Sfold Frequently Asked Questions (and Answers)
1. How is the algorithm for Sfold different from other established algorithms?
- The algorithm generates a statistical sample of RNA secondary structures from the Boltzmann
ensemble of RNA secondary structures. From a statistical mechanics perspective, an RNA
molecule may have a population of structures distributed according to a Boltzmann distribution,
which gives the probability of a secondary structure I at equilibrium as (1/U)exp[-E(I)/RT],
where E(I) is the free energy of the structure, R is the gas constant, T is the absolute
temperature, and U is the partition function for all admissible secondary structures of the RNA
sequence. The algorithm samples secondary structures exactly and rigorously according to the
Boltzmann distribution. The sampling method offers an appealing solution to the problem of
uncertainties in the folding model and in the free energy parameters. For the details of the algorithm and its
unique capabilities listed below, please see our cover article (Ding & Lawrence 2003, Nucleic Acids Res. 31,
2. What are the unique capabilities of the algorithm?
- The Boltzmann ensemble of RNA secondary structures can be efficiently characterized
through significant structural classes by classifying sampled structures.
- The algorithm offers novel probabilistic tools for prediction of RNA target accessibility.
- The algorithm offers novel probabilistic tools for evaluation of RNA/RNA interaction.
- Probabilities of structural motifs such as loops can be easily calculated.
- Free energy distributions are readily available from sampled structures.
3. What is the rationale of Sfold target-accessibility prediction for the design of RNA-targeting nucleic acids?
- Our prediction of accessibility is based on a statistical sample of the Boltzmann ensemble of
secondary structures. This novel approach is appealing for evaluation of target accessibility,
because, as noted by researchers from Sirna Therapeutics (formerly Ribozyme Pharmaceuticals,
Inc.), "In the prediction of
accessible sites, the identification of a single folded structure for a given target mRNA is not of
particular interest. Instead, the objective of this exercise is to assess the likelihood of unpaired
(or substantially unpaired) sites that could be a ribozyme target... The ambiguities in
thermodynamic parameters - and the possibility that each mRNA exists as a population of
different structures - suggest that a stochastic approach to the evaluation of accessible sites
may be appropriate" (Christoffersen, McSwiggen & Konings 1994, J. Mol. Structure
(Theochem) 311, p. 208). The probability profiling approach in Ding and Lawrence
(2001, Nucleic Acids Res. 29, 1034-1046.) reveals target sites that are commonly accessible for
a large number of statistically representative structures for the target RNA. Through assignment
of statistical confidence in predictions, this novel approach bypasses the long-standing difficulty
accessibility evaluation due to limited representation of probable structures.
4. Is there experimental evidence that the potency of siRNAs is influenced by target
accessibility and secondary structure?
- Target accessibility has long been established as an important factor for the potency of
antisense oligonucleotides and trans-cleaving ribozymes. Recently, the importance of target
structure and accessibility in determining the potency of siRNAs has been demonstrated, using a
number of experimental approaches that include oligo library (Lee et al. 2002, Nat. Biotech., 20,
500-505; pp. 502, 503), oligo array (Bohula et al. 2003, J.Biol. Chem. 278, 15991-15997),
antisense evaluation of accessibility (Far and Sczakiel (2003, Nucleic Acids Research, 31, 4417-4424) ), and by targeting the same sequence in both structured and unstructured sites (Vickers et
al. 2003, J. Biol. Chem. 278, 7108-7188; p.7114, left column, top paragraph).
5. How about published empirical rules for siRNA duplex thermodynamics and features?
- siRNA duplex unwinding is a unique feature in RNAi pathway, and the published findings
from Zamore lab, Amgen group, and recently Dharmacon group have important implication for
this step. However, these siRNA duplex rules do not guarantee siRNA function. In the
publications cited above on target structure and accessibility, a number of potent siRNAs do not
meet the key empirical rules, but their function is explained by target accessibility.
It appears to be a consensus view that after the duplex unwinding the antisense strand in the
activated RISC needs to bind to the target sequence through complementary base-paring for
target recognition. This would explain the exquisite specificity by siRNAs. When the target
sequence is in a heavily structured (i.e., helical) region, the large energy barrier will likely
prevent the formation of the hybrid between the antisense siRNA strand and the target sequences.
Zamore lab has shown that a single hydrogen bond can decide which of the duplex strands will
be incorporated into RISC. Likewise, we believe that secondary structure, accessibility and
thermodynamics at the target site are also important.
6. What is Sfold methodology for siRNA design? How is it different from other on-line
- The design method is based on reported scientific evidence on factors that appear to be
important for siRNA function. More specifically, for siRNA screening, Sfold combines target accessibility prediction, siRNA duplex thermodynamic rules as described by Zamore lab and the
Amgen group, and the empirical rules reported by the Dharmacon group. Target accessibility
evaluation is a unique feature of Sfold and is expected to improve the chance of success. For
detailed description of the design tools and design steps using Sfold, please consult the on-line
7. Have the nucleic acid design methods been experimentally validated?
- We have on-going collaborations with "wet" labs at the Wadsworth Center to test our
prediction of target accessibility and our design methods for siRNAs, antisense oligos, and
hammerhead ribozymes. Preliminary results are highly encouraging. Summary of preliminary
testing data can be found in the PDF file from "Validation" page.
8. What else is planned for Sfold?
- More user-friendly tools are under development for each application module.
- By collaborating with experimental labs, we hope to improve the nucleic acid design methods
through experimental feedback. We are currently seeking additional collaborative opportunities
for testing and improving siRNA design.
- Incorporation of DNA parameters for other applications such as design of molecular beacons
and PCR primers and amplicons.
- Development of a new module Stools for folding large number of short RNAs, for designing
control oligos (random, scrambled, mismatched, inverted, etc), for illustrating RNA/RNA
interactions, and for providing other auxiliary tools.
9. How can users contribute to the improvement of the software, for the benefit of the
- By providing experimental feedback.
- By sharing of newly discovered empirical rules, e.g., for the design of siRNAs. Empirical rules
can be easily incorporated into the software for outputting relevant information.
Updated by Ye Ding, April 15, 2004.
For the PDF version of this document, please click here.