The Beal laboratory of chemical biology uses synthetic chemistry and synthetic biology to study and control ribonucleic acids (RNAs).  RNAs are structurally complex biopolymers whose proper function is central to all life including bacteria, viruses and humans. Our work is advancing the understanding of essential basic science principles of RNA structure and recognition and is providing a platform for the development of new medicines targeting RNA or RNA-binding proteins. We create new RNA molecules by chemical synthesis with nucleoside analogs, introducing novel and desirable features.  In addition, new small molecules are synthesized that are capable of perturbing natural RNA function or the function of RNA-modifying enzymes.  Finally, directed evolution carried out in our lab generates new RNA structures and new RNA-modifying enzymes with valuable properties.  We have applied these approaches to the study of RNA-editing adenosine deaminases, RNA interference by short interfering RNAs (siRNAs), the RNA-dependent protein kinase (PKR), and RNA-targeted threading intercalators.  Ongoing projects in the Beal lab are described below, with links to published work.

This is a term that refers to structural changes in an RNA molecule that change its coding properties.  We have an ongoing interest in RNA editing enzymes called ADARs and published a review on this topic in Chemical Reviews in 2006 (Maydanovych '06a).  ADARs are adenosine deaminases that act on RNA and are responsible for editing reactions that occur in eukaryotic mRNAs, including the mRNAs for glutamate and serotonin receptors. The deamination of adenosine in the mRNA results in inosine at that position. Since inosine is translated as guanosine, the ADAR reaction can lead to codon changes in the mRNA strand.  This results in the synthesis of proteins that are structurally and functionally distinct from those encoded in the genome.  Indeed, the diversity of protein structures created by this process appears to be essential for proper functioning of the mammalian central nervous system.  Our understanding of the molecular basis for the editing reactions is still limited. For instance, how specific adenosines in the mRNA are targeted for deamination is not completely understood. The goals of our RNA editing project are 1) to define the molecular mechanism of the RNA-editing adenosine deamination reaction and 2) develop reagents capable of controlling ADAR activity in vivo.

Much of what we have learned about the ADAR reaction mechanism has come from studies with substrates bearing chemically-synthesized nucleoside analogs (Brunnozi '99, Véliz '00, Stephens '00, Easterwood '00, Véliz '01a, Brunnozi '01, Véliz '01b, Véliz '03, Stephens '04, Haudenschild '04, Maydanovych '06b, Maydanovych '07). Through the analysis of a library of these analogs, substrate structure/editing activity relationships have been defined, which in turn led to the generation of a nucleoside analog capable of mechanism-based trapping of ADAR2 (Véliz'03, Haudenschild '04). We found that substitution in RNA of an editing site adenosine with the nucleoside analog 8-azanebularine leads to the trapping of ADAR2 bound to RNA.  This is most likely the result of the formation of a covalent hydrate of the nucleobase in the active site of the enzyme that has structural features of the deamination transition state.

New syntheses are typically required to generate the nucleoside analogs necessary for these studies. For instance, we recently reported the synthesis of several new analogs of 8-azanebularine substituted at C6 as potential inhibitors of the ADAR2 enzyme (Maydanovych '06b, see scheme below).

Another approach taken by our group to study RNA editing falls in the general category of synthetic biology.  We have developed an artificial reporter system in the yeast Saccharomyces cerevisiae that causes the yeast colonies to change color when RNA editing reactions take place inside the yeast cells (Pokharel '06).  This is possible because A to I RNA editing can convert a stop codon into a tryptophan codon.  Thus, expression of a reporter enzyme (a-galactosidase in this case) occurs only if an in-frame stop codon is edited by an ADAR.  This system allows for the rapid screening of libraries of mutant enzymes, ADAR substrates and inhibitor/activator molecules.

Our laboratory has studied another fascinating RNA-binding protein named the RNA-dependent protein kinase or PKR (Spanggord '00, Vuyisich '00, Spanggord '01, Jammi '01, Spanggord '02, Vuyisich '02a, Carlson '02, Vuyisich '02b, Jammi '03, Puthenveetil '04, Véliz '06, Putheveetil '06).  PKR is a component of the interferon signaling system, a collection of pathways that lead to growth inhibition in response to viral infection.  PKR is activated by binding to RNA molecules with extensive duplex secondary structure.  Activated PKR then phosphorylates the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2a).  Phosphorylation of eIF2a has the effect of inhibiting continued initiation of protein synthesis by the eIF2 complex. For the efficient synthesis of its proteins, a virus must inhibit the activity of PKR. Several strategies for viral inhibition of PKR are known, including virally encoded RNA molecules that bind to PKR's RNA-binding domain and block activation. Our efforts in this project have been directed at understanding the binding selectivity of PKR's RNA-binding domain, the differences between activating and inhibiting RNA ligands, the steps in the RNA activation mechanism along with ways to control the activity of PKR using synthetic compounds.  Recently, we also investigated the relationship between PKR activation and off-target effects observed in RNA interference experiments using short interfering RNAs (siRNAs) (see below).

