Overcoming the design, build, test bottleneck for synthesis of nonrepetitive protein-RNA cassettes.

We apply an oligo-library and machine learning-approach to characterize the sequence and structural determinants of binding of the phage coat proteins (CPs) of bacteriophages MS2 (MCP), PP7 (PCP), and Qß (QCP) to RNA. Using the oligo library, we generate thousands of candidate binding sites for each CP, and screen for binding using a high-throughput dose-response Sort-seq assay (iSort-seq). We then apply a neural network to expand this space of binding sites, which allowed us to identify the critical structural and sequence features for binding of each CP. To verify our model and experimental findings, we design several non-repetitive binding site cassettes and validate their functionality in mammalian cells. We find that the binding of each CP to RNA is characterized by a unique space of sequence and structural determinants, thus providing a more complete description of CP-RNA interaction as compared with previous low-throughput findings. Finally, based on the binding spaces we demonstrate a computational tool for the successful design and rapid synthesis of functional non-repetitive binding-site cassettes.

Author Correction: Data storage in DNA with fewer synthesis cycles using composite DNA letters.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Data storage in DNA with fewer synthesis cycles using composite DNA letters.

The density and long-term stability of DNA make it an appealing storage medium, particularly for long-term data archiving. Existing DNA storage technologies involve the synthesis and sequencing of multiple nominally identical molecules in parallel, resulting in information redundancy. We report the development of encoding and decoding methods that exploit this redundancy using composite DNA letters. A composite DNA letter is a representation of a position in a sequence that consists of a mixture of all four DNA nucleotides in a predetermined ratio. Our methods encode data using fewer synthesis cycles. We encode 6.4 MB into composite DNA, with distinguishable composition medians, using 20% fewer synthesis cycles per unit of data, as compared to previous reports. We also simulate encoding with larger composite alphabets, with distinguishable composition deciles, to show that 75% fewer synthesis cycles are potentially sufficient. We describe applicable error-correcting codes and inference methods, and investigate error patterns in the context of composite DNA letters.

An Assay for Quantifying Protein-RNA Binding in Bacteria.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

Synthetic 5' UTRs Can Either Up- or Downregulate Expression upon RNA-Binding Protein Binding.

The construction of complex gene-regulatory networks requires both inhibitory and upregulatory modules. However, the vast majority of RNA-based regulatory "parts" are inhibitory. Using a synthetic biology approach combined with SHAPE-seq, we explored the regulatory effect of RNA-binding protein (RBP)-RNA interactions in bacterial 5' UTRs. By positioning a library of RNA hairpins upstream of a reporter gene and co-expressing them with the matching RBP, we observed a set of regulatory responses, including translational stimulation, translational repression, and cooperative behavior. Our combined approach revealed three distinct states in vivo: in the absence of RBPs, the RNA molecules can be found in either a molten state that is amenable to translation or a structured phase that inhibits translation. In the presence of RBPs, the RNA molecules are in a semi-structured phase with partial translational capacity. Our work provides new insight into RBP-based regulation and a blueprint for designing complete gene-regulatory circuits at the post-transcriptional level.

An in Vivo Binding Assay for RNA-Binding Proteins Based on Repression of a Reporter Gene.

We study translation repression in bacteria by engineering a regulatory circuit that functions as a binding assay for RNA binding proteins (RBP) in vivo. We do so by inducing expression of a fluorescent protein-RBP chimera, together with encoding its binding site at various positions within the ribosomal initiation region (+11-13 nt from the AUG) of a reporter module. We show that when bound by their cognate RBPs, the phage coat proteins for PP7 (PCP) and Qß (QCP), strong repression is observed for all hairpin positions within the initiation region. Yet, a sharp transition to no-effect is observed when positioned in the elongation region, at a single-nucleotide resolution. Employing in vivo Selective 2'-hydroxyl acylation analyzed by primer extension followed by sequencing (SHAPE-seq) for a representative construct, established that in the translationally active state the mRNA molecule is nonstructured, while in the repressed state a structured signature was detected. We then utilize this regulatory phenomena to quantify the binding affinity of the coat proteins of phages MS2, PP7, GA, and Qß to 14 cognate and noncognate binding sites in vivo. Using our circuit, we demonstrate qualitative differences between in vitro to in vivo binding characteristics for various variants when comparing to past studies. Furthermore, by introducing a simple mutation to the loop region for the Qß-wt site, MCP binding is abolished, creating the first high-affinity QCP site that is completely orthogonal to MCP. Consequently, we demonstrate that our hybrid transcriptional-post-transcriptional circuit can be utilized as a binding assay to quantify RNA-RBP interactions in vivo.

