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

PCR products obtained from different on-bead sequences (Table 1) after introduction of primer binding sites. M, base pair ladder. Lane 1, negative control without template. Lane 2, RNA No. 1; Lane 3, RNA No. 2; Lane 4, RNA No. 3; Lane 5, RNA No. 4; Lane 6, RNA No. 5. All PCR products show the expected length.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks004-F3: PCR products obtained from different on-bead sequences (Table 1) after introduction of primer binding sites. M, base pair ladder. Lane 1, negative control without template. Lane 2, RNA No. 1; Lane 3, RNA No. 2; Lane 4, RNA No. 3; Lane 5, RNA No. 4; Lane 6, RNA No. 5. All PCR products show the expected length.

Mentions: As analytical monitoring options are limited for resin-bound RNA, we first optimized the combination of tailing and reverse transcription in solution using a known RNA sequence (data not shown). We found that an anchored primer of 23 thymidines and two constant nucleotides that match the first 2 nt at the 3′-end of the oligonucleotide was ideally suited to produce a single-length reverse transcript starting from the heterogenous poly(A) tail. Finally, this process was applied to RNA being attached to Tentagel beads: it remained to be shown whether both the poly(A) polymerase and the reverse-transcriptase would tolerate the presence of the solid phase in close proximity to the RNA to be modified. To investigate the general applicability of this methodology, we applied it to the five different RNA sequences synthesized on five different pre-loaded resins (10 and 90 µm, high loading and low loading; Table 1). One sequence (No. 5) was designed to include a C-triplet within the variable region, to test if such a triplet would interfere with the adapter ligation. The following steps were applied to three to five single beads out of every sample. Each bead was processed individually since the sequence determination during on-bead screening procedures has to be carried out from a single bead. A bead picked under a light microscope was subjected to polyadenylation and subsequent reverse transcription in a one-pot reaction. Combining two steps in one reaction vessel required buffer adjustment but decreased the number of pipetting steps and thereby reduced the risk of losing the bead. After isolation, the bead was suspended in water in a fresh vial and heated to 95°C to denature the RNA/cDNA hybrid. The adapter ligation was then performed with a ∼50-fold excess of double-stranded adapter over cDNA using T4 DNA ligase. For subsequent amplification, 5% of the crude ligation mixture was subjected to PCR using an adapter-specific primer and an anchored poly(T) primer. Irrespective of sequence, length, bead size and loading capacity, all five reactions yielded a PCR product of the expected length (Figure 3). No differences between individual beads that carry the same sequence could be detected. Even sequence 5, containing an internal C-triplet, produced a full-length PCR product and none of the shorter sequences that would have been expected in case of hybridization of the adapter to the internal C-triplet. Importantly, the efficiency of the method is insensitive to the folding of the RNA molecules. In principle, folding could block sequence segments involved in tailing and adapter ligation. In spite of different folding patterns (prediction by mfold, data not shown), all five sequences were successfully obtained after sequencing. Hence, the method described here seems to be applicable regardless of the folding pattern. In all PCR reactions (except for RNA 4), a single, faint by-product has been observed in some cases that might be caused by adapter amplification. Subsequent gel purification of the PCR product, however, quantitatively removed the by-product, and blunt end cloning and sequencing yielded in all five cases the expected sequences as shown in Table 2, thus demonstrating that this strategy is a reliable and versatile method for the sequencing of unknown on-bead RNA sequences. It should be noted that gel purification of the PCR product is mandatory for a clean sequencing. Omitting this step, e.g. using the crude PCR product and performing T/A cloning, yielded several sequences with extended deletions.Figure 3.


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

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

PCR products obtained from different on-bead sequences (Table 1) after introduction of primer binding sites. M, base pair ladder. Lane 1, negative control without template. Lane 2, RNA No. 1; Lane 3, RNA No. 2; Lane 4, RNA No. 3; Lane 5, RNA No. 4; Lane 6, RNA No. 5. All PCR products show the expected length.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks004-F3: PCR products obtained from different on-bead sequences (Table 1) after introduction of primer binding sites. M, base pair ladder. Lane 1, negative control without template. Lane 2, RNA No. 1; Lane 3, RNA No. 2; Lane 4, RNA No. 3; Lane 5, RNA No. 4; Lane 6, RNA No. 5. All PCR products show the expected length.
Mentions: As analytical monitoring options are limited for resin-bound RNA, we first optimized the combination of tailing and reverse transcription in solution using a known RNA sequence (data not shown). We found that an anchored primer of 23 thymidines and two constant nucleotides that match the first 2 nt at the 3′-end of the oligonucleotide was ideally suited to produce a single-length reverse transcript starting from the heterogenous poly(A) tail. Finally, this process was applied to RNA being attached to Tentagel beads: it remained to be shown whether both the poly(A) polymerase and the reverse-transcriptase would tolerate the presence of the solid phase in close proximity to the RNA to be modified. To investigate the general applicability of this methodology, we applied it to the five different RNA sequences synthesized on five different pre-loaded resins (10 and 90 µm, high loading and low loading; Table 1). One sequence (No. 5) was designed to include a C-triplet within the variable region, to test if such a triplet would interfere with the adapter ligation. The following steps were applied to three to five single beads out of every sample. Each bead was processed individually since the sequence determination during on-bead screening procedures has to be carried out from a single bead. A bead picked under a light microscope was subjected to polyadenylation and subsequent reverse transcription in a one-pot reaction. Combining two steps in one reaction vessel required buffer adjustment but decreased the number of pipetting steps and thereby reduced the risk of losing the bead. After isolation, the bead was suspended in water in a fresh vial and heated to 95°C to denature the RNA/cDNA hybrid. The adapter ligation was then performed with a ∼50-fold excess of double-stranded adapter over cDNA using T4 DNA ligase. For subsequent amplification, 5% of the crude ligation mixture was subjected to PCR using an adapter-specific primer and an anchored poly(T) primer. Irrespective of sequence, length, bead size and loading capacity, all five reactions yielded a PCR product of the expected length (Figure 3). No differences between individual beads that carry the same sequence could be detected. Even sequence 5, containing an internal C-triplet, produced a full-length PCR product and none of the shorter sequences that would have been expected in case of hybridization of the adapter to the internal C-triplet. Importantly, the efficiency of the method is insensitive to the folding of the RNA molecules. In principle, folding could block sequence segments involved in tailing and adapter ligation. In spite of different folding patterns (prediction by mfold, data not shown), all five sequences were successfully obtained after sequencing. Hence, the method described here seems to be applicable regardless of the folding pattern. In all PCR reactions (except for RNA 4), a single, faint by-product has been observed in some cases that might be caused by adapter amplification. Subsequent gel purification of the PCR product, however, quantitatively removed the by-product, and blunt end cloning and sequencing yielded in all five cases the expected sequences as shown in Table 2, thus demonstrating that this strategy is a reliable and versatile method for the sequencing of unknown on-bead RNA sequences. It should be noted that gel purification of the PCR product is mandatory for a clean sequencing. Omitting this step, e.g. using the crude PCR product and performing T/A cloning, yielded several sequences with extended deletions.Figure 3.

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