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An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries.

Wiesmayr A, Fournier P, Jäschke A - Nucleic Acids Res. (2012)

Bottom Line: The cDNA is joined to a DNA adapter by T4 DNA ligase.PCR amplification yielded single-band products that could be cloned and sequenced starting from individual polystyrene beads.The method described here makes the selection of functional RNAs from on-bead RNA libraries more attractive due to increased flexibility in library design, higher yields of full-length sequence on bead and robust sequence determination.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, Heidelberg 69120, Germany.

ABSTRACT
Nucleic acids possess the unique property of being enzymatically amplifiable, and have therefore been a popular choice for the combinatorial selection of functional sequences, such as aptamers or ribozymes. However, amplification typically requires known sequence segments that serve as primer binding sites, which can be limiting for certain applications, like the screening of on-bead libraries. Here, we report a method to amplify and sequence on-bead RNA libraries that requires not more than five known nucleotides. A key element is the attachment of the starting nucleoside to the synthesis resin via the nucleobase, which leaves the 3'-OH group accessible to subsequent enzymatic manipulations. After split-and-mix synthesis of the oligonucleotide library and deprotection, a poly(A)-tail can be efficiently added to this free 3'-hydroxyl terminus by Escherichia coli poly(A) polymerase that serves as an anchored primer binding site for reverse transcription. The cDNA is joined to a DNA adapter by T4 DNA ligase. PCR amplification yielded single-band products that could be cloned and sequenced starting from individual polystyrene beads. The method described here makes the selection of functional RNAs from on-bead RNA libraries more attractive due to increased flexibility in library design, higher yields of full-length sequence on bead and robust sequence determination.

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Related in: MedlinePlus

Loading capacity (µmol/g) of pre-loaded Tentagel resins (10 and 90 µm bead size) after coupling to 2 as determined by absorption measurement (498 nm) of cleaved DMT groups. The different values were obtained by variation of the molar ratio 2/resin amino groups. The nominal loading capacity of both resins before coupling was 220 µmol/g.
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gks004-F1: Loading capacity (µmol/g) of pre-loaded Tentagel resins (10 and 90 µm bead size) after coupling to 2 as determined by absorption measurement (498 nm) of cleaved DMT groups. The different values were obtained by variation of the molar ratio 2/resin amino groups. The nominal loading capacity of both resins before coupling was 220 µmol/g.

Mentions: A solid support suitable for reaction with 2 should not only bear a primary amino group, but also be compatible with solid-phase oligonucleotide synthesis as well as with on-bead screening procedures. Controlled pore glass (CPG), the most commonly used support for nucleic acid synthesis, lacks the mechanical stability and homogeneity in size that is required for on-bead screening procedures (30). In contrast, polystyrene resins are available in different bead sizes with a uniform particle size distribution and a high mechanical stability. We therefore opted for the polystyrene-based, swellable Tentagel resin. This solid support is chemically compatible with all used reagents, and it is equally swellable in all solvents used. This type of resin has frequently been used in the preparation of combinatorial libraries of peptides (30) and has also been applied to the synthesis of OBOC DNA libraries (31). The coupling of 2 was performed on Tentagel-NH2 with two different bead sizes: 10 and 90 µm. After allowing the Tentagel-NH2 to pre-swell in DMF, the beads were suspended in solutions of 2 in DMSO at various concentrations. Addition of 6% DMF facilitated a homogeneous suspension of the beads. After incubation at 40°C overnight and several washing steps, unreacted amino groups were blocked by capping with tert-butylphenoxyacetyl acetic anhydride. The coupling yield of 2 to the resin and the respective loading capacity of each resin were determined by cleavage of the dimethoxytrityl (DMT) group and measurement of its absorption at 498 nm (Figure 1). The 90 µm beads showed better coupling yields than the 10 µm ones. By varying the molar ratio of 2 to resin amino groups, coupling yields could be obtained ranging from 8.3% (Tentagel 10 µm, 1:1 ratio) to quantitative coupling (Tentagel 90 µm, 1:10 ratio). It is therefore possible to adjust the loading capacity of the resin to a desired value depending on the application. For example, a high loading capacity may be beneficial for obtaining high signals during the screening process, while a low loading capacity can be desirable for preventing misfolding, improper binding of a target molecule (32) or quenching of fluorophores (33).Figure 1.


An on-bead tailing/ligation approach for sequencing resin-bound RNA libraries.

