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Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides.

Boos JA, Kirk DW, Piccolotto ML, Zuercher W, Gfeller S, Neuner P, Dattler A, Wishart WL, Von Arx F, Beverly M, Christensen J, Litherland K, van de Kerkhof E, Swart PJ, Faller T, Beyerbach A, Morrissey D, Hunziker J, Beuvink I - Nucleic Acids Res. (2013)

Bottom Line: Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides.Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image.Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research (NIBR), Novartis Pharma AG, Basel, Basel-Stadt CH-4056, Switzerland and NIBR, Novartis Pharma AG, Cambridge, Massachusetts, MA 02139, USA.

ABSTRACT
Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

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Validation of the biodistribution obtained for AMO-miR-16 using CL-qPCR. (a) Chemical reaction that occurs during the side-by-side target hybridization of a DNA-oligo containing a 3′-phosphorothioate group and a DNA-oligo containing a 5′-biphenylsulphonlyl group. The template-mediated, proximity-dependent reaction results in displacement of the biphenylsulphonyl leaving group and the formation of a carbon–sulphur bond. (BASE represents Adenosine, Guanosine, Thymidine or Cytidine). (b) Quantification of AMO-miR-16 in plasma isolated from AMO-miR-16- (black) and PBS-treated (grey) mice. Values are the average of triplicate measurements from five animals. Error bars, s.e.m (n = 15). *P < 0.05 (Mann–Whitney Rank Sum test). (c) The biodistribution of miR-16 and AMO-miR-16 were determined in mouse whole body sections, treated either with AMO-miR-16 (left panel) or with PBS (right panel) and normalized against genomic 18S. Tissue annotation: Eye (E), Brain (B), Lung (Lu), Heart (H), Liver (Li), Stomach (St), Kidney (K), Bone (Bo) and Gastro-Intestinal tract (GI). (d–f) Quantification of AMO-miR-16 (d), miR-16 (e) and miR-191 (f) in tissues isolated from mice treated either with AMO-miR-16 (black) or PBS (grey). Values are the average of triplicate measurements from four animals. Error bars, s.e.m. (n = 12). *P < 0.05 [Mann–Whitney Rank Sum test (d and e), t-test (f), n.s.: not significant].
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gkt515-F4: Validation of the biodistribution obtained for AMO-miR-16 using CL-qPCR. (a) Chemical reaction that occurs during the side-by-side target hybridization of a DNA-oligo containing a 3′-phosphorothioate group and a DNA-oligo containing a 5′-biphenylsulphonlyl group. The template-mediated, proximity-dependent reaction results in displacement of the biphenylsulphonyl leaving group and the formation of a carbon–sulphur bond. (BASE represents Adenosine, Guanosine, Thymidine or Cytidine). (b) Quantification of AMO-miR-16 in plasma isolated from AMO-miR-16- (black) and PBS-treated (grey) mice. Values are the average of triplicate measurements from five animals. Error bars, s.e.m (n = 15). *P < 0.05 (Mann–Whitney Rank Sum test). (c) The biodistribution of miR-16 and AMO-miR-16 were determined in mouse whole body sections, treated either with AMO-miR-16 (left panel) or with PBS (right panel) and normalized against genomic 18S. Tissue annotation: Eye (E), Brain (B), Lung (Lu), Heart (H), Liver (Li), Stomach (St), Kidney (K), Bone (Bo) and Gastro-Intestinal tract (GI). (d–f) Quantification of AMO-miR-16 (d), miR-16 (e) and miR-191 (f) in tissues isolated from mice treated either with AMO-miR-16 (black) or PBS (grey). Values are the average of triplicate measurements from four animals. Error bars, s.e.m. (n = 12). *P < 0.05 [Mann–Whitney Rank Sum test (d and e), t-test (f), n.s.: not significant].

Mentions: Most therapeutic oligonucleotides contain various degrees of chemical modifications to confer appropriate characteristics such as nuclease resistance, affinity, specificity, safety, distribution and cellular uptake. Although chemical modifications can improve the pharmacokinetic and dynamic properties of these oligonucleotides, they also pose analytical challenges. For example, a 2′-o-Methyl-modified anti-miRNA-16 oligonucleotide (AMO-miR-16) is readily detectable by RT-qPCR (30) whereas a 2′-o-(2-methoxyethyl)-modified sequence is not (Supplementary Figure S6). For this reason, we developed the CL-qPCR assay; a two-step assay that uses qPCR to quantify the amount of product formed in a self-directed chemical-ligation of two oligodeoxynucleotides templated by the fully complementary analyte (11,31) (Supplementary Figure S7a), independently of the sequence (Supplementary Figure S7b) or the chemical modifications present in the template (Supplementary Figure S8). During the first step, binding of the two DNA oligonucleotides, side-by-side, on the target initiates the reaction of the nucleophilic phosphorothioate group, located on the 3′-end of one oligonucleotide, with the electrophilic carbon at the 5′-end of the adjacent oligonucleotide. This proximity-dependent reaction results in displacement of the sulphonate leaving group and the formation of a carbon–sulphur bond, covalently linking the two oligonucleotides into a single unique DNA-oligonucleotide (Figure 4a), which can be quantified by PCR, despite the carbon–sulphur bond being somewhat longer than the carbon–oxygen bond of regular DNA.Figure 4.


Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides.

