Our group is working on a research program Chemical Approaches to Control the Function of Regulatory RNAs supported by an NIGMS Maximizing Investigators' Research Award (MIRA R35 GM130207). The two research projects of this program focus on using synthetic organic chemistry to explore and modulate structure and function of biologically significant RNA molecules. Research Assistant positions for new graduate students and postdocs are available on both projects.
- Amide-Modified RNA: Synthesis, Structure and Potential for RNA Interference
- Sequence Selective Recognition of Double-Stranded Non-Coding RNA via Triplex Forming PNA also supported by National Science Foundation, Chemistry Division (CHE-1708761).
Amide-Modified RNA: Synthesis, Structure and Potential for RNA Interference
RNA interference (RNAi) has become one of the most powerful and widely used research tools in molecular biology and functional genomics. There is also an intriguing potential that RNAi may become a new therapeutic approach. Despite the remarkable progress, RNAi is far from being a perfect tool and needs significant improvement for in vivo research and therapeutic applications. In particular, the potency, delivery and cellular uptake, biodistribution, and enzymatic stability of short interfering RNAs (siRNAs) must be improved. The long-term goal of this project is to develop chemical modifications that optimize the properties of siRNAs while studying structure and function of RNA in a broader context.
The project builds on our recent discoveries that amide linkage (AM1) is an excellent structural replacement for phosphate in RNA. Currently, we are testing further hypotheses that amides may also mimic the phosphate-amino acid interactions in RNA-protein complexes and may significantly improve the properties of siRNAs. The central innovative aspect of this research is in replacing the negatively charged and polar phosphate with the neutral and relatively hydrophobic amide linkage. Such a dramatic modification of RNA’s backbone has little precedent in the RNAi field and, if successful, will initiate a paradigm shift in our thinking of what can and what cannot be tolerated in siRNAs. Reduction of siRNA’s charge will allow design of novel siRNA-cell penetrating peptide conjugates, which has not been possible with unmodified RNA. Our recent results strongly support the hypothesis that replacement of phosphates with amides will be well tolerated in siRNAs. Current focus in on challenging the paradigm that negatively charged phosphates are required for recognition of RNA by proteins. The specific aims of the project are to:
- Study the RNAi activity of siRNAs having individual phosphates of the guide strand systematically replaced with amides. We hypothesize that 1) amide modifications in the guide strand will not significantly decrease the RNAi activity and 2) the oxygen of amide carbonyl may mimic the hydrogen bonds formed by the non-bridging oxygens of phosphate in RNA-Argonaute (Ago) complexes. We will test these hypotheses in collaboration with colleagues at Dharmacon (part of GE Healthcare) by assaying the silencing activity of amide-modified siRNAs in cell culture. We expect that not all positions of the guide strand will tolerate the amide modification equally well. The differences in sensitivity to amide modifications will provide insights into protein-RNA recognition and RNAi mechanism.
- Study the recognition of amide-modified RNA by proteins using X-ray crystallography, NMR and calorimetric techniques. We hypothesize that the oxygen of amide carbonyl may mimic the hydrogen bonds formed by the non-bridging oxygens of phosphate with amino acids of Piwi. This is similar to hypothesis #2 in Aim 1, except that we will use a structural (X-ray and NMR) rather than a functional (RNAi activity) approach. We will study interactions of amide-modified RNA with proteins in collaboration with Martin Egli (X-ray crystallography, Vanderbilt University) and Scott Kennedy (NMR, University of Rochester).
- Study the cellular uptake and activity of siRNAs having multiple consecutive amide linkages. We hypothesize that multiple amide modifications 1) will be well tolerated in the passenger strand because its phosphates do not interact with Ago and 2) amides will enable (because of the reduced charge) conjugation of siRNA with cationic CPP that was not possible with the negatively charged RNA. We anticipate that CPP conjugation will significantly enhance cellular uptake, which is a major bottleneck in the siRNA field.