Wiesmayr A, Fournier P, Jäschke A - Nucleic Acids Res. (2012)

Loading capacity (µmol/g) of pre-loaded Tentagel resins (10 and 90 µm bead size) after coupling to 2 as determined by absorption measurement (498 nm) of cleaved DMT groups. The different values were obtained by variation of the molar ratio 2/resin amino groups. The nominal loading capacity of both resins before coupling was 220 µmol/g.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351178&req=5

gks004-F1: Loading capacity (µmol/g) of pre-loaded Tentagel resins (10 and 90 µm bead size) after coupling to 2 as determined by absorption measurement (498 nm) of cleaved DMT groups. The different values were obtained by variation of the molar ratio 2/resin amino groups. The nominal loading capacity of both resins before coupling was 220 µmol/g.
Mentions: A solid support suitable for reaction with 2 should not only bear a primary amino group, but also be compatible with solid-phase oligonucleotide synthesis as well as with on-bead screening procedures. Controlled pore glass (CPG), the most commonly used support for nucleic acid synthesis, lacks the mechanical stability and homogeneity in size that is required for on-bead screening procedures (30). In contrast, polystyrene resins are available in different bead sizes with a uniform particle size distribution and a high mechanical stability. We therefore opted for the polystyrene-based, swellable Tentagel resin. This solid support is chemically compatible with all used reagents, and it is equally swellable in all solvents used. This type of resin has frequently been used in the preparation of combinatorial libraries of peptides (30) and has also been applied to the synthesis of OBOC DNA libraries (31). The coupling of 2 was performed on Tentagel-NH2 with two different bead sizes: 10 and 90 µm. After allowing the Tentagel-NH2 to pre-swell in DMF, the beads were suspended in solutions of 2 in DMSO at various concentrations. Addition of 6% DMF facilitated a homogeneous suspension of the beads. After incubation at 40°C overnight and several washing steps, unreacted amino groups were blocked by capping with tert-butylphenoxyacetyl acetic anhydride. The coupling yield of 2 to the resin and the respective loading capacity of each resin were determined by cleavage of the dimethoxytrityl (DMT) group and measurement of its absorption at 498 nm (Figure 1). The 90 µm beads showed better coupling yields than the 10 µm ones. By varying the molar ratio of 2 to resin amino groups, coupling yields could be obtained ranging from 8.3% (Tentagel 10 µm, 1:1 ratio) to quantitative coupling (Tentagel 90 µm, 1:10 ratio). It is therefore possible to adjust the loading capacity of the resin to a desired value depending on the application. For example, a high loading capacity may be beneficial for obtaining high signals during the screening process, while a low loading capacity can be desirable for preventing misfolding, improper binding of a target molecule (32) or quenching of fluorophores (33).Figure 1.

Bottom Line: The cDNA is joined to a DNA adapter by T4 DNA ligase.PCR amplification yielded single-band products that could be cloned and sequenced starting from individual polystyrene beads.The method described here makes the selection of functional RNAs from on-bead RNA libraries more attractive due to increased flexibility in library design, higher yields of full-length sequence on bead and robust sequence determination.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, Heidelberg 69120, Germany.

ABSTRACT
Nucleic acids possess the unique property of being enzymatically amplifiable, and have therefore been a popular choice for the combinatorial selection of functional sequences, such as aptamers or ribozymes. However, amplification typically requires known sequence segments that serve as primer binding sites, which can be limiting for certain applications, like the screening of on-bead libraries. Here, we report a method to amplify and sequence on-bead RNA libraries that requires not more than five known nucleotides. A key element is the attachment of the starting nucleoside to the synthesis resin via the nucleobase, which leaves the 3'-OH group accessible to subsequent enzymatic manipulations. After split-and-mix synthesis of the oligonucleotide library and deprotection, a poly(A)-tail can be efficiently added to this free 3'-hydroxyl terminus by Escherichia coli poly(A) polymerase that serves as an anchored primer binding site for reverse transcription. The cDNA is joined to a DNA adapter by T4 DNA ligase. PCR amplification yielded single-band products that could be cloned and sequenced starting from individual polystyrene beads. The method described here makes the selection of functional RNAs from on-bead RNA libraries more attractive due to increased flexibility in library design, higher yields of full-length sequence on bead and robust sequence determination.

Show MeSH
Related in: MedlinePlus