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.

The combinatorics of transcriptional regulation

The ability to regulate gene expression is of central importance for the adaptability of living organisms to changes in their internal and external environment. At the transcriptional level, binding of transcription factors (TFs) in the vicinity of promoters can modulate the rate at which transcripts are produced, and as such play an important role in gene regulation. TFs with regulatory action at multiple promoters is the rule rather than the exception, with examples ranging from TFs like the cAMP receptor protein (CRP) in E. coli that regulates hundreds of different genes, to situations involving multiple copies of the same gene, such as on plasmids, or viral DNA. When the number of TFs heavily exceeds the number of binding sites, TF binding to each promoter can be regarded as independent. However, when the number of TF molecules is comparable to the number of binding sites, TF titration will result in coupling ("entanglement") between transcription of different genes. The last few decades have seen rapid advances in our ability to quantitatively measure such effects, which calls for biophysical models to explain these data. Here we develop a statistical mechanical model which takes the TF titration effect into account and use it to predict both the level of gene expression and the resulting correlation in transcription rates for a general set of promoters. To test these predictions experimentally, we create genetic constructs with known TF copy number, binding site affinities, and gene copy number; hence avoiding the need to use free fit parameters. Our results clearly prove the TF titration effect and that the statistical mechanical model can accurately predict the fold change in gene expression for the studied cases. We also generalize these experimental efforts to cover systems with multiple different genes, using the method of mRNA fluorescence in situ hybridization (FISH). Interestingly, we can use the TF titration affect as a tool to measure the plasmid copy number at different points in the cell cycle, as well as the plasmid copy number variance. Finally, we investigate the strategies of transcriptional regulation used in a real organism by analyzing the thousands of known regulatory interactions in E. coli. We introduce a "random promoter architecture model" to identify overrepresented regulatory strategies, such as TF pairs which coregulate the same genes more frequently than would be expected by chance, indicating a related biological function. Furthermore, we investigate whether promoter architecture has a systematic effect on gene expression by linking the regulatory data of E. coli to genome-wide expression censuses.