Engineering nucleic acid mechanisms for regulation and readout of gene expression : conditional Dicer substrate formation and sensitive multiplexed northern blots

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

A hybrid-parallel framework for the nonlinear seismic analysis of very tall buildings

FRAME3D, a program for the nonlinear seismic analysis of steel structures, has previously been used to study the collapse mechanisms of steel buildings up to 20 stories tall. The present thesis is inspired by the need to conduct similar analysis for much taller structures. It improves FRAME3D in two primary ways.

First, FRAME3D is revised to address specific nonlinear situations involving large displacement/rotation increments, the backup-subdivide algorithm, element failure, and extremely narrow joint hysteresis. The revisions result in superior convergence capabilities when modeling earthquake-induced collapse. The material model of a steel fiber is also modified to allow for post-rupture compressive strength.

Second, a parallel FRAME3D (PFRAME3D) is developed. The serial code is optimized and then parallelized. A distributed-memory divide-and-conquer approach is used for both the global direct solver and element-state updates. The result is an implicit finite-element hybrid-parallel program that takes advantage of the narrow-band nature of very tall buildings and uses nearest-neighbor-only communication patterns.

Using three structures of varied sized, PFRAME3D is shown to compute reproducible results that agree with that of the optimized 1-core version (displacement time-history response root-mean-squared errors are ~〖10〗^(-5) m) with much less wall time (e.g., a dynamic time-history collapse simulation of a 60-story building is computed in 5.69 hrs with 128 cores—a speedup of 14.7 vs. the optimized 1-core version). The maximum speedups attained are shown to increase with building height (as the total number of cores used also increases), and the parallel framework can be expected to be suitable for buildings taller than the ones presented here.

PFRAME3D is used to analyze a hypothetical 60-story steel moment-frame tube building (fundamental period of 6.16 sec) designed according to the 1994 Uniform Building Code. Dynamic pushover and time-history analyses are conducted. Multi-story shear-band collapse mechanisms are observed around mid-height of the building. The use of closely-spaced columns and deep beams is found to contribute to the building's “somewhat brittle” behavior (ductility ratio ~2.0). Overall building strength is observed to be sensitive to whether a model is fracture-capable.

Some uses of the spontaneous polarization method

As a partial fulfillment of the requirements in obtaining a Professional Degree in Geophysical Engineering at the California Institute of Technology. Spontaneous Polarization method of electrical exploration was chosen as the subject of this thesis. It is also known as "self potential electrical prospecting" and "natural currents method."

The object of this thesis is to present a spontaneous polarization exploration work done by the writer, and to apply analytical interpretation methods to these field results.

The writer was confronted with the difficulty of finding the necessary information in a complete paper about this method. The available papers are all too short and repeat the usual information, giving the same examples. The decision was made to write a comprehensive paper first, including the writer's experience, and then to present the main object of the thesis.

The following paper comprises three major parts:

1 - A comprehensive treatment of the spontaneous polarization method.

2 - Report of the field work.

3 - Analytical interpretation of the field work results.

The main reason in choosing this subject is that this method is the most reliable, easiest and requires the least equipment in prospecting for sulphide orebodies on unexplored, rough terrains.

The intention of the writer in compiling the theoretical and analytical information has been mainly to prepare a reference paper about this method.

The writer wishes to express his appreciation to Dr. G. W. Potapenko, Associate Professor of Physics at California Institute of Technology, for his generous help.

Decoding cosets of first-order Reed-Muller code

Proper encoding of transmitted information can improve the performance of a communication system. To recover the information at the receiver it is necessary to decode the received signal. For many codes the complexity and slowness of the decoder is so severe that the code is not feasible for practical use. This thesis considers the decoding problem for one such class of codes, the comma-free codes related to the first-order Reed-Muller codes.

A factorization of the code matrix is found which leads to a simple, fast, minimum memory, decoder. The decoder is modular and only n modules are needed to decode a code of length 2ⁿ. The relevant factorization is extended to any code defined by a sequence of Kronecker products.

The problem of monitoring the correct synchronization position is also considered. A general answer seems to depend upon more detailed knowledge of the structure of comma-free codes. However, a technique is presented which gives useful results in many specific cases.

