Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

Abstract

The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first time. The main mechanism for LNA ODNs binding plasmid DNA is demonstrated to be by strand displacement. LNA ODNs are more stably bound to plasmid DNA than similar peptide nucleic acid (PNA) 'clamps' for procedures such as particle-mediated DNA delivery (gene gun). It is shown that LNA ODNs remain associated with plasmid DNA after cationic lipid-mediated transfection into mammalian cells. LNA ODNs can bind to DNA in a sequence-specific manner so that binding does not interfere with plasmid conformation or gene expression. Attachment of CpG-based immune adjuvants to plasmid by 'hybrid' phosphorothioate-LNA ODNs induces tumour necrosis factor-alpha production in the macrophage cell line RAW264.7. This observation exemplifies an important new, controllable methodology for adding functionality to plasmids for gene delivery and DNA vaccination.

Figure 1

Binding of ODNs to plasmid and restriction mapping of the binding site. (A) Agarose gel (without EtBr) of 0.023 µM plasmid gWiz incubated with Alexa Fluor 568- (or rhodamine for LNA4) labelled LNA ODNs at high, (H, 4 µM) or low (L, 0.5 µM) ODN concentrations. (B) As (A) with EtBr. Lane M, 1 kb DNA ladder; lane 1, gWiz without ODN. (C) Agarose gel (without EtBr) of plasmids bound with Alexa Fluor 568- labelled LNA ODNs digested with restriction enzymes. (D) As (C) with EtBr. Lane M, 1 kb DNA ladder; lane 1, 0.024 µM pGG2XGFP + 4 µM LNA7 digested with NdeI; lane 2, 0.023 µM gWiz + 4 µM LNA8 digested with BsaI and SphI.

Figure 1

Binding of ODNs to plasmid and restriction mapping of the binding site. (A) Agarose gel (without EtBr) of 0.023 µM plasmid gWiz incubated with Alexa Fluor 568- (or rhodamine for LNA4) labelled LNA ODNs at high, (H, 4 µM) or low (L, 0.5 µM) ODN concentrations. (B) As (A) with EtBr. Lane M, 1 kb DNA ladder; lane 1, gWiz without ODN. (C) Agarose gel (without EtBr) of plasmids bound with Alexa Fluor 568- labelled LNA ODNs digested with restriction enzymes. (D) As (C) with EtBr. Lane M, 1 kb DNA ladder; lane 1, 0.024 µM pGG2XGFP + 4 µM LNA7 digested with NdeI; lane 2, 0.023 µM gWiz + 4 µM LNA8 digested with BsaI and SphI.

Figure 2

Binding of LNA ODNs to plasmid allows complementary DNA ODNs to bind. (A) Agarose gel (without EtBr) of 0.023 µM plasmid gWiz incubated with rhodamine-labelled LNA ODNs at high (H, 4 µM) or low (L, 0.5 µM) ODN concentrations. (B) As (A) with EtBr. Lane M, 1 kb DNA ladder. (C) Agarose gel (without EtBr) of 0.023 µM plasmid gWiz incubated with LNA ODNs at high (H, 4 µM) or low (L, 0.5 µM) ODN concentrations. Unbound LNA ODN was removed, the plasmid–LNA ODN complex was incubated with the fluorescein-labelled DNA primer KH2 (4 µM) and free ODN was again removed prior to loading on a gel (except lane 4). (D) As (C) with EtBr. Lane 1, gWiz + LNA6 H + KH2; lane 2, gWiz + LNA6 L + KH2; lane 3, empty; lane 4, gWiz + KH2.

