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280 Direct Sequencing of Nucleic Acids without Clonal Amplification or Synthesis for the Molecular Characterization of Cancer

Number of anticipated awards: 2

(Fast-Track proposals will be accepted.)

Budget (total costs): Phase I: $150,000;
Phase II: $1,000,000

(Note: Proposals with budgets exceeding the above amounts will be returned without review. Phase I project periods may last a maximum of 9 months. Proposals with durations exceeding 9 months will be returned without review.)

The deadline for receipt of all contract proposals submitted in response to this solicitation is: November 9, 2009.

Nucleotides in DNA and RNA are frequently modified by exogenous and endogenous insults, leading to a broad spectrum of chemical modifications (e.g., oxidation, alkylation, formation of cyclobutane pyrimidine dimers, etc). These chemical modifications can significantly alter the functional expression of genes in vivo, resulting in genome instability and variations in the sequence of a transcript and/or polypeptide. Moreover, knowledge of these sequence variations in normal, premalignant, and malignant cells in a human biospecimen may provide critical information in understanding the molecular basis of cancer. Unfortunately, interrogating such sequence variations is presently intractable because the currently available high-throughput methods for nucleic acid sequencing depend on enzymatic clonal amplification using high-fidelity polymerases. In particular, these methods conceal many DNA lesions inherent to the original nucleic acid sequence by incorporating a non-damaged nucleotide opposite the adduct (mismatch, etc), followed by amplification of this undamaged sequence. In effect, this amplification step masks the underlying information contained in the original sequence. Therefore, to understand the true functional nature of the individual genome and/or transcriptome, new high-throughput sequencing techniques are required that are highly sensitive and specific at the single molecule level. Such methods must also be capable of detecting nucleic acid adducts to obtain an accurate read of the non-amplified genome or transcriptome.

Project Goals:
The primary goal of this topic is to encourage small businesses to develop novel high-throughput technologies that will enable researchers to directly capture nucleic acid sequence information without the need for amplification. Such technologies are expected to aid researchers in identifying specific and predominant variants involved in the development of disease phenotype(s). In particular, sequence information of DNA, messenger RNA (mRNA) and/or microRNA (miRNA), which is obtained from human cancer biospecimens, is expected to provide new insights into cancer onset and progression. Future benefits of these technologies (which are beyond the scope of the current topic) may include new approaches for risk assessment in cancer epidemiology, and potentially new cancer prevention and/or therapeutic strategies.

Examples of novel technologies and approaches that might be developed under this topic include the following: near-field optical techniques in combination with nanotip-enhanced vibrational spectroscopy, high throughput molecular imaging techniques, novel hybridization techniques that increase the sensitivity of detection to the single molecule level, and novel nucleic acid stabilization methods. These approaches are not meant to be exhaustive. Offerors are encouraged to develop any appropriate innovative technology to meet the goals of this contract topic. Technologies and approaches aimed at sequencing all of the sequence and splice variants in a cell are of particular interest to NCI.

Phase I activities and expected deliverables:
  • Develop a novel technique to directly capture nucleic acid sequence information without the need for amplification (i.e., develop an approach to obtain sequence information at the single molecule level).
  • Show proof-of-concept by applying the technology to obtain sequence information for one or more of the following: DNA, mRNA and/or miRNA.
  • Demonstrate the ability to successfully detect both unaltered (i.e., non-damaged) nucleotides, as well as cancer-relevant, chemically-altered (i.e., adducted) nucleotides.

Phase II activities and expected deliverables:
  • Apply the technique(s) to biospecimens, such as cancer cell lines.
  • Adapt the techniques for the high-resolution, high-throughput screening of cancer biospecimens.
  • Characterize and demonstrate the accuracy and specificity of the technique/assay.
  • Develop a commercially viable assay that is capable of directly obtaining sequence information for one or more of the following in a cancer biospecimen: DNA, mRNA, and/or miRNA.

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