Elvin A. Alemán

Exploring RNA folding one molecule at a time.

RNA molecules fold into stable native structures to perform their biological function. RNA folding can be influenced by ions, co-factors, and proteins through numerous mechanisms. Understanding these mechanisms at the molecular level is important for elucidating the structure-function relationship in biologically important RNAs. Recent developments in single molecule spectroscopy have provided new approaches to investigate RNA folding and have allowed identification of kinetic intermediates that would otherwise remain hidden in ensemble-averaged experiments. Here we summarize some of these developments, which provide new insight into the effect of Mg(2+) ions in RNA folding landscapes, the role of cooperativity in RNA tertiary folding, the stepwise folding of RNA during transcription, and the hierarchical assembly of RNA-protein complexes.

Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle.

Fluorescent nucleotide analogues, such as 2-aminopurine (2AP) and pyrrolo-C (PyC), have been extensively used to study nucleic acid local conformational dynamics in bulk experiments. Here we present a proof-of-principle approach using 2AP and PyC fluorescence at the single-molecule level. Our data show that ssDNA, dsDNA, or RNA containing both 2AP and PyC can be monitored using single-molecule fluorescence and a click chemistry immobilization method. We demonstrate that this approach can be used to monitor DNA and RNA in real time. This is the first reported assay using fluorescent nucleotide analogs at the single-molecule level. We anticipate that single 2AP or PyC fluorescence will have numerous applications in studies of DNA and RNA, including protein-induced base-flipping dynamics in protein–nucleic acid complexes.

Laser-Assisted Single-Molecule Refolding (LASR)

To assemble into functional structures, biopolymers search for global minima through their folding potential energy surfaces to find the native conformation. However, this process can be hindered by the presence of kinetic traps. Here, we present a new single-molecule technique, termed laser-assisted single-molecule refolding (LASR), to characterize kinetic traps at the single-molecule level. LASR combines temperature-jump kinetics and single-molecule spectroscopy. We demonstrate the use of LASR to measure single-molecule DNA melting curves with ∼1°C accuracy and to determine the activation barrier of a model kinetic trap. We also show how LASR, in combination with mutagenesis, can be used to estimate the yields of competing pathways, as well as to generate and characterize transient, unstable complexes.

Photoinduced electron-transfer within osmium(II) and ruthenium(II) bis-terpyridine donor acceptor dyads

The synthesis and photophysical characterization of two different series of electron donor-acceptor dyads containing Ru(II) and Os(II) bis-terpyridines (M(tpy)(2)(2+)) were prepared and studied in order to compare the oft-studied Ru(tpy)(2)(2+) chromophore with the less studied Os(tpy)(2)(2+) chromophore. The first series of dyads incorporates a benzoquinone (BQ) group as the electron acceptor, whereas the second contains a substituted pyromellitimide (PI) group as the electron acceptor. Steady-state emission experiments indicated efficient quenching of the 3MLCT emission of the electronically excited Os(II)-BQ complexes (7-8) compared to both model complexes (3-4) and the Os(II)-PI complex 10. Femtosecond pump-probe absorption experiments on 7-8 yielded ultrafast electron transfer rate constants (kET) of approximately 2.0 x 10(11) s(-1) (7) and 1.3 x 10(10) s(-1) (8) that were in good agreement with the low emission quantum yield results. Charge-recombination (kCR) in these complexes was also quite rapid, with rate constants of approximately 6.7 x 10(10) s(-1) (7) and 1.2 x 10(10) s(-1) (8). The analogous Ru(II) complexes underwent charge separation with rate constants of 7.6 x 10(10) s(-1) (5) and approximately 2.3 x 10(10) s(-1) (6), while charge recombination in these complexes occurred with rate constants of approximately 2.1 x 10(10) s(-1) (5) and approximately 5.3 x 10(10) s(-1) (6). Electron transfer in the pyromellitimide-containing complexes occurred only for Os(II)-PI (10), which exhibited significantly slower electron transfer (approximately 4.3 x 10(6) s(-1)) and charge recombination (approximately 7.7 x 10(6) s(-1)) rate constants. The nearly thermoneutral free energy of electron transfer and short excited state lifetime in the case of Ru(II)-PI (9) presumably prevents electron transfer in this compound.

Covalent‐Bond‐Based Immobilization Approaches for Single‐Molecule Fluorescence

The streptavidin-biotin bridge is commonly used in single-molecule studies to surface immobilize biomolecules onto microscope slides. However, the presence of tryptophanes impedes utilization of UV light and numerous fluorescent nucleotide analogs, such as 2-aminopurine. We present two new approaches to surface-immobilize nucleic acids for single molecule fluorescence experiments using covalent bonds and self-assembled monolayers instead of the traditional avidin-biotin linkage. The first approach takes advantage of a click-chemistry reaction between an azide and an alkyne to surface-immobilize nucleic acids through the resulting triazole linkage. The second approach uses disulfide bond bridges for immobilization. We have characterized the properties of the resulting surface-immobilized fluorophore-labeled DNA molecules and single-molecule fluorescence detection. We find that both approaches are specific and yield comparable surface densities and low fluorescence background to the avidin-biotin linkage, but offer new surface chemical properties that might be advantageous to prevent non-specific binding of biopolymers to the surface and to expand the range of fluorescent probes that can be employed in single molecule studies.