Figure 2

Binding of LNA ODNs to plasmid allows complementary DNA ODNs to bind. (A) Agarose gel (without EtBr) of 0.023 µM plasmid gWiz incubated with rhodamine-labelled LNA ODNs at high (H, 4 µM) or low (L, 0.5 µM) ODN concentrations. (B) As (A) with EtBr. Lane M, 1 kb DNA ladder. (C) Agarose gel (without EtBr) of 0.023 µM plasmid gWiz incubated with LNA ODNs at high (H, 4 µM) or low (L, 0.5 µM) ODN concentrations. Unbound LNA ODN was removed, the plasmid–LNA ODN complex was incubated with the fluorescein-labelled DNA primer KH2 (4 µM) and free ODN was again removed prior to loading on a gel (except lane 4). (D) As (C) with EtBr. Lane 1, gWiz + LNA6 H + KH2; lane 2, gWiz + LNA6 L + KH2; lane 3, empty; lane 4, gWiz + KH2.

Figure 3

Sequencing strategy and results to show specific DNA strand displacement upon LNA ODN binding. (A) Sequencing strategy: GeneGrip site 2 on pGG2XGFP, plus bound LNA7 and DNA sequencing primer Cy5 RevGG2B (Table 1). Data displayed in (B–E) start from the marked T* and finish at the end of the arrow (A). (B) Sequence from pGG2XGFP by dsDNA sequencing. Further sequence data by ALF ssDNA sequencing kit, manually deciphered chromatograms: (C) 0.024 µM pGG2XGFP + 0.5 µM LNA7 (3 µg of template, primer annealed at 42°C); (D) 0.024 µM pGG2XGFP + 0.5 µM LNA7 (1 µg of template, primer annealed at 37°C); (E) pGG2XGFP alone (1 µg of template, primer annealed at 37°C).

Figure 3

Sequencing strategy and results to show specific DNA strand displacement upon LNA ODN binding. (A) Sequencing strategy: GeneGrip site 2 on pGG2XGFP, plus bound LNA7 and DNA sequencing primer Cy5 RevGG2B (Table 1). Data displayed in (B–E) start from the marked T* and finish at the end of the arrow (A). (B) Sequence from pGG2XGFP by dsDNA sequencing. Further sequence data by ALF ssDNA sequencing kit, manually deciphered chromatograms: (C) 0.024 µM pGG2XGFP + 0.5 µM LNA7 (3 µg of template, primer annealed at 42°C); (D) 0.024 µM pGG2XGFP + 0.5 µM LNA7 (1 µg of template, primer annealed at 37°C); (E) pGG2XGFP alone (1 µg of template, primer annealed at 37°C).

Figure 4

Binding stability of LNA and PNA ODNs to plasmid in gene gun preparations. (A) Agarose gel (without EtBr) of 0.024 µM plasmid pGG2XGFP incubated with 4 µM Alexa Fluor 568 (LNA) or rhodamine-labelled (PNA) ODNs before (b) and after (a) plasmid binding to gold beads. (B) As (A) with EtBr. Lane M, 100 bp DNA ladder. (C) Agarose gel (without EtBr) of 0.023 µM plasmid gWiz incubated with 4 µM rhodamine-labelled LNA or PNA ODNs after (a) plasmid binding to gold beads, unless stated as before (b). (D) As (C) with EtBr. Lane M, 1 kb DNA ladder; Mirus, pGL3CMV rhodamine labelled with Mirus Label IT kit.

Figure 4

Binding stability of LNA and PNA ODNs to plasmid in gene gun preparations. (A) Agarose gel (without EtBr) of 0.024 µM plasmid pGG2XGFP incubated with 4 µM Alexa Fluor 568 (LNA) or rhodamine-labelled (PNA) ODNs before (b) and after (a) plasmid binding to gold beads. (B) As (A) with EtBr. Lane M, 100 bp DNA ladder. (C) Agarose gel (without EtBr) of 0.023 µM plasmid gWiz incubated with 4 µM rhodamine-labelled LNA or PNA ODNs after (a) plasmid binding to gold beads, unless stated as before (b). (D) As (C) with EtBr. Lane M, 1 kb DNA ladder; Mirus, pGL3CMV rhodamine labelled with Mirus Label IT kit.