Boos JA, Kirk DW, Piccolotto ML, Zuercher W, Gfeller S, Neuner P, Dattler A, Wishart WL, Von Arx F, Beverly M, Christensen J, Litherland K, van de Kerkhof E, Swart PJ, Faller T, Beyerbach A, Morrissey D, Hunziker J, Beuvink I - Nucleic Acids Res. (2013)

Validation of the biodistribution obtained for AMO-miR-16 using CL-qPCR. (a) Chemical reaction that occurs during the side-by-side target hybridization of a DNA-oligo containing a 3′-phosphorothioate group and a DNA-oligo containing a 5′-biphenylsulphonlyl group. The template-mediated, proximity-dependent reaction results in displacement of the biphenylsulphonyl leaving group and the formation of a carbon–sulphur bond. (BASE represents Adenosine, Guanosine, Thymidine or Cytidine). (b) Quantification of AMO-miR-16 in plasma isolated from AMO-miR-16- (black) and PBS-treated (grey) mice. Values are the average of triplicate measurements from five animals. Error bars, s.e.m (n = 15). *P < 0.05 (Mann–Whitney Rank Sum test). (c) The biodistribution of miR-16 and AMO-miR-16 were determined in mouse whole body sections, treated either with AMO-miR-16 (left panel) or with PBS (right panel) and normalized against genomic 18S. Tissue annotation: Eye (E), Brain (B), Lung (Lu), Heart (H), Liver (Li), Stomach (St), Kidney (K), Bone (Bo) and Gastro-Intestinal tract (GI). (d–f) Quantification of AMO-miR-16 (d), miR-16 (e) and miR-191 (f) in tissues isolated from mice treated either with AMO-miR-16 (black) or PBS (grey). Values are the average of triplicate measurements from four animals. Error bars, s.e.m. (n = 12). *P < 0.05 [Mann–Whitney Rank Sum test (d and e), t-test (f), n.s.: not significant].
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Related In: Results  -  Collection

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gkt515-F4: Validation of the biodistribution obtained for AMO-miR-16 using CL-qPCR. (a) Chemical reaction that occurs during the side-by-side target hybridization of a DNA-oligo containing a 3′-phosphorothioate group and a DNA-oligo containing a 5′-biphenylsulphonlyl group. The template-mediated, proximity-dependent reaction results in displacement of the biphenylsulphonyl leaving group and the formation of a carbon–sulphur bond. (BASE represents Adenosine, Guanosine, Thymidine or Cytidine). (b) Quantification of AMO-miR-16 in plasma isolated from AMO-miR-16- (black) and PBS-treated (grey) mice. Values are the average of triplicate measurements from five animals. Error bars, s.e.m (n = 15). *P < 0.05 (Mann–Whitney Rank Sum test). (c) The biodistribution of miR-16 and AMO-miR-16 were determined in mouse whole body sections, treated either with AMO-miR-16 (left panel) or with PBS (right panel) and normalized against genomic 18S. Tissue annotation: Eye (E), Brain (B), Lung (Lu), Heart (H), Liver (Li), Stomach (St), Kidney (K), Bone (Bo) and Gastro-Intestinal tract (GI). (d–f) Quantification of AMO-miR-16 (d), miR-16 (e) and miR-191 (f) in tissues isolated from mice treated either with AMO-miR-16 (black) or PBS (grey). Values are the average of triplicate measurements from four animals. Error bars, s.e.m. (n = 12). *P < 0.05 [Mann–Whitney Rank Sum test (d and e), t-test (f), n.s.: not significant].
Mentions: Most therapeutic oligonucleotides contain various degrees of chemical modifications to confer appropriate characteristics such as nuclease resistance, affinity, specificity, safety, distribution and cellular uptake. Although chemical modifications can improve the pharmacokinetic and dynamic properties of these oligonucleotides, they also pose analytical challenges. For example, a 2′-o-Methyl-modified anti-miRNA-16 oligonucleotide (AMO-miR-16) is readily detectable by RT-qPCR (30) whereas a 2′-o-(2-methoxyethyl)-modified sequence is not (Supplementary Figure S6). For this reason, we developed the CL-qPCR assay; a two-step assay that uses qPCR to quantify the amount of product formed in a self-directed chemical-ligation of two oligodeoxynucleotides templated by the fully complementary analyte (11,31) (Supplementary Figure S7a), independently of the sequence (Supplementary Figure S7b) or the chemical modifications present in the template (Supplementary Figure S8). During the first step, binding of the two DNA oligonucleotides, side-by-side, on the target initiates the reaction of the nucleophilic phosphorothioate group, located on the 3′-end of one oligonucleotide, with the electrophilic carbon at the 5′-end of the adjacent oligonucleotide. This proximity-dependent reaction results in displacement of the sulphonate leaving group and the formation of a carbon–sulphur bond, covalently linking the two oligonucleotides into a single unique DNA-oligonucleotide (Figure 4a), which can be quantified by PCR, despite the carbon–sulphur bond being somewhat longer than the carbon–oxygen bond of regular DNA.Figure 4.

Bottom Line: Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides.Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image.Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research (NIBR), Novartis Pharma AG, Basel, Basel-Stadt CH-4056, Switzerland and NIBR, Novartis Pharma AG, Cambridge, Massachusetts, MA 02139, USA.

ABSTRACT
Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

Show MeSH