A Synthetic Oligo Library and Sequencing Approach Reveals an Insulation Mechanism Encoded within Bacterial σ54 Promoters.

We use an oligonucleotide library of >10,000 variants to identify an insulation mechanism encoded within a subset of σ54 promoters. Insulation manifests itself as reduced protein expression for a downstream gene that is expressed by transcriptional readthrough. It is strongly associated with the presence of short CT-rich motifs (3-5 bp), positioned within 25 bp upstream of the Shine-Dalgarno (SD) motif of the silenced gene. We provide evidence that insulation is triggered by binding of the ribosome binding site (RBS) to the upstream CT-rich motif. We also show that, in E. coli, insulator sequences are preferentially encoded within σ54 promoters, suggesting an important regulatory role for these sequences in natural contexts. Our findings imply that sequence-specific regulatory effects that are sparsely encoded by short motifs may not be easily detected by lower throughput studies. Such sequence-specific phenomena can be uncovered with a focused oligo library (OL) design that mitigates sequence-related variance, as exemplified herein.

Using synthetic bacterial enhancers to reveal a looping-based mechanism for quenching-like repression.

We explore a model for 'quenching-like' repression by studying synthetic bacterial enhancers, each characterized by a different binding site architecture. To do so, we take a three-pronged approach: first, we compute the probability that a protein-bound dsDNA molecule will loop. Second, we use hundreds of synthetic enhancers to test the model's predictions in bacteria. Finally, we verify the mechanism bioinformatically in native genomes. Here we show that excluded volume effects generated by DNA-bound proteins can generate substantial quenching. Moreover, the type and extent of the regulatory effect depend strongly on the relative arrangement of the binding sites. The implications of these results are that enhancers should be insensitive to 10-11 bp insertions or deletions (INDELs) and sensitive to 5-6 bp INDELs. We test this prediction on 61 σ(54)-regulated qrr genes from the Vibrio genus and confirm the tolerance of these enhancers' sequences to the DNA's helical repeat.

Therapeutics of hearing loss: expectations vs reality.

With the completion of the sequencing of the human genome, the field of medicine is undergoing a dramatic and fundamental change. The identification of our genes and the proteins they encode and the mechanisms of mutations that are pathogenic will allow us to devise revolutionary new ways to diagnose, treat and prevent the thousands of disorders that affect us. Certainly, disorders of the auditory system are no exception. Revealing the molecular mechanisms of hearing and understanding the role of each player in the intricate auditory network could enable us to employ gene- or cell-based therapy to cure or prevent hearing loss. To this end, much emphasis has been placed on the identification and characterization of genes involved in human deafness, as well as research on mouse models for deafness. Ultimately, the effect of genomics on medicine will be dramatic, providing us with the ability to cure sensory defects, a tangible goal that is now within our reach.

PU.1 silencing leads to terminal differentiation of erythroleukemia cells.

The transcription factor PU.1 plays a central role in development and differentiation of hematopoietic cells. Evidence from PU.1 knockout mice indicates a pivotal role for PU.1 in myeloid lineage and B-lymphocyte development. In addition, PU.1 is a key player in the development of Friend erythroleukemia disease, which is characterized by proliferation and differentiation arrest of proerythrocytes. To study the role of PU.1 in erythroleukemia, we have used murine erythroleukemia cells, isolated from Friend virus-infected mice. Expression of PU.1 small interfering RNA in these cells led to significant inhibition of PU.1 levels. This was accompanied by inhibition of proliferation and restoration in the ability of the proerythroblastic cells to produce hemoglobin, i.e., reversion of the leukemic phenotype. The data suggest that overexpression of PU.1 gene is the immediate cause for maintaining the leukemic phenotype of the disease by retaining the self-renewal capacity of transformed erythroblastic cells and by blocking the terminal differentiation program towards erythrocytes.