Spectroscopy of Free-Base N-Confused Tetraphenylporphyrin Radical Anion and Radical Cation

The radical anions and radical cations of the two tautomers (1e and 1i) of 5,10,15,20-tetraphenyl N-confused free-base porphyrin have been studied using a combination of cyclic voltammetry, steady state absorption spectroscopy, and computational chemistry. N-Confused porphyrins (NCPs), alternatively called 2-aza-21-carba-5,10,15,20-tetraphenylporphyrins or inverted porphyrins, are of great interest for their potential as building blocks in assemblies designed for artificial photosynthesis, and understanding the absorption spectra of the corresponding radical ions is paramount to future studies in multicomponent arrays where electron-transfer reactions are involved. NCP 1e was shown to oxidize at a potential of E(ox) 0.65 V vs Fc(+)|Fc in DMF and reduce at E(red) -1.42 V, while the corresponding values for 1i in toluene were E(ox) 0.60 V and E(red) -1.64 V. The geometries of these radical ions were computed at the B3LYP/6-31+G(d)//B3LYP/6-31G(d) level in the gas phase and in solution using the polarizable continuum model (PCM). From these structures and that of H(2)TPP and its corresponding radical ions, the computed redox potentials for 1e and 1i were calculated using the Born-Haber cycle. While the computed reduction potentials and electron affinities were in excellent agreement with the experimental reduction potentials, the calculated oxidation potentials displayed a somewhat less ideal relationship with experiment. The absorption spectra of the four radical ions were also measured experimentally, with radical cations 1e(•+) and 1i(•+) displaying significant changes in the Soret and Q-band regions as well as new low energy absorption bands in the near-IR region. The changes in the absorption spectra of radical anions 1e(•-) and 1i(•-) were not as dramatic, with the changes occurring only in the Soret and Q-band regions. These results were favorably modeled using time-dependent density functional calculations at the TD-B3LYP/6-31+G(d)//B3LYP/6-31G(d) level. These results were also compared to the existing data of free base tetraphenylporphyrin and free base tetraphenylchlorin.

Solvent Effects on the UV–Vis Absorption Properties and Tautomerism of N-Confused Tetraphenylporphyrin

The two tautomeric forms of N-confused tetraphenylporphyrin (NCTPP) show distinctly different absorption spectra. The existence of each tautomer in solution has been shown to be strongly solvent-dependent. In the present work, we have studied the tautomerization using absorption spectroscopy in 15 different solvents. While changes in the two tautomers are not large in the Soret band region, the distinct spectral changes between the two tautomers in the Q-band region provide a convenient way to measure the concentration of each tautomer. The resulting data shows a strong correlation between the tautomer and the H-bond accepting ability of the solvent. The anomaly in this data is for the alcoholic solvents ethanol and methanol, for which we observe evidence for H-bonding, presumably between the exterior N2 nitrogen of the NCTPP and the O-H proton of the solvent.

Characterization of Fluorescent Base Analogs to Study DNA Base Flipping at the Single Molecule Level

Chemical damage to DNA bases can result in mutations, block replication, and lead to cancer. It has been suggested that the phenomenon of base flipping take place by some enzymes during the repair of DNA damages. However, it still remains to be answered if the enzyme “pushes” the nucleotide out of the helix (active mechanism) or if the enzyme binds to a provisional flipped base (passive mechanism). Single molecule fluorescence has demonstrated to be a powerful technique to determine the formation of one or more intermediates, and to study the kinetics of the processes from the instant before an enzyme interact with the DNA until the release of the enzymatic product, one molecule at a time. Therefore, in order to optimize and maximize the repair of damaged DNA, new single molecule approaches to fully assess the kinetic mechanism of the base flipping process are needed.In previous work, the adenine fluorescent base analog 2-aminopurine (2AP) has been extensively used to study base flipping in ensemble average experiments. In addition, a novel 2AP single molecule approach was recently developed.1 In order to generate single molecule fluorescence assays to probe base flipping in different DNA-enzyme complexes, we need to study fluorescent base analogs (FBA) for all the natural bases. Several FBA molecules have been synthesized during the last four decades and we have selected one FBA molecule for each DNA base to probe base flipping. We have characterized the fluorescent properties of different FBA-substituted DNA molecules that mimic the different states proposed for the base flipping process.1.Aleman, E.A., Patrick, E., de Silva, C., Musier-Forsyth, K. & Rueda, D. Single-molecule dynamics with fluorescent nucleotide analogues. In preparationto be submitted