EvOligo: A Novel Software to Design and Group Libraries of Oligonucleotides Applicable for Nucleic Acid-Based Experiments

Abstract

Experimental methods based on DNA and RNA hybridization, such as multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, or microarray analysis, require the use of mixtures of multiple oligonucleotides (primers or probes) in a single test tube. To provide an optimal reaction environment, minimal self- and cross-hybridization must be achieved among these oligonucleotides. To address this problem, we developed EvOligo, which is a software package that provides the means to design and group DNA and RNA molecules with defined lengths. EvOligo combines two modules. The first module performs oligonucleotide design, and the second module performs oligonucleotide grouping. The software applies a nearest-neighbor model of nucleic acid interactions coupled with a parallel evolutionary algorithm to construct individual oligonucleotides, and to group the molecules that are characterized by the weakest possible cross-interactions. To provide optimal solutions, the evolutionary algorithm sorts oligonucleotides into sets, preserves preselected parts of the oligonucleotides, and shapes their remaining parts. In addition, the oligonucleotide sets can be designed and grouped based on their melting temperatures. For the user's convenience, EvOligo is provided with a user-friendly graphical interface. EvOligo was used to design individual oligonucleotides, oligonucleotide pairs, and groups of oligonucleotide pairs that are characterized by the following parameters: (1) weaker cross-interactions between the non-complementary oligonucleotides and (2) more uniform ranges of the oligonucleotide pair melting temperatures than other available software products. In addition, in contrast to other grouping algorithms, EvOligo offers time-efficient sorting of paired and unpaired oligonucleotides based on various parameters defined by the user.

The RNA–RNA base pairing potential of human Dicer and Ago2 proteins

“…In these assays, in addition to the above-characterized three pairs of substrates, we also used two short RNAs that were differentially base paired with guide RNA, R21. One 21-nt RNA, called "mR21", was designed using EvOligo software computation core [26] to form a double-mismatched duplex with R21, that mimicked a miRNA duplex. The other 21-nt RNA, called "cR21", formed a perfect duplex with R21 that mimicked an siRNA duplex (based on Ameres et al [21]).…”

Short 2′-O-methyl/LNA oligomers as highly-selective inhibitors of miRNA production in vitro and in vivo

“…The lower (more negative) the value, the stronger the interaction between the oligonucleotides. Using EvOligo ( 57 ) we calculated ΔGs for the duplexes formed by unmodified AL-ASOs and their respective miR-181a precursors, as well as AL-ASOs and other pre-miRNAs (potential off-targets identified within known X. laevis precursors). We found that theoretical Δ G for AL:pre-miR-181a-1/-2 duplexes were all below –21 kcal/mol ( Supplementary Table S1 ), which was at least 1.5 times lower than for AL-ASOs pairs with any other pre-miRNA.…”

“…One of the input PTS from the last alignment round was selected and converted into reverse-complement to obtain a control oligomer sequence (Ctrl and h Ctrl). The self- and cross-hybridization free Gibbs energies of all designed oligomers were calculated using EvOligo ( 57 ).…”

Fluorescent Protein Variants Generated by Reassembly between Skeleton and Chromophore

“…In addition, homologous sequences between FPs were preferentially selected as the cutting sites to increase the fragment recombination. Theoretically, the total reassemble clones were A 360 20 + A 450 25 = 1.08 × 10 66 , provided that all of the sections can be recombined freely. Actually, the chances of premature reassembled products, invalid combination products, and wildtype recombination reduced the final obtained clones.…”

“…Oligonucleotide design for synthesis was the key part of this study. Although several algorithms have now been made to design combinatorial oligonucleotide pools, few computationally designed oligonucleotide pools have been characterized experimentally. − DNAworks algorithm was used as it has advantages in the balance of highly homogeneous melting temperatures, minimization of hairpin formation, ease of handling, and experimental verification . Here, 18 widely used FPs were selected as input templates for computer-based oligonucleotide design, and thermally balanced oligonucleotides were massively synthesized in parallel in our light-directed microfluidic arrays.…”