Finite-Element Simulations of Earthquakes

This thesis discusses simulations of earthquake ground motions using prescribed ruptures and dynamic failure. Introducing sliding degrees of freedom led to an innovative technique for numerical modeling of earthquake sources. This technique allows efficient implementation of both prescribed ruptures and dynamic failure on an arbitrarily oriented fault surface. Off the fault surface the solution of the three-dimensional, dynamic elasticity equation uses well known finite-element techniques. We employ parallel processing to efficiently compute the ground motions in domains containing millions of degrees of freedom.

Using prescribed ruptures we study the sensitivity of long-period near-source ground motions to five earthquake source parameters for hypothetical events on a strike-slip fault (M_w 7.0 to 7.1) and a thrust fault (M_w 6.6 to 7.0). The directivity of the ruptures creates large displacement and velocity pulses in the ground motions in the forward direction. We found a good match between the severity of the shaking and the shape of the near-source factor from the 1997 Uniform Building Code for strike-slip faults and thrust faults with surface rupture. However, for blind thrust faults the peak displacement and velocities occur up-dip from the region with the peak near-source factor. We assert that a simple modification to the formulation of the near-source factor improves the match between the severity of the ground motion and the shape of the near-source factor.

For simulations with dynamic failure on a strike-slip fault or a thrust fault, we examine what constraints must be imposed on the coefficient of friction to produce realistic ruptures under the application of reasonable shear and normal stress distributions with depth. We found that variation of the coefficient of friction with the shear modulus and the depth produces realistic rupture behavior in both homogeneous and layered half-spaces. Furthermore, we observed a dependence of the rupture speed on the direction of propagation and fluctuations in the rupture speed and slip rate as the rupture encountered changes in the stress field. Including such behavior in prescribed ruptures would yield more realistic ground motions.

Proton magnetic resonance studies of ribonucleic acid complexes. I. Complexes of biological bases and oligonucleotides with RNA. II. Template recognition and the degeneracy of the genetic code

Part I. Complexes of Biological Bases and Oligonucleotides with RNA

The physical nature of complexes of several biological bases and oligonucleotides with single-stranded ribonucleic acids have been studied by high resolution proton magnetic resonance spectroscopy. The importance of various forces in the stabilization of these complexes is also discussed.

Previous work has shown that purine forms an intercalated complex with single-stranded nucleic acids. This complex formation led to severe and stereospecific broadening of the purine resonances. From the field dependence of the linewidths, T₁ measurements of the purine protons and nuclear Overhauser enhancement experiments, the mechanism for the line broadening was ascertained to be dipole-dipole interactions between the purine protons and the ribose protons of the nucleic acid.

The interactions of ethidium bromide (EB) with several RNA residues have been studied. EB forms vertically stacked aggregates with itself as well as with uridine, 3'-uridine monophosphate and 5'-uridine monophosphate and forms an intercalated complex with uridylyl (3' → 5') uridine and polyuridylic acid (poly U). The geometry of EB in the intercalated complex has also been determined.

The effect of chain length of oligo-A-nucleotides on their mode of interaction with poly U in D₂0 at neutral pD have also been studied. Below room temperatures, ApA and ApApA form a rigid triple-stranded complex involving a stoichiometry of one adenine to two uracil bases, presumably via specific adenine-uracil base pairing and cooperative base stacking of the adenine bases. While no evidence was obtained for the interaction of ApA with poly U above room temperature, ApApA exhibited complex formation of a 1:1 nature with poly U by forming Watson-Crick base pairs. The thermodynamics of these systems are discussed.

Part II. Template Recognition and the Degeneracy of the Genetic Code

The interaction of ApApG and poly U was studied as a model system for the codon-anticodon interaction of tRNA and mRNA in vivo. ApApG was shown to interact with poly U below ~20°C. The interaction was of a 1:1 nature which exhibited the Hoogsteen bonding scheme. The three bases of ApApG are in an anti conformation and the guanosine base appears to be in the lactim tautomeric form in the complex.

Due to the inadequacies of previous models for the degeneracy of the genetic code in explaining the observed interactions of ApApG with poly U, the "tautomeric doublet" model is proposed as a possible explanation of the degenerate interactions of tRNA with mRNA during protein synthesis in vivo.