The aims of the project are achieved through a comprehensive and multidisciplinary study involving collaborative efforts in synthetic and biophysical chemistry, structural biochemistry, RNA biology, and cell biochemistry and fluorescence microscopy. The main impact of successfully reaching the proposed aims will be improved chemically modified siRNAs for in vivo applications as research tools and, potentially, as lead compounds for drug development. The ultimate goal is to solve the delivery and cellular uptake problems, which would dramatically expand the ability to use RNAi in animals to study physiology of disease. The proposed studies will also advance fundamental knowledge on RNA-protein interactions and provide unique insights into RNAi mechanism.
Sequence Selective Recognition of Double-Stranded Non-Coding RNA via Triplex Forming PNA
The protein-coding mRNA represents only a small fraction of RNA transcribed from genomic DNA. The majority of cellular RNA consists of the so-called non-coding RNAs that play important yet not fully understood roles in regulation of gene expression. Molecular recognition of such regulatory RNAs would be highly useful for fundamental biology and practical applications in biotechnology. However, most non-coding RNAs fold in double-stranded conformations and molecular recognition of such structures is a formidable problem. Designing small molecules that selectively recognize RNA using hydrophobic or electrostatic interactions has been a challenging process. On the other hand, hydrogen bond mediated base pairing, which is the key feature of nucleic acids, has been underutilized in molecular recognition of RNA. This presents a gap in both fundamental knowledge and practical techniques that limit progress in biochemistry and molecular biology. Since the nucleobases of RNA are already base paired in the double helix, the most selective and straightforward sequence readout for double-stranded RNA would be the major groove triple helix formation. However, RNA triple helices have been little studied.
This project builds on our recent discoveries that peptide nucleic acid (PNA) forms highly stable and sequence selective triple helices with double-stranded RNA at physiologically relevant conditions and will test the hypothesis that modified nucleobases (Figure) will allow sequence selective recognition of a vide variety of double-stranded RNAs. The long-term goals of our research are to (1) discover new modes of sequence-selective recognition of double-stranded RNA (dsRNA) and (2) develop novel tools for recognition of and functional interference with biologically relevant non-coding dsRNA. The objectives of this proposal are to:
- Study the triple helical recognition of dsRNA using nucleobase-modified PNA (Figure) at physiologically relevant conditions. We propose that further development and optimization of the modified nucleobases, such as M, P and E, will expand the scope of recognition to sequences common in biologically significant non-coding dsRNA. We will explore the exciting possibility that triple helix formation could be used to recognize any dsRNA sequence.
- Study the conjugation of PNA with cationic peptides to enhance the cellular uptake of PNA. Poor cellular uptake is the most significant bottleneck limiting the applications and impact of PNA technology. We hypothesize that the cationic peptides will increase cellular permeability of PNA while providing additional enhancement of the triple helix stability by binding selectively in the major groove of RNA.
- Study the molecular structure of PNA-RNA triple helices using NMR in collaboration with Pauld Agris (University at Albany). We hypothesize that the strong preference of PNA for binding to dsRNA over dsDNA (observed in our earlier studies) is a result of favorable hydrophobic interactions in the narrow major groove of RNA. Comparison of molecular structure of PNA-dsRNA with PNA-dsDNA triplex will enhance our understanding of nucleic acid recognition and explain the unique affinity of PNA to RNA as well as provide critical information for future design of better RNA binders.
Exploring the sequence selective recognition of RNA duplex will advance fundamental knowledge on molecular recognition of nucleic acids. In contrast to extensive research on DNA triple helices, RNA has received relatively little attention. Surprisingly, before our studies there were no reports on stable RNA triple helices using PNA as the third strand. This represents a significant knowledge gap for molecular recognition of RNA. Development of sequence selective RNA binders is important for understanding the biochemistry of non-coding RNAs and may strongly impact fundamental RNA biology and practical applications in biotechnology. Our results suggest that PNA is an unexpectedly well-suited ligand for recognition of biologically important RNA species and has unique RNA over DNA selectivity.