Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

<div><p>Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining <i>in vitro</i> transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.</p></div

Removal of universal PCR primer sequences.

<p>(A) Experimental design. (B,C) 2% agarose gel electrophoresis. An RNA ladder is used as single-stranded ladder. lane A – RNA, lane B – sodium hydroxide degradation of RNA, lane C – cDNA (step 1), lane D – exonuclease I hydrolysis of cDNA, lane E – 50 bp ssDNA (step 5; b), and lane E – 50 bp ssDNA (step 6; c), and lane F – exonuclease I hydrolysis of ssDNA.</p

<i>In vitro</i> transcription and reverse transcription (IVT-RT).

<p>(A) Experimental design. (B) 2% agarose gel electrophoresis. An RNA ladder is used as single-stranded ladder. lane A – RNA (step 3), lane B – cDNA (step 5), and lane C – spin-column purified cDNA (step 5).</p

Primer sequences.

<p>*The restriction enzyme sequence is underlined.</p><p>**T7 promoter sequence is in bold.</p

Exonucleolytic hydrolysis of 5<b>′</b>-phosphorylated strand.

<p>(A) Experimental design. (B) lane A – emulsion PCR product (step 2), lane B – exonuclease I hydrolysis of PCR product, lane C – ssDNA product of the lambda exonuclease treatment after removal of PBS (fig. 4 step 6; c), and lane D – exonuclease I hydrolysis of PBS-free ssDNA.</p

Alkaline denaturation method.

<p>(A) Experimental design. (B) lane A – emulsion PCR product (step2), lane B – Nb.BtsI nicked ssDNA (step 3), lane C – nicked DNA (step 4), lane D alkali-melting of nicked ssDNA (step 6), lane E – one of negative selection (step 7), lane F – exonuclease I hydrolysis step 7 products.</p

Determination of library coverage.

<p>(a) Experimental design. (b) Distribution of cDNA signal intensity. (c) Venn diagram – Present call per array.</p

From Design to Screening: A New Antimicrobial Peptide Discovery Pipeline

<div><p>Antimicrobial peptides (AMPs) belong to a class of natural microbicidal molecules that have been receiving great attention for their lower propensity for inducing drug resistance, hence, their potential as alternative drugs to conventional antibiotics. By generating AMP libraries, one can study a large number of candidates for their activities simultaneously in a timely manner. Here, we describe a novel methodology where <i>in silico</i> designed AMP-encoding oligonucleotide libraries are cloned and expressed in a cellular host for rapid screening of active molecules. The combination of parallel oligonucleotide synthesis with microbial expression systems not only offers complete flexibility for sequence design but also allows for economical construction of very large peptide libraries. An application of this approach to discovery of novel AMPs has been demonstrated by constructing and screening a custom library of twelve thousand plantaricin-423 mutants in <i>Escherichia coli</i>. Analysis of selected clones by both Sanger-sequencing and 454 high-throughput sequencing produced a significant amount of data for positionally important residues of plantaricin-423 responsible for antimicrobial activity and, moreover, resulted in identification of many novel variants with enhanced specific activities against <i>Listeria innocua</i>. This approach allows for generation of fully tailored peptide collections in a very cost effective way and will have countless applications from discovery of novel AMPs to gaining fundamental understanding of their biological function and characteristics.</p> </div

Antimicrobial activity of wild-type and mutant plantaricin peptides against <i>Listeria innocua</i> 33090.

<p>ND; not determined.</p

Activities and amino acid sequences of ten Pln-423 mutants tested against <i>L. innocua</i> 33090.

<p>(<b>A</b>) Clear zones indicate growth inhibition of the tester strain due to presence of active peptides produced by host colonies in the center. Wild-type Pln-423 (WT) and NC (negative control with no AMP expression) was included as controls. (<b>B</b>) Corresponding amino acid sequences of the mutants tested in A (mutations are shown in red).</p