Figure 5

Co-localisation of plasmid and bound LNA ODNs when transfected into CHO cells and demonstration that plasmid with bound LNA ODNs shows unmodified gene expression. (A) Co-localisation in CHO cells, 36 h post-transfection, of LNA bound to plasmid, visualised by confocal microscopy. 1, gWiz labelled with fluorescein Mirus Label IT kit, FITC channel; 2, DAPI-stained nuclei, UV light; 3, LNA9R, TRITC channel; 4, overlay of 1, 2 and 3. (B) As (A), but control of gWiz mixed with Alexa Fluor 568-labelled, unbound LNA ODN. 1, gWiz labelled with fluorescein Mirus Label IT kit, FITC channel; 2, DAPI-stained nuclei, UV light; 3, LNA11, TRITC channel; 4, overlay of 1, 2 and 3. (C) Luciferase activity, 24 h post-transfection, in MC5-7 cells transfected by gene gun with gWiz ± LNA or PNA ODNs, or pGL3CMV ± rhodamine Mirus Label IT. Values are a mean of six pooled, independent gene gun transfections. (D) Luciferase activity, 24 h post-transfection, in HeLa cells lipofected by DMRIE-C with gWiz ± LNA or PNA ODNs. Values are a mean of four independent transfections, with standard deviations shown.

Figure 5

Co-localisation of plasmid and bound LNA ODNs when transfected into CHO cells and demonstration that plasmid with bound LNA ODNs shows unmodified gene expression. (A) Co-localisation in CHO cells, 36 h post-transfection, of LNA bound to plasmid, visualised by confocal microscopy. 1, gWiz labelled with fluorescein Mirus Label IT kit, FITC channel; 2, DAPI-stained nuclei, UV light; 3, LNA9R, TRITC channel; 4, overlay of 1, 2 and 3. (B) As (A), but control of gWiz mixed with Alexa Fluor 568-labelled, unbound LNA ODN. 1, gWiz labelled with fluorescein Mirus Label IT kit, FITC channel; 2, DAPI-stained nuclei, UV light; 3, LNA11, TRITC channel; 4, overlay of 1, 2 and 3. (C) Luciferase activity, 24 h post-transfection, in MC5-7 cells transfected by gene gun with gWiz ± LNA or PNA ODNs, or pGL3CMV ± rhodamine Mirus Label IT. Values are a mean of six pooled, independent gene gun transfections. (D) Luciferase activity, 24 h post-transfection, in HeLa cells lipofected by DMRIE-C with gWiz ± LNA or PNA ODNs. Values are a mean of four independent transfections, with standard deviations shown.

Figure 5

Co-localisation of plasmid and bound LNA ODNs when transfected into CHO cells and demonstration that plasmid with bound LNA ODNs shows unmodified gene expression. (A) Co-localisation in CHO cells, 36 h post-transfection, of LNA bound to plasmid, visualised by confocal microscopy. 1, gWiz labelled with fluorescein Mirus Label IT kit, FITC channel; 2, DAPI-stained nuclei, UV light; 3, LNA9R, TRITC channel; 4, overlay of 1, 2 and 3. (B) As (A), but control of gWiz mixed with Alexa Fluor 568-labelled, unbound LNA ODN. 1, gWiz labelled with fluorescein Mirus Label IT kit, FITC channel; 2, DAPI-stained nuclei, UV light; 3, LNA11, TRITC channel; 4, overlay of 1, 2 and 3. (C) Luciferase activity, 24 h post-transfection, in MC5-7 cells transfected by gene gun with gWiz ± LNA or PNA ODNs, or pGL3CMV ± rhodamine Mirus Label IT. Values are a mean of six pooled, independent gene gun transfections. (D) Luciferase activity, 24 h post-transfection, in HeLa cells lipofected by DMRIE-C with gWiz ± LNA or PNA ODNs. Values are a mean of four independent transfections, with standard deviations shown.

