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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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(A) Synthesis of the activated uridine analogue 2: (i) 1. DMT-Cl, pyridine; 2. TBDMS-Cl, imidazole, DMF; (ii) 1,2,4-triazole, POCl3, NEt3, acetonitrile. (B) Coupling scheme of protected 4-triazolyluridine (2) to amino-functionalized resin (for conditions, see ‘Materials and Methods’ section). The covalent linkage is formed between the base moiety of 2 and the amino-groups of the resin, thereby leaving the 3′-OH accessible for subsequent modifications.
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gks004-SCH1: (A) Synthesis of the activated uridine analogue 2: (i) 1. DMT-Cl, pyridine; 2. TBDMS-Cl, imidazole, DMF; (ii) 1,2,4-triazole, POCl3, NEt3, acetonitrile. (B) Coupling scheme of protected 4-triazolyluridine (2) to amino-functionalized resin (for conditions, see ‘Materials and Methods’ section). The covalent linkage is formed between the base moiety of 2 and the amino-groups of the resin, thereby leaving the 3′-OH accessible for subsequent modifications.


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

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

(A) Synthesis of the activated uridine analogue 2: (i) 1. DMT-Cl, pyridine; 2. TBDMS-Cl, imidazole, DMF; (ii) 1,2,4-triazole, POCl3, NEt3, acetonitrile. (B) Coupling scheme of protected 4-triazolyluridine (2) to amino-functionalized resin (for conditions, see ‘Materials and Methods’ section). The covalent linkage is formed between the base moiety of 2 and the amino-groups of the resin, thereby leaving the 3′-OH accessible for subsequent modifications.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks004-SCH1: (A) Synthesis of the activated uridine analogue 2: (i) 1. DMT-Cl, pyridine; 2. TBDMS-Cl, imidazole, DMF; (ii) 1,2,4-triazole, POCl3, NEt3, acetonitrile. (B) Coupling scheme of protected 4-triazolyluridine (2) to amino-functionalized resin (for conditions, see ‘Materials and Methods’ section). The covalent linkage is formed between the base moiety of 2 and the amino-groups of the resin, thereby leaving the 3′-OH accessible for subsequent modifications.
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