Reversible Oligonucleotide Chain Blocking Enables Bead Capture and Amplification of T-Cell Receptor α and ß Chain mRNAs.

Next-generation sequencing (NGS) has proven to be an exceptionally powerful tool for studying genetic variation and differences in gene expression profiles between cell populations. However, these population-wide studies are limited by their inability to detect variation between individual cells within a population, inspiring the development of single-cell techniques such as Drop-seq, which add a unique barcode to the mRNA from each cell prior to sequencing. Current Drop-seq technology enables capture, amplification, and barcoding of the entire mRNA transcriptome of individual cells. NGS can then be used to sequence the 3'-end of each message to build a cell-specific transcriptional landscape. However, current technology does not allow high-throughput capture of information distant from the mRNA poly-A tail. Thus, gene profiling would have much greater utility if beads could be generated having multiple transcript-specific capture sequences. Here we report the use of a reversible chain blocking group to enable synthesis of DNA barcoded beads having capture sequences for the constant domains of the T-cell receptor α and ß chain mRNAs. We demonstrate that these beads can be used to capture and pair TCRα and TCRß sequences from total T-cell RNA, enabling reverse transcription and PCR amplification of these sequences. This is the first example of capture beads having more than one capture sequence, and we envision that this technology will be of high utility for applications such as pairing the antigen receptor chains that give rise to autoimmune diseases or measuring the ratios of mRNA splice variants in cancer stem cells.

Rigidification of a flexible protease inhibitor variant upon binding to trypsin.

The Tyr35-->Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by X-ray crystallography and isothermal titration calorimetry (ITC). The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 A resolution X-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 A data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical with that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side-chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity.

Identification of a residue critical for maintaining the functional conformation of BPTI.

The effects of amino acid replacements on the backbone dynamics of bovine pancreatic trypsin inhibitor (BPTI) were examined using 15N NMR relaxation experiments. Previous studies have shown that backbone amide groups within the trypsin-binding region of the wild-type protein undergo conformational exchange processes on the micros time scale, and that replacement of Tyr35 with Gly greatly increases the number of backbone atoms involved in such motions. In order to determine whether these mutational effects are specific to the replacement of this residue with Gly, six additional replacements were examined in the present study. In two of these, Tyr35 was replaced with either Ala or Leu, and the other four were single replacements of Tyr23, Phe33, Asn43 or Asn44, all of which are highly buried in the native structure and conserved in homologous proteins. The Y35A and Y35L mutants displayed dynamic properties very similar to those of the Y35G mutant, with the backbone segments including residues 10-19 and 32-44 undergoing motions revealed by enhanced 15N transverse relaxation rates. On the other hand, the Y23L, N43G and N44A substitutions caused almost no detectable changes in backbone dynamics, on either the ns-ps or ms-micros time scales, even though each of these replacements significantly destabilizes the native conformation. Replacement of Phe33 with Leu caused intermediate effects, with several residues that have previously been implicated in motions in the wild-type protein displaying enhanced transverse relaxation rates. These results demonstrate that destabilizing amino acid replacements can be accommodated in a native protein with dramatically different effects on conformational dynamics and that Tyr35 plays a particularly important role in defining the conformation of the trypsin-binding site of BPTI.