Figure 6

‘Hybrid’ CpG PTO–LNA ODNs bind to plasmid and retain CpG activity in vitro. (A) Agarose gel (without EtBr) of 0.027 µM plasmid pGG2XEMPTY incubated with ODNs, labelled either by Ulysis Alexa Fluor 488 or by Alexa Fluor 568 at the 5′ end. (B) As (A) with EtBr. Lane M, 1 kb DNA ladder; lane 1, pGG2xEMPTY; lane 2, pGG2xEMPTY + 4 µM 5′ Alexa Fluor 568-labelled LNA8; lane 3, pGG2xEMPTY + 4 µM Ulysis-labelled LNA8; lane 4, pGG2xEMPTY + 9 µM Ulysis-labelled PTOCpG-LNA; lane 5, pGG2xEMPTY + 9 µM Ulysis-labelled PTOGpC-LNA. (C) Agarose gel (without EtBr) of plasmid incubated with Ulysis-labelled ‘hybrid’ PTO–LNA ODNs before and after S400HR gel exclusion chromatography. (D) As (C) with EtBr. Lane M, 1 kb DNA ladder; 0.027 µM pGG2xEMPTY + 9 µM PTOCpG-LNA before (lane 1) and after S400HR separation (lane 2). (E) TNF-α ELISA data for the dose response curve of gWiz plasmid ± bound PTOCpG–LNA or PTOGpC–LNA, transfected into RAW264.7.

Figure 6

‘Hybrid’ CpG PTO–LNA ODNs bind to plasmid and retain CpG activity in vitro. (A) Agarose gel (without EtBr) of 0.027 µM plasmid pGG2XEMPTY incubated with ODNs, labelled either by Ulysis Alexa Fluor 488 or by Alexa Fluor 568 at the 5′ end. (B) As (A) with EtBr. Lane M, 1 kb DNA ladder; lane 1, pGG2xEMPTY; lane 2, pGG2xEMPTY + 4 µM 5′ Alexa Fluor 568-labelled LNA8; lane 3, pGG2xEMPTY + 4 µM Ulysis-labelled LNA8; lane 4, pGG2xEMPTY + 9 µM Ulysis-labelled PTOCpG-LNA; lane 5, pGG2xEMPTY + 9 µM Ulysis-labelled PTOGpC-LNA. (C) Agarose gel (without EtBr) of plasmid incubated with Ulysis-labelled ‘hybrid’ PTO–LNA ODNs before and after S400HR gel exclusion chromatography. (D) As (C) with EtBr. Lane M, 1 kb DNA ladder; 0.027 µM pGG2xEMPTY + 9 µM PTOCpG-LNA before (lane 1) and after S400HR separation (lane 2). (E) TNF-α ELISA data for the dose response curve of gWiz plasmid ± bound PTOCpG–LNA or PTOGpC–LNA, transfected into RAW264.7.

Figure 6

‘Hybrid’ CpG PTO–LNA ODNs bind to plasmid and retain CpG activity in vitro. (A) Agarose gel (without EtBr) of 0.027 µM plasmid pGG2XEMPTY incubated with ODNs, labelled either by Ulysis Alexa Fluor 488 or by Alexa Fluor 568 at the 5′ end. (B) As (A) with EtBr. Lane M, 1 kb DNA ladder; lane 1, pGG2xEMPTY; lane 2, pGG2xEMPTY + 4 µM 5′ Alexa Fluor 568-labelled LNA8; lane 3, pGG2xEMPTY + 4 µM Ulysis-labelled LNA8; lane 4, pGG2xEMPTY + 9 µM Ulysis-labelled PTOCpG-LNA; lane 5, pGG2xEMPTY + 9 µM Ulysis-labelled PTOGpC-LNA. (C) Agarose gel (without EtBr) of plasmid incubated with Ulysis-labelled ‘hybrid’ PTO–LNA ODNs before and after S400HR gel exclusion chromatography. (D) As (C) with EtBr. Lane M, 1 kb DNA ladder; 0.027 µM pGG2xEMPTY + 9 µM PTOCpG-LNA before (lane 1) and after S400HR separation (lane 2). (E) TNF-α ELISA data for the dose response curve of gWiz plasmid ± bound PTOCpG–LNA or PTOGpC–LNA, transfected into RAW264.7.