Efficient synthesis and replication of diverse sequence libraries composed of biostable nucleic acid analogues

Abstract

Functional nucleic acids can be evolved in vitro using cycles of selection and amplification, starting from diverse-sequence libraries, which are typically restricted to natural or partially-modified polymer chemistries. Here, we describe the efficient DNA-templated synthesis and reverse transcription of libraries entirely composed of serum nuclease resistant alternative nucleic acid chemistries validated in nucleic acid therapeutics; locked nucleic acid (LNA), 2'-O-methyl-RNA (2'OMe-RNA), or mixtures of the two. We evaluate yield and diversity of synthesised libraries and measure the aggregate error rate of a selection cycle. We find that in addition to pure 2'-O-methyl-RNA and LNA, several 2'OMe-RNA/LNA blends seem suitable and promising for discovery of biostable functional nucleic acids for biomedical applications.

Fig. 1. Efficient templated synthesis of pure and mixed-chemistry diverse-sequence 2′OMe-RNA and LNA oligonucleotides. (a) Chemical structures and (b) nucleoside triphosphate (xNTP) combinations of 2′OMe-RNA and LNA used in the study. (c) Diagram and (d) denaturing acrylamide (PAGE) and (e) agarose (AAGE) gels showing DNA-templated synthesis of N₄₀ XNA libraries using the chemistries shown in (b), catalysed by a blend of engineered XNA polymerases “pol3M” and “pol6G12[I521L]”. Primer extensions were visualised by FITC fluorescence; unlabelled molecular weight standards (Mwt) were run on the same gels and revealed by staining with Sybr Gold. Gels are representative of at least three replicate reactions per chemistry.

Fig. 2. Efficient reverse transcription of pure and mixed-chemistry diverse-sequence 2′OMe-RNA and LNA oligonucleotides. (a) Diagram showing strategies for assessment of XNA RT reactions. (b) Urea-PAGE gel showing XNA-templated (FITC, green) cDNA synthesis (Cy5, red). (c) Agarose gels showing amplification of cDNA by RT-PCR. Gels are representative of at least three replicate reactions per chemistry. RTs were catalysed by a blend of engineered XNA polymerases “RT-C8” and “RT521L” primed either by (a)(1a) a Cy5 fluorophore-labelled DNA primer (‘Test’ RT) in order to directly visualise cDNA synthesis (b), or alternatively RTs were primed by (a)(1b) a biotinylated DNA primer (‘X-SELEX’ RT) enabling purification of cDNA and amplification (c) via a two-step PCR strategy: firstly (a)(2) an ‘out-nested’ PCR dependent on a reverse primer site derived from the X-SELEX_RT primer generates templates for, secondly, either (a)(3a) a PCR to generate sequencing libraries preserving a diagnostic mismatched base derived from the RT primer, or instead (a)(3b) an ‘in-nested’ PCR that regenerates the XNA synthesis template (although here is used only as a proof-of-concept demonstration).

Fig. 3. Pure and mixed-chemistry 2′OMe-RNA and LNA synthetic genetic systems enable synthesis and replication of biostable oligonucleotide libraries without substantial biases or loss of library diversity. Graphs show (a) diversity of N₄₀ libraries (DNA shown in ESI Fig. S5), and (b) aggregate error and (c) errors per base following a full cycle of synthesis of pure and mixed-chemistry 2′OMe-RNA and LNA oligonucleotides, reverse transcription and cDNA amplification (DNA → XNA → DNA → DNA sequenced). See also ESI Tables S2 and S3 for further breakdown of errors and numbers of sequences analysed.