Name: Eriks Rozners
Position: Assistant professor, chemistry/chemical biology, Northeastern University
Background: Postdoc, organic chemistry, University of Michigan, Ann Arbor — 1999-2000
Postdoc, organic chemistry, University of Wisconsin, Madison — 1997-1999
Postdoc, bio-organic chemistry, Karolinska Institute — 1996-1997
Postdoc, bio-organic chemistry, Stockholm University — 1994-1996
PhD, organic chemistry, Riga Technical University — 1993
At Northeastern University, Eriks Rozners investigates the chemistry and biochemistry of nucleic acids, focusing on RNA structure and function. Recently, he was awarded a five-year grant from the National Institute of General Medical Sciences to research whether internucleoside amides can be used to modify siRNAs, improving their enzymatic stability and cellular uptake.
Though in its earliest stages, his work has already garnered the attention of at least one major RNAi player, Dharmacon, which is a collaborator on the NIH-funded project.
This week, RNAi News spoke with Rozners about his efforts with RNAi modifications.
Let’s start with a general overview of your lab and the research you do there.
We are organic chemists here at Northeastern, and we have a relatively small group — we usually have a couple of graduate students, a postdoc, and several undergraduate students.
We are interested in using chemical approaches to explore RNA’s structure and function. Along with that goes our interest in RNAi, [which] would be a practical application for the chemical modifications of RNA that we develop.
We have two interests that drive our research. One is a fundamental academic interest in how chemical modifications affect RNA’s structure and biophysical properties.
[Under our NIH grant], we will develop amide linkages as substitutes for phosphodiesters in RNA. One can think about that as combining the structural elements of proteins and nucleic acids to create an RNA mimic. So we are taking the amide linkage, which is the backbone of proteins and replacing the phosphodiester backbone of RNA. In that way, we’d like to impose some protein-like properties on RNA.
In our lab we do organic synthesis and some biophysical studies — mostly ultraviolet spectroscopy combined with [some] thermodynamic methods such as osmotic stress to look at thermal stability and hydration of nucleic acids. We use these methods to synthesize modified RNAs and to study their biophysical properties, essentially.
[My collaborator on the grant at Vanderbilt University], Martin [Egli], brings [the RNA work] to the next level using crystallography techniques where we can look at the detailed molecular structure [of the modified RNAs]. Hopefully, we’ll be able to compare what we get with spectroscopic and thermodynamic methods with the structural data to get better insight into how chemical modification affects RNA. We’re really excited about this possibility.
These amide modifications, can you talk about what they involve and why they are appealing for RNAi?
My interest in amides started somewhere in the mid 90s when I was a postdoc at Stockholm University. That was a time when antisense was very popular.
A lot of modifications were tried in DNA — altogether something like 200 modifications have been reviewed in the literature — but at that time almost no work was done in RNA. No one was really interested in chemical modifications of RNA and RNA interference was not yet discovered.
Out of the 200 modifications tried, relatively few supported the structure of DNA, and amides were among the best. At that point I was working on RNA chemistry and was interested in seeing what this modification would do in RNA, so I started my own independent project on checking the properties of amide-modified RNA.
It turned out to be very interesting that the properties of amides in RNA were different than in DNA. In DNA, amides were structurally and thermally fairly neutral — they seemed to have surprisingly little effect on DNA structure and thermal stability. In RNA, amides turned out to be even better substitutes for phosphodiesters than in DNA. The amides did not change the overall structure of A-type RNA. Moreover, in contrast to DNA, one of the isomeric amides remarkably increased the thermal stability of RNA.
Why would that be important for RNA interference? The current wisdom is that for RNAi the overall structure of the siRNA’s duplex is very important. The identity of the functional groups, especially 2’ hydroxyl, seems to be less important. There are a couple of fairly successful modifications that actually replace 2’-hydroxyl with 2’-fluorine or with a 2’-methoxy group, and they seem to be well-tolerated in RNA interference.
So we hypothesized that maybe a phosphodiester linkage could be replaced by amides and would not change the overall structure of the RNA duplex, [while being] quite well tolerated in RNA interference. Hydrophobic non-ionic linkages, such as amides, may offer several advantages for siRNAs. First, the absence of the natural phosphate will confer high nuclease resistance to such RNA analogues. Second, the reduction of the negative charge may facilitate crossing of cellular membranes. Third, the increased hydrophobicity may favor binding to serum transport proteins, which would improve the biodistribution and pharmacokinetics of the modified siRNAs.
At this point, what’s the extent of the testing done with these amides in RNAi?
We are in early stages of the project. Our first aim is to develop efficient synthetic methods to introduce amide linkages in RNA. The challenge there is that we’d like to have the ability for an automated synthesizer to switch during RNA synthesis between phosphodiesters and amides at will. So instead of having four monomers we use to prepare RNA, we’d have eight, and we’d be able to introduce phosphodiesters and amides at any position we’d like. It will probably take us one or two years to get that working. However, we hope to have the first amide modified siRNAs tested even before the synthesis is fully optimized.
We are very interested in structural studies not just [to satisfy our] fundamental interest in RNA, but also to understand the molecular basis of RNA interference. We’d like to understand why the chemical modifications are or are not accepted by RNAi proteins.
We’d like to test the modified RNAs as soon as possible but of course that goal depends on the chemistry, and I must admit we are at the early stages there.
You’re also collaborating with Dharmacon on this project, right?
That’s right. [Dharmacon’s director of biology research and development], Devin Leake, is a consultant on our grant. They are interested in testing amide-modified RNAs.
Is there an arrangement giving them rights to the commercial applications of amides in RNAi, or has that not yet been envisioned in the arrangement?
We have not discussed it yet. At this stage we are focused on getting the compounds synthesized and tested. Before the project received the NIH funding even the synthesis of the modified siRNAs was only a proposal and a challenging one without substantial support. We have the NIH support now, [and] I expect we will get to the practical aspects and potential commercialization soon, especially if the amides are RNAi-friendly. There are other companies [that] might be interested in this area as well.
Can’t put the cart before the horse, as they say.
This is a five-year grant?
Yes, this is a five-year grant, and is my first NIH grant. It is a very exciting time in my laboratory. We are looking for new students and postdocs to join the group. I am also very interested in establishing new collaborations, both on structural aspects of chemically modified RNA and on biology of RNA interference.
How much is the grant worth?
I would not be able to give you precise numbers because the NIH only gives money for the first year, and all the other years are pending. The budgets of the federal funding agencies have been a very sensitive matter lately. We expect that the overall expenses will be somewhere around $1.25 million for the five years, but that’s a very rough estimate.