Joshua H. Baraban

In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo

The combination of emergent RNA interference (RNAi) technology with in ovo electroporation in the chick embryo has the potential to provide a powerful and rapid means for functional analyses of novel genes in vivo. In this study, we show that electroporation of short 21‐bp RNA duplexes (siRNAs) is a quick and simple method for silencing exogenous and endogenous gene expression in vivo. Quantitative comparisons with two other RNAi protocols that use long double‐stranded RNA duplexes and endonuclease‐digested duplexes (esiRNAs) demonstrate that siRNAs are significantly more effective at reducing gene expression. Furthermore, we also find that much higher amounts of siRNA are required for silencing of endogenous gene expression relative to plasmid‐borne reporter constructs. In short, these results demonstrate that siRNAs are the most effective type of double‐stranded RNA duplex for silencing gene expression and suggest that there might be important differences between silencing endogenous and exogenous genes. Finally, we review the parameters for each of these RNA‐based methods of RNAi and the controls required to analyze RNAi data in the context of the developing vertebrate embryo. Developmental Dynamics 231:592–600, 2004.

DISCOVERY OF SiCSi IN IRC+10216: A MISSING LINK BETWEEN GAS AND DUST CARRIERS OF Si–C BONDS

We report the discovery in space of a disilicon species, SiCSi, from observations between 80 and 350 GHz with the IRAM 30m radio telescope. Owing to the close coordination between laboratory experiments and astrophysics, 112 lines have now been detected in the carbon-rich star CW Leo. The derived frequencies yield improved rotational and centrifugal distortion constants up to sixth order. From the line profiles and interferometric maps with the Submillimeter Array, the bulk of the SiCSi emission arises from a region of 6″ in radius. The derived abundance is comparable to that of SiC2. As expected from chemical equilibrium calculations, SiCSi and SiC2 are the most abundant species harboring a Si-C bond in the dust formation zone and certainly both play a key role in the formation of SiC dust grains.

High-accuracy estimates for the vinylidene-acetylene isomerization energy and the ground state rotational constants of :C═CH2.

Highly accurate calculations are reported for properties of vinylidene (H2C═C:), specifically the position of its zero-point vibrational level relative to that of acetylene and its equilibrium structure and ground state rotational constants. The isomerization energy of vinylidene calculated at the HEAT-456QP level of theory is 43.53 ± 0.15 kcal mol(-1), in agreement with the previous best estimate, but associated with a much smaller uncertainty. In addition, the thermochemical calculations presented here also allow a determination of the H2CC-H bond energy of the vinyl radical at the HEAT-345(Q) level of theory, which is 77.7 ± 0.3 kcal mol(-1). The equilibrium structure of vinylidene, estimated with an additivity scheme that includes treatment of correlation effects beyond CCSD(T) as well as relativistic and adiabatic (diagonal Born-Oppenheimer correction) contributions, is rCC = 1.2982 ± 0.0003 Å, rCH = 1.0844 ± 0.0003 Å, and θCCH = 120.05 ± 0.05°, with zero-point rotational constants (including vibrational contributions and electronic contributions to the moment of inertia) estimated to be A0 = 9.4925 ± 0.0150 cm(-1), B0 = 1.3217 ± 0.0017 cm(-1), and C0 = 1.1602 ± 0.0016 cm(-1).

Reduced dimension discrete variable representation study of cis–trans isomerization in the S1 state of C2H2

Isomerization between the cis and trans conformers of the S(1) state of acetylene is studied using a reduced dimension discrete variable representation (DVR) calculation. Existing DVR techniques are combined with a high accuracy potential energy surface and a kinetic energy operator derived from FG theory to yield an effective but simple Hamiltonian for treating large amplitude motions. The spectroscopic signatures of the S(1) isomerization are discussed, with emphasis on the vibrational aspects. The presence of a low barrier to isomerization causes distortion of the trans vibrational level structure and the appearance of nominally electronically forbidden à (1)A(2)←X̃ (1)Σ(g)(+) transitions to vibrational levels of the cis conformer. Both of these effects are modeled in agreement with experimental results, and the underlying mechanisms of tunneling and state mixing are elucidated by use of the calculated vibrational wavefunctions.

Cis-trans isomerization in the S1 state of acetylene: identification of cis-well vibrational levels.

A systematic analysis of the S(1)-trans (Ã(1)A(u)) state of acetylene, using IR-UV double resonance along with one-photon fluorescence excitation spectra, has allowed assignment of at least part of every single vibrational state or polyad up to a vibrational energy of 4200 cm(-1). Four observed vibrational levels remain unassigned, for which no place can be found in the level structure of the trans-well. The most prominent of these lies at 46 175 cm(-1). Its (13)C isotope shift, exceptionally long radiative lifetime, unexpected rotational selection rules, and lack of significant Zeeman effect, combined with the fact that no other singlet electronic states are expected at this energy, indicate that it is a vibrational level of the S(1)-cis isomer (Ã(1)A(2)). Guided by ab initio calculations [J. H. Baraban, A. R. Beck, A. H. Steeves, J. F. Stanton, and R. W. Field, J. Chem. Phys. 134, 244311 (2011)] of the cis-well vibrational frequencies, the vibrational assignments of these four levels can be established from their vibrational symmetries together with the (13)C isotope shift of the 46 175 cm(-1) level (assigned here as cis-3(1)6(1)). The S(1)-cis zero-point level is deduced to lie near 44 900 cm(-1), and the ν(6) vibrational frequency of the S(1)-cis well is found to be roughly 565 cm(-1); these values are in remarkably good agreement with the results of recent ab initio calculations. The 46 175 cm(-1) vibrational level is found to have a 3.9 cm(-1) staggering of its K-rotational structure as a result of quantum mechanical tunneling through the isomerization barrier. Such tunneling does not give rise to ammonia-type inversion doubling, because the cis and trans isomers are not equivalent; instead the odd-K rotational levels of a given vibrational level are systematically shifted relative to the even-K rotational levels, leading to a staggering of the K-structure. These various observations represent the first definite assignment of an isomer of acetylene that was previously thought to be unobservable, as well as the first high resolution spectroscopic results describing cis-trans isomerization.

