Limits...
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.

Show MeSH

Related in: MedlinePlus

Post-synthetic introduction of primer binding sites for amplification. (A) Polyadenylation of the 3′-terminus of RNA on bead. After introduction of the poly(A)-tail, reverse transcription can be performed using an anchored poly(T) primer (grey arrow). (B) Adapter ligation to the 3′-end of the generated cDNA with T4 DNA ligase. The 3′-CCC overhang of the double-stranded adapter (in blue) hybridizes only to full length product exhibiting three guanosines at the 3′-terminus. Subsequent amplification by PCR using an adapter-specific and an anchored poly(T)-primer (grey arrows) yields a product that can be subjected to blunt end cloning and sequencing.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351178&req=5

gks004-F2: Post-synthetic introduction of primer binding sites for amplification. (A) Polyadenylation of the 3′-terminus of RNA on bead. After introduction of the poly(A)-tail, reverse transcription can be performed using an anchored poly(T) primer (grey arrow). (B) Adapter ligation to the 3′-end of the generated cDNA with T4 DNA ligase. The 3′-CCC overhang of the double-stranded adapter (in blue) hybridizes only to full length product exhibiting three guanosines at the 3′-terminus. Subsequent amplification by PCR using an adapter-specific and an anchored poly(T)-primer (grey arrows) yields a product that can be subjected to blunt end cloning and sequencing.

Mentions: To amplify unknown RNA sequences, it is necessary to introduce a first primer binding site at the 3′-terminus to enable reverse transcription, and then a second primer binding site at the 3′-end of the respective cDNA to perform PCR. Our strategy consists of adding a homopolymeric tail to the 3′-terminus of the resin-bound RNA sequence by E. coli poly(A) polymerase, to allow for the use of a poly(T) primer during reverse transcription (Figure 2A). For the introduction of the second primer binding site at the 3′-end of the respective cDNA, a specific problem had to be taken into account: solid phase RNA synthesis always yields incomplete sequences due to non-quantitative coupling efficiencies. This leads to beads that carry shorter, abortive sequences next to the full-length product. To ensure that only the full-length product is amplified, we introduced three cytidines at the 5′-end of the RNA strands, which during RT are converted to three deoxyguanosines at the 3′-end of the cDNA. These deoxyguanosines serve as anchor for the 3′-overhang of a double-stranded DNA-adapter which in turn is ligated by T4 DNA ligase (Figure 2B). Hence, only the full length sequences will carry the second primer, and will therefore be amplified. The three additional cytidines have a double role: to allow for the introduction of the second primer, and to exclude abortive sequences. The PCR product obtained by using the two introduced primers can then be subjected to blunt end cloning and sequencing.Figure 2.


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

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

Post-synthetic introduction of primer binding sites for amplification. (A) Polyadenylation of the 3′-terminus of RNA on bead. After introduction of the poly(A)-tail, reverse transcription can be performed using an anchored poly(T) primer (grey arrow). (B) Adapter ligation to the 3′-end of the generated cDNA with T4 DNA ligase. The 3′-CCC overhang of the double-stranded adapter (in blue) hybridizes only to full length product exhibiting three guanosines at the 3′-terminus. Subsequent amplification by PCR using an adapter-specific and an anchored poly(T)-primer (grey arrows) yields a product that can be subjected to blunt end cloning and sequencing.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks004-F2: Post-synthetic introduction of primer binding sites for amplification. (A) Polyadenylation of the 3′-terminus of RNA on bead. After introduction of the poly(A)-tail, reverse transcription can be performed using an anchored poly(T) primer (grey arrow). (B) Adapter ligation to the 3′-end of the generated cDNA with T4 DNA ligase. The 3′-CCC overhang of the double-stranded adapter (in blue) hybridizes only to full length product exhibiting three guanosines at the 3′-terminus. Subsequent amplification by PCR using an adapter-specific and an anchored poly(T)-primer (grey arrows) yields a product that can be subjected to blunt end cloning and sequencing.
Mentions: To amplify unknown RNA sequences, it is necessary to introduce a first primer binding site at the 3′-terminus to enable reverse transcription, and then a second primer binding site at the 3′-end of the respective cDNA to perform PCR. Our strategy consists of adding a homopolymeric tail to the 3′-terminus of the resin-bound RNA sequence by E. coli poly(A) polymerase, to allow for the use of a poly(T) primer during reverse transcription (Figure 2A). For the introduction of the second primer binding site at the 3′-end of the respective cDNA, a specific problem had to be taken into account: solid phase RNA synthesis always yields incomplete sequences due to non-quantitative coupling efficiencies. This leads to beads that carry shorter, abortive sequences next to the full-length product. To ensure that only the full-length product is amplified, we introduced three cytidines at the 5′-end of the RNA strands, which during RT are converted to three deoxyguanosines at the 3′-end of the cDNA. These deoxyguanosines serve as anchor for the 3′-overhang of a double-stranded DNA-adapter which in turn is ligated by T4 DNA ligase (Figure 2B). Hence, only the full length sequences will carry the second primer, and will therefore be amplified. The three additional cytidines have a double role: to allow for the introduction of the second primer, and to exclude abortive sequences. The PCR product obtained by using the two introduced primers can then be subjected to blunt end cloning and sequencing.Figure 2.

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