A Chemical Screen for Biological Small Molecule-RNA Conjugates Reveals CoA-Linked RNA

Compared with the rapidly expanding set of known biological roles for RNA, the known chemical diversity of cellular RNA has remained limited primarily to canonical RNA, 3'-aminoacylated tRNAs, nucleobase-modified RNAs, and 5'-capped mRNAs in eukaryotes. We developed two methods to detect in a broad manner chemically labile cellular small molecule-RNA conjugates. The methods were validated by the detection of known tRNA and rRNA modifications. The first method analyzes small molecules cleaved from RNA by base or nucleophile treatment. Application to Escherichia coli and Streptomyces venezuelae RNA revealed an RNA-linked hydroxyfuranone or succinyl ester group, in addition to a number of other putative small molecule-RNA conjugates not previously reported. The second method analyzes nuclease-generated mononucleotides before and after treatment with base or nucleophile and also revealed a number of new putative small molecule-RNA conjugates, including 3'-dephospho-CoA and its succinyl-, acetyl-, and methylmalonyl-thioester derivatives. Subsequent experiments established that these CoA species are attached to E. coli and S. venezuelae RNA at the 5' terminus. CoA-linked RNA cannot be generated through aberrant transcriptional initiation by E. coli RNA polymerase in vitro, and CoA-linked RNA in E. coli is only found among smaller (approximately < 200 nucleotide) RNAs that have yet to be identified. These results provide examples of small molecule-RNA conjugates and suggest that the chemical diversity of cellular RNA may be greater than previously understood.Chemistry and Chemical Biolog

Reversible oligonucleotide chain blocking enables bead capture and amplification of T-Cell receptor alpha and beta chain mRNAs

Next-generation sequencing (NGS) has proven to be an exceptionally powerful tool for studying genetic variation and differences in gene expression profiles between cell populations. However, these population-wide studies are limited by their inability to detect variation between individual cells within a population, inspiring the development of single-cell techniques such as Drop-seq, which add a unique barcode to the mRNA from each cell prior to sequencing. Current Drop-seq technology enables capture, amplification, and barcoding of the entire mRNA transcriptome of individual cells. NGS can then be used to sequence the 3′-end of each message to build a cell-specific transcriptional landscape. However, current technology does not allow high-throughput capture of information distant from the mRNA poly-A tail. Thus, gene profiling would have much greater utility if beads could be generated having multiple transcript-specific capture sequences. Here we report the use of a reversible chain blocking group to enable synthesis of DNA barcoded beads having capture sequences for the constant domains of the T-cell receptor α and β chain mRNAs. We demonstrate that these beads can be used to capture and pair TCRα and TCRβ sequences from total T-cell RNA, enabling reverse transcription and PCR amplification of these sequences. This is the first example of capture beads having more than one capture sequence, and we envision that this technology will be of high utility for applications such as pairing the antigen receptor chains that give rise to autoimmune diseases or measuring the ratios of mRNA splice variants in cancer stem cells

Learning from the unexpected in life and DNA self-assembly

The greatest lessons in life and science often arise from the unexpected. Thus, rather than viewing these experiences as hindering our progress, they should be embraced and appreciated for their ability to lead to new discoveries. In this perspective, I will discuss the unexpected events that have shaped my career path and the early stages of my independent research program

Convenient and Scalable Synthesis of Fmoc-Protected Peptide Nucleic Acid Backbone

The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA

Templated Synthesis of Peptide Nucleic Acids via Sequence-Selective Base-Filling Reactions

Templated Synthesis of Peptide Nucleic Acids via Sequence-Selective Base-Filling Reaction

Evaluating the Effect of Ionic Strength on Duplex Stability for PNA Having Negatively or Positively Charged Side Chains

<div><p>The enhanced thermodynamic stability of PNA:DNA and PNA:RNA duplexes compared with DNA:DNA and DNA:RNA duplexes has been attributed in part to the lack of electrostatic repulsion between the uncharged PNA backbone and negatively charged DNA or RNA backbone. However, there are no previously reported studies that systematically evaluate the effect of ionic strength on duplex stability for PNA having a charged backbone. Here we investigate the role of charge repulsion in PNA binding by synthesizing PNA strands having negatively or positively charged side chains, then measuring their duplex stability with DNA or RNA at varying salt concentrations. At low salt concentrations, positively charged PNA binds more strongly to DNA and RNA than does negatively charged PNA. However, at medium to high salt concentrations, this trend is reversed, and negatively charged PNA shows higher affinity for DNA and RNA than does positively charged PNA. These results show that charge screening by counterions in solution enables negatively charged side chains to be incorporated into the PNA backbone without reducing duplex stability with DNA and RNA. This research provides new insight into the role of electrostatics in PNA binding, and demonstrates that introduction of negatively charged side chains is not significantly detrimental to PNA binding affinity at physiological ionic strength. The ability to incorporate negative charge without sacrificing binding affinity is anticipated to enable the development of PNA therapeutics that take advantage of both the inherent benefits of PNA and the multitude of charge-based delivery technologies currently being developed for DNA and RNA.</p> </div

Chemical structures of backbone-modified PNA.

<p>Chemical structures of backbone-modified PNA.</p

Synthesis of negatively charged PNA monomer.

<p>Synthesis of negatively charged PNA monomer.</p

T_m vs [NaCl] for PNA:RNA 1 duplexes.

<p>(A) PNA (<b>nf</b>/<b>1neg</b>/<b>1pos</b>):RNA <b>1</b>. (B) PNA (<b>nf</b>/<b>3neg</b>/<b>3pos</b>):RNA <b>1</b>. Conditions: 3 µM PNA, 3 µM RNA, 10 mM sodium phosphate buffer with added NaCl, pH 7.2. Error bars represent standard deviation of three or four independent trials.</p