzle-skimmer dissociation and conventional in-cell IRMPD reveals a significant improvement in signal-to-noise ratio and fragment yield, particularly for larger, more highly charged fragment ions.

Multipole storage assisted dissociation, a novel in-source dissociation technique for electrospray ionization generated ions.

In this work we present a novel in-source dissociation scheme referred to as multipole storage assisted dissociation (MSAD) for electrospray ionization (ESI) generated ions in which dissociation is effected by employing extended ion accumulation intervals in a high pressure rf-only hexapole assembly prior to mass analysis. Following an extended ion accumulation interval in which ions are confined in the rf-only hexapole, ions are gated out of the hexapole, trapped, and mass analyzed in the trapped ion cell of a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The accumulation region is comprised of an rf-only hexapole ion guide which separates two electrodes, a biased skimmer cone, and an auxiliary 'gate' electrode at the low pressure end of the hexapole. This technique should be applicable to other mass spectrometry platforms compatible with pulsed ionization sources including quadrupole ion traps, and time-of-flight mass analyzers. This concept is demonstrated with the dissociation of a small protein in which selective fragmentation is observed at labile amino acid linkages producing primarily y-type fragment ions.

Hydrogen/deuterium exchange of nucleotides in the gas phase.

Gas-phase hydrogen/deuterium exchange reactions have been performed on the 5'- and 3'-nucleotide monophosphates and on the 3'5'-cyclic nucleotides. Following negative mode electrospray ionization and transport to a Fourier transform ion cyclotron resonance cell, each nucleotide was reacted with gaseous D2O for up to 600 s. Extensive deuterium exchange was observed for the 3'- and 5'-nucleotides in negative ion mass spectra with relative rates of exchange following the trend 5'dCMP > 5'-dAMP > 5'dTMP >> 5'-dGMP and 3'-dGMP > 3'-dAMP approximately equal to 3'-dCMP approximately equal to 3'-dTMP. At least two classes of exchanging protons are observed. The more facile class is assigned to the amino protons of the bases, with a slower class attributed to the phosphate and/or hydroxyl proton. Overall, the 3'-nucleotides exchange more quickly than the 5'-oligonucleotides. The cyclic nucleotides did not undergo deuterium exchange, suggesting that a charged phosphate group proximate to the base is required to catalyze the exchange reaction. Exchange through tautomerization of the bases is no observed, although molecular modeling suggests an energy barrier of < 30 kcal.

The opening of a single base without perturbations of neighboring nucleotides: a study on crystal B-DNA duplex d(CGCGAATTCGCG)2.

In this work we explore the possibility of the opening of a single base without perturbation of its neighboring nucleotides. Low energy base opening into the grooves can be accomplished by rotation of the relevant backbone and glycosidic bond torsion angles. The pathway has been determined by identifying zeta torsion angle as the reaction coordinate together with the accompanying geometric requirement that guides the displacement of other torsion angles. Our study on Dickerson dodecamer duplex d(CGCGAATTCGCG)2 showed that all bases with normal equilibrium zeta can be rotated by approximately 30 degrees, corresponding to approximately 3.5A base displacement, towards the major groove. Such an opening extent is comparable with estimated amplitudes of local angular motions in DNA bases from NMR experiments, which might facilitate proton exchange. The computed base opening energy barrier is also comparable with measured base pair opening enthalpy. These results indicate possible relevance of the pathway studied in this work with experimentally observed base pair opening process. Our analysis also showed a preference for base opening along the major groove and an abnormal opening behavior for bases with unusual equilibrium zeta torsion angle.

Characterization of oligonucleotide metabolism in vivo via liquid chromatography/electrospray tandem mass spectrometry with a quadrupole ion trap mass spectrometer.

The pattern of nuclease degradation observed for an antisense phosphorothioate oligonucleotide in pig kidney was determined using liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) and LC/ESI-MS/MS with a quadrupole ion trap mass spectrometer. Metabolites were separated by length using reversed-phase high-performance liquid chromatography with aqueous hexafluoropropan-2-ol-triethylamine and a methanol gradient. The individual masses of metabolites in each LC peak were determined via deconvolution and converted into potential nucleotide compositions. The nucleotide composition was used to locate metabolites within the known oligomer sequence. The identity of metabolites was confirmed using on-line LC/MS/MS to generate fragment ions suitable for sequence verification. A limited number of shorter oligonucleotide fragments were observed, suggesting that metabolism in vivo may be sequence dependent.