On the HCN – HNC Energy Difference

The value for the HCN → HNC 0 K isomerization energy has been investigated by combining state-of-the-art electronic structure methods with the Active Thermochemical Tables (ATcT) approach. The directly computed energy difference between HCN and HNC at the HEAT-456QP level of theory is 5236 ± 50 cm(-1). This is substantially lower (by ∼470 cm(-1) or ∼1.3 kcal/mol) than the recently proposed high-level multireference configuration interaction value of 5705 ± 20 cm(-1) of Barber et al. ( Mon. Not. R. Astron. Soc. 2014, 437, 1828-1835 ). The discrepancy was analyzed by the ATcT approach, using several distinct steps, which (a) independently corroborated the current single-reference HEAT-456QP result, (b) independently found that the recent multireference-based value is highly unlikely to be correct within its originally stated uncertainty, and (c) produced a recommended value of 5212 ± 30 cm(-1) for the HCN → HNC isomerization energy at 0 K, based on all currently available knowledge. The ATcT standard enthalpies of formation at 0 and 298 K for HCN, HNC, and their cations and anions are also presented.

The Ã1Au state of acetylene: ungerade vibrational levels in the region 45,800–46,550 cm−1

The ungerade vibrational levels of the 1Au (S1-trans) state of C2H2 lying in the region 45,800–46,550 cm−1 have been assigned from IR–UV double resonance spectra. The aim has been to classify the complete manifold of S1-trans levels in this region, so as to facilitate the assignment of the bands of S1-cis C2H2. The rotational structure is complicated because of the overlapping of vibrational polyads with different Coriolis and Darling–Dennison parameters, but assignments have been possible with the help of predictions based on the properties of polyads at lower energy. An important result is that the analysis of the (1141, 1161) polyad determines the anharmonicity constants x 14 and x 16, which will be needed to proceed to higher energies. Some regions of impressive complexity occur. Among these is the band given by the 3361, K = 1 state at 45,945 cm−1, where a three-level interaction within the S1 state is confused by triplet perturbations. Several probable S1-cis states have been observed, including cis-62, K = 1; this vibrational level appears to show a K-staggering, of the type that arises when quantum mechanical tunnelling through the barrier to cis-trans isomerization is possible. The total number of identified cis vibrational states is now 6 out of an expected 10 up to the energies discussed in this paper.

Ab initio effective rotational and rovibrational Hamiltonians for non-rigid systems via curvilinear second order vibrational Møller–Plesset perturbation theory

We present a perturbative method for ab initio calculations of rotational and rovibrational effective Hamiltonians of both rigid and non-rigid molecules. Our approach is based on a curvilinear implementation of second order vibrational Møller-Plesset perturbation theory extended to include rotational effects via a second order contact transformation. Though more expensive, this approach is significantly more accurate than standard second order vibrational perturbation theory for systems that are poorly described to zeroth order by rectilinear normal mode harmonic oscillators. We apply this method to and demonstrate its accuracy on two molecules: Si2C, a quasilinear triatomic with significant bending anharmonicity, and CH3NO2, which contains a completely unhindered methyl rotor. In addition to these two examples, we discuss several key technical aspects of the method, including an efficient implementation of Eckart and quasi-Eckart frame embedding that does not rely on numerical finite differences.

Discovery of a Missing Link: Detection and Structure of the Elusive Disilicon Carbide Cluster.

The rotational spectrum of the elusive but fundamentally important silicon carbide SiCSi has been detected using sensitive microwave techniques aided by high-level ab initio methods. Its equilibrium structure has been determined to very high precision using isotopic substitution and vibrational corrections calculated quantum-chemically: it is an isosceles triangle with a Si-C bond length of 1.693(1) Å, and an apex angle of 114.87(5)°. Now that all four Si(m)C(n) clusters with m + n = 3 have been observed experimentally, their structure and chemical bonding can be rigorously compared. Because Si2C is so closely linked to other Si-bearing molecules that have been detected in the evolved carbon star IRC+10216, it is an extremely promising candidate for detection with radio telescopes.

Anharmonic force fields of cis- and trans-S1 C2H2

We calculate second-order vibrational perturbation theory (VPT2) anharmonic force fields for the cis and trans conformers of S1 C2H2, and compare the results to experiment. The vibrational assignments of recently observed levels belonging to the cis well are of particular interest. A refined estimate of the cis origin position (44,870 ± 10 cm−1) is proposed, and preliminary low-energy fits to the global J = K = 0 trans level structure are also described. The performance of perturbation theory in this isomerizing system is examined, and both surprising successes and failures are encountered. We examine these and their causes, and offer practical suggestions for avoiding the pitfalls of applying perturbation theory to systems with large amplitude motions.