PKR (and other related dsRNA-binding proteins) do not bind RNA sequence-specifically.  This can complicate analysis of PKR-RNA interactions, since multiple complexes often exist in solution when PKR is added to an RNA ligand.  Therefore, we adapted the technique of affinity cleaving to the study of PKR-RNA interactions (Spanggord '01, Spanggord '02, Vuyisich '02a, Carlson '02).  This technique had been used for other ligand-nucleic acid interactions and can provide useful low-resolution structural information, even when multiple complexes exist.  This allowed us to suggest binding modes for PKR with various RNA ligands including those that activate the kinase and those that block activation.  During these studies, we also developed a method to site-specifically modify a PKR ligand to block binding at the specified site (Puthenveetil '04).  This work was published in 2004 in the March issue of the journal ChemBioChem and featured on its cover.  Since it was known that PKR binds duplex RNA through minor groove contacts, we synthesized RNA with a sterically occluded minor groove at certain sites.  This was accomplished by adding a benzyl group to the minor groove at the N2 position of a pre-selected guanosine residue.  When presented with such a modified RNA structure, PKR fails to bind at the benzylated binding site.   Ongoing studies involve exploring the extent to which different modifications control binding by PKR and other duplex RNA binding proteins.

The studies described above provided a method for identifying PKR binding sites on an RNA ligand and a method for blocking the protein from interactions with the RNA at specified sites.  We then turned our attention to the study of PKR binding to siRNAs.  Several labs had shown that PKR could bind siRNAs.  Furthermore, this interaction appeared to lead to PKR activation, an effect that would be undesirable in most RNA interference (RNAi) experiments.  Indeed, activation of PKR can be placed in the category of unwanted "off-target" effects that limit the use of RNAi.  In a paper published in 2006 in Nucleic Acids Research (Puthenveetil '06), we confirmed that siRNAs activate PKR in vitro, albeit much more weakly than a long duplex RNA polymer (polyI•polyC).  Based on where PKR bound the siRNA duplexes and how benzyl modifications effected binding and activation, we proposed a model for activation of PKR by siRNAs that involved the assembly of a PKR dimer on a single siRNA duplex. We continue to study the effect on PKR binding, RNAi potency and off-target effects for different chemical modifications of siRNAs.  For instance, 2-aminopurine derivatives have been prepared with various alkyl amines substituted for the 2-amino group (see scheme below). These compounds form base pairs with uridine in siRNAs and project various functional groups into the siRNA minor groove.

A common goal for many chemical biologists is the discovery of small molecule ligands for target biomolecules (e.g. proteins, DNA, RNA, oligosaccharides etc.).  Once identified, these compounds become powerful tools in the study of the function of their targets in their native environments.  In addition, since the biological targets are often of therapeutic interest, new medicines can be developed from small molecule ligands for these targets.  In our laboratory, we have pursued small molecules capable of tight, selective binding to RNA Carlson '00a , Carlson '00b , Carlson '02a, Carlson '03, Krishnamurthy '04, Gooch '04, Carlson '05, Gooch '05, Krishnamurthy '06, Krishnamurthy '07).  Through these studies, we aim to define principles that can guide the design of RNA-binding small molecules that can control the function of target RNAs inside living cells.

Compounds designed in our labs for this purpose are chimeric molecules with peptide-like domains and heterocyclic domains.  The heterocycle is present to allow for stacking on or between RNA base pairs whereas the peptide domain contains amino acids with varying side chain structures providing different functional groups for hydrogen bonding or hydrophobic interactions with target RNAs.  In work partially supported by a Camille Dreyfus Teacher/Scholar Award to PAB, we used in vitro evolution of RNA (SELEX) to discover RNA motifs that are predisposed to bind to molecules with this general design Carlson '03).  From these studies, we found these compounds bind to certain RNA targets in a threading intercalation mode and prefer duplex RNA sites adjacent to helix defects, such as internal loops and bulges.  These studies led to the identification of target sites in naturally occurring RNAs including a site in helix 22 of E. coli 16S ribosomal RNA (Gooch '05).  Our current work in this area is focused on the generation and screening of libraries of macrocyclic helix-threading peptides. These macrocylces are generated using ring-closing metathesis reactions with peptides containing 2-(p-allylphenyl)quinoline residues (Krishnamurthy '07) (see schemes below).